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Advances in Experimental Medicine and Biology 931

Advances in Microbiology, Infectious Diseases and Public Health

ChristineImbert Editor

and Related
Volume 3
Advances in Experimental Medicine
and Biology
Advances in Microbiology, Infectious Diseases
and Public Health

Volume 931

Editorial Board
Irun R. Cohen, The Weizmann Institute of Science, Rehovot, Israel
N.S. Abel Lajtha, Kline Institute for Psychiatric Research, Orangeburg, NY, USA
John D. Lambris, University of Pennsylvania, Philadelphia, PA, USA
Rodolfo Paoletti, University of Milan, Milan, Italy

Subseries Editor
Gianfranco Donelli, Microbial Biofilm Laboratory, Fondazione Santa Lucia
IRCCS, Rome, Italy
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Christine Imbert

Fungal Biofilms and

Related Infections
Advances in Microbiology,
Infectious Diseases and
Public Health Volume 3
Christine Imbert
University of Poitiers
Laboratory Ecology & Biology of Interactions
Poitiers, France

ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-319-42359-3 ISBN 978-3-319-42360-9 (eBook)
DOI 10.1007/978-3-319-42360-9

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Aspergillus Biofilms in Human Disease . . . . . . . . . . . . . . . . . . . . . 1

Craig Williams, Ranjith Rajendran, and Gordon Ramage
Candida albicans in Multispecies Oral Communities;
A Keystone Commensal? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Marleen M. Janus, Hubertine M.E. Willems, and Bastiaan P. Krom
The Extracellular Matrix of Fungal Biofilms . . . . . . . . . . . . . . . . 21
Kaitlin F. Mitchell, Robert Zarnowski, and David R. Andes
Fungal Biofilms: Update on Resistance . . . . . . . . . . . . . . . . . . . . . 37
Elisa Borghi, Francesca Borgo, and Giulia Morace
Fungi, Water Supply and Biofilms . . . . . . . . . . . . . . . . . . . . . . . . 49
Catherine KauffmannLacroix, Damien Costa, and Christine Imbert
Diagnostic of Fungal Infections Related to Biofilms . . . . . . . . . . . 63
Maurizio Sanguinetti and Brunella Posteraro
Disinfectants to Fight Oral Candida Biofilms . . . . . . . . . . . . . . . . 83
M. Elisa Rodrigues, Mariana Henriques, and Sonia Silva
Updates on Therapeutic Strategies Against Candida
(and Aspergillus) Biofilm Related Infections . . . . . . . . . . . . . . . . . 95
Fuad Kamel Muakkassa and Mahmoud Ghannoum
Natural Sources as Innovative Solutions Against
Fungal Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Marion Girardot and Christine Imbert

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2016) 3: 111
DOI 10.1007/5584_2016_4
# Springer International Publishing Switzerland 2016
Published online: 7 June 2016

Aspergillus Biofilms in Human Disease

Craig Williams, Ranjith Rajendran, and Gordon Ramage

The biofilm phenotype of Aspergillus species is an important and accepted
clinical entity. While industrially these biofilms have been used exten-
sively in important biofermentations, their role in clinical infection is less
well defined. A recent flurry of activity has demonstrated that these
interesting filamentous moulds have the capacity to form biofilms both
in vitro and in vivo, and through various investigations have shown that
these are exquisitely resistant to antifungal therapies through a range of
adaptive resistance mechanisms independent of defined genetic changes.
This review will explore the clinical importance of these biofilms and
provide contemporary information with respect to their clinical

Aspergillus biofilm Filamentous moulds Fungal infections
Aspergillosis Antifungal drugs

1 Introduction broad-spectrum antibiotics, and an aging and

more immuno-compromised patient population,
Fungal biofilms are an important clinical prob- has created an opportunity for yeasts and moulds
lem (Ramage and Williams. 2013). The wide- to form infections in the form of biofilms. This
spread use of indwelling medical devices, chapter will discuss the anatomical areas where
Aspergillus biofilms may be important and the
C. Williams (*) evidence that they exist and discuss the treatment
Institute of Healthcare Policy and Practice, University of of aspergillus infection.
West of Scotland, High St, Paisley PA1 2BE, UK A biofilm is composed of microorganisms
attached to surfaces or one another and enclosed
R. Rajendran and G. Ramage within an extrapolymeric matrix. The biofilm
Infection and Immunity Research Group, Glasgow Dental
mode of growth is the preferred form of growth
School and Hospital, School of Medicine, College of
Medical, Veterinary and Life Sciences, University of of microorganisms and accounts for up to 65 %
Glasgow, Glasgow, Scotland of all clinical infections. This mode of growth

2 C. Williams et al.

gives the organism a number of advantages antifungal agents. Within the biofilm there are a
including high level antimicrobial resistance range of niches and hypoxic areas, which influ-
which may cause problems for the clinician ence filamentation (Bonhomme et al. 2011).
attempting to treat such infections (Donlan and Flow of fluids across the surface of the biofilm
Costerton. 2002). Over recent years there has may then result in the dispersion of daughter
been a growing appreciation that pathogenic fun- cells, which attach to a new substrate and the
gal species both have the ability to form biofilms cycle starts again (Uppuluri et al. 2010). This
and that these biofilms may impact clinical prac- entire process is controlled by various transcrip-
tice (Ramage et al. 2009; Sayed et al. 2012; Fan- tion factors, such as Bcr1p, Ace2p, Efg1p and
ning and Mitchell. 2012; Williams and Ramage. Zap1p, which are involved in precisely regulated
2015). molecular pathways (Finkel and Mitchell 2011;
Fungi can be broadly divided into yeasts and Nobile et al. 2006; Zhao et al. 2006; Fanning
moulds. In terms of the number of infections, et al. 2012). The molecular biology of Aspergil-
C. albicans, a normal commensal of human lus biofilms is less well understood, but it is a
mucosal surfaces and opportunistic pathogen in rapidly developing area of microbiology
immunocompromised patients, is the most clini- (Beauvais et al. 2007; Bom et al. 2015; Gravelat
cally important of fungi species in terms of the et al. 2008; Gravelat et al. 2010; Mowat
production of clinically relevant biofilms. These et al. 2007; Ramage et al. 2011; Winkelstroter
biofilms have much in common with Aspergillus et al. 2015). Figure 1 illustrates the morphologi-
biofilms, which are the subject of this chapter. A cal complexity of these filamentous moulds.
candidal biofilm begins with yeast cells attaching
to a relevant surface using defined adhesins,
followed by the formation of a microcolony
2 Where May Aspergillus
with yeast cells undergoing morphological
Biofilms Be Important?
switching to pseudo- and true-hyphae, which
results in the rapid formation of a meshwork of
2.1 Upper Airways
hyphae interspersed with budding yeast cells
(Ramage et al. 2002). As the biofilm matures it
Sinusitis (or rhinosinusitis) is defined as an
becomes enclosed in a glucan rich polymeric
inflammation of the mucous membrane lining
matrix (Nett et al. 2010) which provides protec-
the paranasal sinuses. It may be acute or chronic,
tion from host defenses and treatment with
however, subacute and acute exacerbation of

Fig. 1 Scanning electron micrographs of biofilms formed by (a) an Aspergillus fumigatus and (b) an co-culture of
Aspergillus fumigatus and Staphylococcus epidermidis
Aspergillus Biofilms in Human Disease 3

chronic diseases has also been described and as however that fungal biofilms present in the lung
all types have similar symptoms it is often clini- may also contribute to infection.
cally difficult to distinguish these. Around ninety Filamentous fungi, mainly A. fumigatus, may
percent of adults have had some symptoms of cause a spectrum of respiratory disease including
sinusitis at some time. There is a growing appre- a discrete lesion in a pre-existing cavity,
ciation that chronic rhinosinusitis is typified by aspergilloma, wheezing mediated by an immune
biofilm growth (Foreman et al. 2011; Keir response, allergic bronchopulmonary aspergillo-
et al. 2011; Ebbens et al. 2009a). While there is sis (ABPA) and invasive aspergillosis
increasing evidence for the role of bacterial (IA) (Denning. 1998). A bronchopulmonary
biofilms in this infection, there role of fungi lavage (BAL) of these individuals often reveals
remains controversial. (Ebbens et al. 2009b). the presence of numerous intertwined hyphae in
Paranasal sinus fungus balls have been described the form of a complex multicellular structure
(Grosjean and Weber. 2007; Karkas et al. 2013), when examined histologically (Jayshree
which share some of the features of fungal et al. 2006), this is indicative of a biofilm pheno-
biofilms (Harding et al. 2009; Mowat type (Harding et al. 2009; Mowat et al. 2008a, b).
et al. 2008a, b). In a recent study of 118 patients The recently described Aspergillus bronchitis
with chronic sinusitis, nasal discharge, headache may also be biofilm associated and is
and visual disturbance, over a 14 year period characterized by bronchial casts containing
23.7 % had a sphenoidal fungus ball in which mycelia forming compact masses (Young
Aspergillus fumigatus and Aspergillus nidulans et al. 1970). It is clear that Aspergillus species
hyphae were observed microscopically (Karkas form medically important biofilms (Ramage
et al. 2013). In terms of infections associated et al. 2011; Gutierrez-Correa et al. 2012) and
with foreign bodies A. fumigatus infection within understanding their clinical role in is crucial, as
the maxillary sinus associated with a zygomatic with all biofilms, these structures are highly
implant has been reported (Sato et al. 2010). resistant to antifungal therapy (Mowat
Experimental studies have shown that et al. 2008a, b; Seidler et al. 2008).
A. fumigatus biofilms form in a primary human Infection in CF patients is also commonly
sinonasal epithelial model (Singhal et al. 2011) associated with S. aureus and Haemophilus
and in a sheep model of induced sinus biofilms influenzae, and recent advances in culture-
A. fumigatus readily forms biofilms often independent, next generation sequencing
associated with Staphylococcus aureus (Boase technologies, have revealed that the microbiome
et al. 2011). These data suggest that fungal of the CF lung is much richer than previously
biofilms, alone or more likely in mixed species appreciated comprising of a diverse range of
biofilms with other organisms, may play a role in bacterial and fungal pathogens, of which
sinus infection however there is little evidence to A. fumigatus is the most prevalent filamentous
support the role of fungi in other upper airway fungi (Ramage et al. 2011). A. fumigatus has a
biofilm infections such as otitis media (Bakaletz. prevalence rate of between 10 and 57 % (Pihet
2007; Martin et al. 2005; Yao and Messner et al. 2009; Bauernfeind et al. 1987), though
2001). other fungi have been isolated from the lungs
including, Scedosporium species, Aspergillus
niger, Aspergillus flavus, Aspergillus nidulans,
Aspergillus terreus (Sudfeld et al. 2010; Cimon
2.2 Lower Airways
et al. 2003). Lungs of CF sufferers are lined with
a thick viscous mucus layer susceptible to
Lower respiratory tract infection may be due to
polymicrobial infections, leading to recurrent
biofilm infection, the archetype of which is Pseu-
infections and continuous inflammation (Rowe
domonas aeruginosa in cystic fibrosis patients
et al. 2005). The interplay between the pathogens
(Singh et al. 2000). It is also now recognised
residing in the lung may be responsible for the
4 C. Williams et al.

acute exacerbations associated with CF, where understood and requires further investigation
the balance is tipped towards an environment into their polymicrobial interactions. Indeed,
with excess inflammatory, oxidative and proteo- these studies highlight potential battles going on
lytic activity. Several studies have identified an within a polymicrobial environment such as the
association between A. fumigatus and CF lung, which plays a crucial role in the overall
P. aeruginosa, whereby co-infection saw pathogenesis of disease (Peters et al. 2012),
decreased pulmonary function in comparison to exemplified by studies in a Drosophila model of
those with a mono-infection (Amin et al. 2010), a polymicrobial infection in which
phenomenon also reported with Candida species microorganisms from CF showed a different out-
and P. aeruginosa (Chotirmall et al. 2010). Evi- come depending on the presence or absence of
dence is therefore increasing for the improved P. aeruginosa (Sibley et al. 2008a, b). Collec-
clinical management of these patients (Delhaes tively, these provide evidence for the need to
et al. 2012). Indeed, interkingdom interactions of consider earlier use of antifungals to improve
the CF lung, and elsewhere, may lead to adverse clinical management of these patients (Delhaes
clinical outcomes (Leclair and Hogan 2010). The et al. 2012).
ability of these microbes to form strong mixed
species biofilms likely contributes towards their
persistence, making it extremely difficult to erad- 3 Wounds
icate the infection (Seidler et al. 2008; Lutz
et al. 2012). P. aeruginosa has also been shown Non-healing wounds, such as diabetic foot ulcers
to inhibit A. fumigatus filamentation via the (Seth et al. 2012) represent a significant clinical
release of molecules involved in intra-cellular burden to patients, and are associated with the
communication (Mowat et al. 2010). presence of microbial biofilms. S. aureus and
Investigations into the interactions between P. aeruginosa are often isolated together in
these two are limited, however the release of these patients and have been shown to have a
small molecules designed to inhibit fungal non-random association within the wound site
growth appear to be the primary form of interac- (Fazli et al. 2009). Evidence is emerging that
tion. One particular group of metabolites known pathogenic fungal species may play a role in
as phenazines, have been reported to inhibit these infections (Branski et al. 2009).
A. fumigatus biofilm formation, however it was Wounds acquired in combat situations espe-
also found that A. fumigatus was able to convert cially with persistent evidence of wound necrosis
these metabolites released by P. aeruginosa to often contain fungi with mould isolates found in
produce fungal siderophores, which may in turn 83 % of cases (Mucorales, n 16; Aspergillus
influence CF progression (Moree et al. 2012). spp, n 16; Fusarium spp, n 9), commonly
Furthermore, P. aeruginosa releases the with multiple mould species among infected
metalloprotease elastase, which has been shown wounds (28 %). Clinical outcomes included
to be toxic to host cells (Smith et al. 2015). It was 3 related deaths (8.1 %), frequent debridements
found that elastase production was constitutive, and amputation revisions (58 %) (Warkentien
but became significantly increased in the pres- et al. 2012).
ence of A. fumigatus during biofilm co-culture. A next generation sequencing approach to
Furthermore, elastase was cytotoxic to human venous leg ulcers reveals that C. albicans,
lung adenocarcinoma cells, and therefore the C. glabrata and Aspergillus species are present,
presence of both of these pathogens could con- but intriguingly the authors report that
tribute towards enhanced pathogenicity (Smith individuals seem to have unique microbial
et al. 2015). Thus, in general, evidence suggests profiles, (Wolcott et al. 2009). A further retro-
that the co-isolation of both of these organisms spective molecular analysis of 915 chronic
indicates a poorer prognosis; however the rela- wound infections, pressure ulcers, diabetic foot
tionship between the two remains poorly ulcers, non-healing surgical wounds and venous
Aspergillus Biofilms in Human Disease 5

leg ulcers, showed that 208 (23 %) of these deemed failures and an alternative regimen used.
contained pathogenic fungi (Dowd et al. 2011). Micafungin, Caspofungin Liposomal
Yeasts were the most abundant fungi (Candida Amphotericin and Amphotericin B (AmB)
spp.), but Aureobasidium, Cladosporium, deoxycholate are also recommended.
Curvularia, Engodontium, Malessezia, For Invasive aspergillus three major sets of
Trichtophyton, and Ulocladium were also pres- guidelines are available, ECIL (Maertens
ent. Overall, fungal species represented over et al. 2011), ESCMID (Ullmann et al. 2012),
50 % of the microbial burden in the majority of and IDSA (Walsh et al. 2008). Again, none spe-
specimens examined but direct evidence that the cifically reference the presence of biofilm and
fungi were present as biofilms is lacking. they all recommend either azoles, AmB or
echinocandins with different grades of evidence.
A useful review by Leroux and Ullmann (2013)
4 Medical Devices highlight the methodological differences
between these studies and tabulates the
Broad-spectrum antibiotics, parenteral nutrition, recommendations (Leroux and Ullmann 2013).
immuno-suppression due to chemotherapy and So when considering aspergillus infection it is
radiotherapy, and disruption of mucosal barriers clear that AmB, including a variety of lipid
due to surgery, are among the most important formulations and members of the azole class,
predisposing factors for invasive fungal infection which includes fluconazole, itraconazole,
(Odds 1988). Candida species predominate and voriconazole (VRZ) and posaconazole, are effec-
are the fourth most common cause of blood- tive in the treatment of invasive aspergillosis
stream infection in patients requiring intensive (IA) and are the mainstay treatment for the dis-
care and the most common etiologic agent of ease (Denning et al. 1989; Oren et al. 2006; Raad
fungal related biofilm infection. However, other et al. 2008; Sambatakou et al. 2006).
filamentous fungi biofilm related infections have One consideration in the choice of treatment
also been increasingly described, including is the presence of triazole resistance. High rates
Aspergillus (Escande et al. 2011). Aspergillus of triazole resistance in A. fumigatus were first
species have been reported to cause serious bio- reported in the Netherlands and in the
material related biofilm infections, involving UK. Subsequently the rate of triazole resistance
catheters, joint replacements, cardiac pace in A. fumigatus has been reported in Europe at
makers, heart valves, and breast augmentation rates from 1.7 to 29.6 %. More worrying is a
implants (Escande et al. 2011; Langer reported increase in the yearly rate of resistance
et al. 2003; Rosenblatt and Pollock 1997; Jeloka by 6 % per annum in patients without prior expo-
et al. 2011; Golmia et al. 2011). Fungal biofilms sure to antifungal therapy (Goncalves
are also associated with building fabrics and hos- et al. 2016).
pital infrastructure (Short et al. 2011; Siqueira Although the triazoles have proven efficacy
et al. 2011; Richardson 2009; Anaissie with good safety profiles, they have been shown
et al. 2002). to be associated with resistance through their
continuous use (Howard et al. 2009; Meneau
et al. 2005; Mosquera and Denning 2002).
5 Antifungal Treatments Azoles actively target the 14--demethylase
enzyme, blocking ergosterol biosynthesis and
Guidelines exist for a number of Aspergillus destabilizing the cell membranes of actively
infections. The guidance for Chronic Pulmonary growing cells. Mutations within the ergosterol
aspergillosis (Denning et al. 2016), which does biosynthesis pathway have been reported to
not mention biofilms, recommends, a minimum cause azole cross-resistance through mutations
of 46 months oral triazole therapy initially and within the cyp51A gene (Howard et al. 2009;
patients who deteriorate in this period should be Mellado et al. 2007; Snelders et al. 2008;
6 C. Williams et al.

Snelders et al. 2010). However, a recent study which was coincidental with a strain-dependent
reported that 43 % of azole-resistant isolates did increase in azole resistance. This demonstrates
not carry the cyp51A mutation, indicating that that efflux pumps are expressed in complex
other mechanisms of resistance were responsible A. fumigatus biofilm populations and that they
(Bueid et al. 2010). In addition to this mechanism contribute to azole resistance. Moreover, VRZ
of resistance the presence of biofilm may require treatment induces efflux pump expression
consideration of other mechanisms of resistance (Rajendran et al. 2013).
(Ramage et al. 2011; Rajendran et al. 2013;
Ramage et al. 2012; Robbins et al. 2011).
7 Extracellular Matrix
6 Efflux Pumps
Biofilms are encased in an extracellular matrix
(ECM). In A fumigatus the ECM is composed
Azole resistance may be mediated by multidrug
mainly of polysaccharides, hydrophobin, and
resistance (MDR) pumps, which are involved in
melanin (Beauvais et al. 2014). This matrix is
the active extrusion of antimicrobial molecules,
considered to be an important virulence factor
including azole (Rajendran 2011). MDR efflux
and is also related to their high resistance to
transporter genes of the ATP-binding cassette
antifungal agents. Previous studies with cultures
(ABC) and the major facilitator superfamily
of A. fumigatus maintained under static aerial
(MFS) classes have been shown to be clinically
conditions have demonstrated the presence of
important in different pathogenic fungi (Cannon
an ECM on the colony surface of colonial
et al. 2009; Morschhauser 2010). Sequence anal-
mycelia that colonies encased with ECM are
ysis suggests that A. fumigatus has 278 different
extremely hydrophobic and display more resis-
MFS and 49 ABC transporters (Nierman
tance to antifungal polyenes AmB and nystatin
et al. 2005). A. fumigatus MDR (AfuMDR)
(Beauvais et al. 2007). It is thought that ECM
pumps have been described in several studies
may act as a physical barrier that decreases the
and have been shown to be associated with
access of antifungals to cells embedded in the
increased resistance to itraconazole (da Silva
biofilm community. The penetration of the drugs
Ferreira et al. 2004; Nascimento et al. 2003).
is a function of the amount and nature of ECM, as
Recently, it has been shown that non-cyp51a
well as the physicochemical properties of the
mediated itraconazole resistance may be
antifungal agents. Thus anything that disrupts
associated with efflux pump activity through
the biofilm may increase the sensitivity to anti-
cdr1B (Fraczek et al. 2013).
fungal agents. cspA encodes a repeat-rich
In an A fumigatus biofilm model azole resis-
glycophosphatidylinositol-anchored cell wall
tance was shown to increase 16128-fold in the
protein in A. fumigatus. A deletion of cspA
12 h phase and >512-fold at the 24 h phase
resulted in a rougher conidial surface, reduced
compared to 8-h germlings (Rajendran
biofilm formation and decreased resistance to
et al. 2013). An Ala-Nap uptake assay
antifungal agents (Fan et al. 2015). Another
demonstrated a significant increase in efflux
example is the oligosaccharide OligoG which is
pump activity in the 12-h and 24-h phases
an alginate derived from seaweed, SEM and
(P < 0.0001). In addition efflux pump activity
AFM both showed that OligoG (2 %) markedly
of the 8-h germling cells was significantly
disrupted fungal biofilm formation, both alone,
induced by VRZ. Inhibition of efflux pump activ-
and in combination with fluconazole. Calculation
ity with the competitive substrate MC-207,110
of Fractional Inhibitory Concentration Index
reduced the VRZ MIC values for the
showed that for A. fumigatus at the higher
A. fumigatus germling cells by 28-fold . Quan-
concentrations of OligoG used synergy occurred
titative expression analysis of AfuMDR4 mRNA
between the compound and AmB and VRZ
transcripts also showed a phase-dependent
(Tondervik et al. 2014).
increase as the mycelial complexity increased,
Aspergillus Biofilms in Human Disease 7

Aspergillus ECM was initially thought to be VRZ-exposed cells by eight-fold, which visually
composed of galactomannan, alpha-1,3 glucans, could be explained by destabilisation of the bio-
melanin and other proteins including film when examined microscopically. Pharmaco-
hydrophobins (Beauvais et al. 2007). However logical inhibition of Hsp90 by GDA also
our work has demonstrated the presence of extra- significantly improved biofilm susceptibility to
cellular DNA (eDNA) in A. fumigatus biofilm AmB by 48-fold. Suggesting that A. fumigatus
ECM, which is released upon fungal autolysis pre-exposure to VRZ concomitantly induces
(Rajendran et al. 2013). We suggested that the eDNA release and activates the stress response,
role of eDNA is maintenance and stability of which collectively confers AmB resistance
biofilms and biofilm resistance to antifungal in vitro (Rajendran et al. 2013).
drugs (Krappmann and Ramage 2013). When
A. fumigatus biofilms are treated with DNAse
there is improved antifungal susceptibility to 9 Other Approaches
AmB or caspofungin. These findings together
demonstrated the important role of eDNA in Undoubtedly the most effective and logical way
Aspergillus ECM biofilm. The role of eDNA in of dealing with clinically important fungal
biofilm formation and stability has been con- biofilms is to either inhibit their development,
firmed by another group who added exogenous use mechanical force to disrupt them or simply
eDNA in an in vitro biofilm model (Shopova remove and replace an implicated medical
et al. 2013). They also showed that eDNA device. Wound fungal biofilms are managed
improved surface adhesion of fungal spores and with surgical debridement (Warkentien
also co-localised with ECM biofilm et al. 2012). In severe wounds, such as those
polysaccharides, becoming part of the ECM occurring from combat trauma, liposomal AmB,
surrounding the biofilm cells. VRZ and posaconazole have been used, often as
Other components of the ECM may also be combinational therapy, although the clinical
targeted to improve therapeutic response to outcomes were variable. Nevertheless, it has
antifungals. When biofilms are treated with algi- been reported that in the management of a case
nate lyase (AlgL) both fractional inhibitory con- of fungal osteomyelitis combined use of VRZ
centration index values and time kill analyses and terbinafine along with surgical debridement
show synergy between AlgL and amphotericin. was able to successfully control a Scedosporium
In addition a combination of AlgL and inflatum infection and salvage the limb (Cetrulo
amphotericin showed a reduction in hyphal et al. 2012).
thicknesses (Bugli et al. 2013). These studies suggest that wound fungal
biofilms may have a different structural compo-
sition, as they respond to azoles more effectively
8 Previous Therapy than other fungal biofilms. Many of these
infections are polymicrobial, and undergo
As described earlier triazoles are the mainstay of repeated debridement with topical antiseptics.
treatment for aspergillosis. Failure to respond Moreover, wound dressings containing antimi-
clinically or refractory infections may necessitate crobial molecules are used, so it is not surprising
a switch to other antifungal agents, including that fungal wound biofilms respond to azole ther-
AmB. One study explored the possibility that in apy in this context.
A. fumigatus biofilms sequential antifungal ther-
apy may impact adaptive resistance mechanisms
(Rajendran et al. 2015). A. fumigatus sensitivity 10 Concluding Remarks
to AmB was decreased when it was tested in
combination with VRZ. The mechanism of this From review of the available literature it is evi-
increase may be twofold. Depletion of eDNA by dent that Aspergillus biofilms may play a signifi-
DNase treatment enhanced AmB activity against cant role in clinical medicine. These fungi have
8 C. Williams et al.

been shown to form biofilms in both hard and Aspergillus species and other opportunistic molds.
soft tissue, and upon implanted medical devices. Clin Infect Dis 35:E86E88
Bakaletz LO (2007) Bacterial biofilms in otitis media:
Diagnosing the presence of a fungal biofilm is evidence and relevance. Pediatr Infect Dis J 26:S17
difficult, however recently published guidelines S19
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Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2016) 3: 1320
DOI 10.1007/5584_2016_5
# Springer International Publishing Switzerland 2016
Published online: 7 June 2016

Candida albicans in Multispecies Oral

Communities; A Keystone Commensal?

Marleen M. Janus, Hubertine M.E. Willems,

and Bastiaan P. Krom

The complexity of the oral cavity, in which many hundreds of microbial
species interact represents a challenge for modern microbiologists. What
are all these species doing there? And why do we accept so many
opportunistic pathogens to be part of our health (commensal) microflora?
While the role of bacteria are often being studied, the role of fungi in the
interactions within the oral cavity are understudied. This is partly because
fungi in the oral cavity are generally considered as pathogens and related
to diseases. In this chapter we will explore mechanisms of interaction
between bacteria and fungi in the oral cavity that are involved in mainte-
nance of oral health. We will argue that fungi in general and
C. albicans specifically, should be regarded a keystone commensal in
the oral cavity.

cavity is colonized by billions of microorganisms

1 Introduction ranging from viruses, bacteria to eukaryotes such
as fungi and protozoa (Filoche et al. 2010). These
1.1 The Oral Cavity microorganisms mostly reside in 3-dimensional
communities termed biofilms with large species
The oral cavity is the beginning of the gastroin- diversity. The oral cavity is a very diverse
testinal tract and digestion of our food starts here. environment with many distinct ecological
Like other parts of this digestive system, the oral niches (Simon-Soro et al. 2013) that probably

M.M. Janus and B.P. Krom (*)

Top Institute Food and Nutrition, Wageningen, The H.M.E. Willems
Netherlands Department of Preventive Dentistry, Academic Centre for
Dentistry Amsterdam (ACTA), University of Amsterdam
Department of Preventive Dentistry, Academic Centre for
and VU University Amsterdam, Gustav Mahlerlaan 3004,
Dentistry Amsterdam (ACTA), University of Amsterdam
1081 LA Amsterdam, The Netherlands
and VU University Amsterdam, Gustav Mahlerlaan 3004,
1081 LA Amsterdam, The Netherlands Department of Clinical Pharmacy, University of
e-mail: Tennessee Health Science Center, Memphis, TN, USA

14 M.M. Janus et al.

differentiate based on the nature of the surface attention (Krom et al. 2014). Candida albicans is
(e.g. hard tooth surfaces or softer mucosal probably the most commonly encounter oral fun-
surfaces), but also on varying nutrient availabil- gus. It is linked to common diseases such as oral
ity and dynamic occurrence of oxygen limitation. thrush in infants and HIV-positive patients. In
addition, it is commonly found associated with
biomaterials such as dentures where it causes
1.2 Oral Ecology denture stomatitis (ODonnell et al. 2015).
More recently it has been suggested that
Upon birth humans are colonized by bacteria, C. albicans is related to development of tooth
starting in the oral cavity. While passing through caries (Metwalli et al. 2013) and might be linked
the birth canal the infant gets inoculated with to endodontitis (Waltimo et al. 2003). In all cases
resident bacteria. However, recently bacterial C. albicans is rarely found alone; there is strong
presence was shown in the placenta and it is evidence for complicated physical interactions
possible that the fetus is trained to recognize with a range of oral bacteria. For instance,
commensal flora during its time in the womb S. mutans and C. albicans interact physically
(Zaura et al. 2015). Surprisingly, these placental and are found in high numbers in caries, espe-
bacteria were closely related to oral bacteria. cially in early childhood caries (Metwalli
After initial colonization of the oral cavity, the et al. 2013).
downstream parts of the gastrointestinal tracts
are slowly colonized, initially with these oral
bacteria. After some time the gut ecology 1.4 Oral Fungi in Health
differentiates into a specific composition and
remains relatively stable over time. The oral eco- Compared to the attention given to the role of
system however is relatively dynamic; it changes bacteria in the healthy oral cavity and compared
over time with its ever-changing host. Hour-to- to the involvement of fungi in pathology, there is
hour variation, monthly cycles as well as life- little to no information on the role of fungi
time changes such as tooth eruption and tooth in healthy oral ecologies. Ghannoum and
loss change the playing field for adhesion. Also coworkers for the first time described the
hormonal changes during puberty and pregnancy mycobiome (the total of all fungi) of healthy
are reflected in the composition of the saliva and volunteers (Ghannoum et al. 2010). Subsequently
strongly influences oral composition (Kumar several other studies have shown that health oral
2013). In general it is believed that a healthy ecologies contain many fungal species (Dupuy
oral ecology is highly diverse in species compo- et al. 2014; Monteiro-da-Silva et al. 2013;
sition. This diversity is lost when pathology Monteiro-da-Silva et al. 2014). It is becoming
arises and/or vice versa, pathology arises when clear that over 100 fungal species can be found
this diversity is lost. The role of oral bacteria in as part of the oral flora. Due to the highly mobile
oral pathologies is well studied. This is not nature of fungal spores some of these species
surprising as they are the cause of the most com- might be environmental contaminants and be
mon infectious diseases of today: caries and gin- transiently present, but many others are likely
givitis. In contrast, the role of bacteria in to be part of the resident oral flora. It should be
maintaining health has only recently attracted taken into account that while fungal counts might
attention. remain extremely low compared to bacterial
counts (less than 0.1 %), their size compensates
for this. Lets considered bacteria to be spheres
1.3 Oral Fungi Focussed on Disease with a radius of 0.5 m (very realistic) and fungi
to have a radius of 2.5 m (realistic for yeasts,
In contrast to the role of bacteria in oral diseases, but certainly not for filamentous forms). The
the role of fungi has received considerably less calculated volumes according to the formula
Candida albicans in Multispecies Oral Communities; A Keystone Commensal? 15

V 4/3**r3 are 0.5 m3 and 65.4 m3, respec- interactions can occur on different levels; mainly
tively. This means that if the total number of physical, metabolic and chemical interactions
fungi is only 0.1 % of the total microbial load, (Fig. 2.1).
they represent at least 10 % of the biovolume
(ergo biomass). Therefor they are to be consid-
ered a significant part of the healthy oral ecol- 2.1 Physical Interactions
ogy and should not be disregarded!
In this chapter we will explore mechanisms of Within the biofilm microbes are by definition
interaction between bacteria and fungi in the oral closely packed. However, on a single cell level,
cavity that are involved in maintenance of oral there are positive and negative interactions
health. We will argue that fungi in general and resulting in attraction and repulsion of individual
C. albicans specifically, should be regarded a cells. As a consequence of non-specific (mostly
keystone commensal in the oral cavity. hydrophobic and electrostatic) and specific
(protein-protein) interactions certain species are
commonly found together. Both mechanisms
2 Type of Bacterium-Fungus play a role in the adhesion of bacteria to fungi,
Interactions Related to Health as has been illustrated recently for the
interactions between the Gram-negative bacte-
It is generally accepted that a healthy ecosystem rium Pseudomonas aeruginosa and C. albicans.
is a balanced ecosystem. Such balance is P. aeruginosa strongly adheres to and kills
maintained by the interaction of forces within hyphae of C. albicans, but not yeasts cells. This
the ecosystem. These forces are derived from difference in adhesion to and subsequent killing
interactions between individuals within the eco- of the hyhal and yeast morphology is utilized by
system and balance is the overall result of all of C. albicans as a defense mechanism (for more
these interactions (Krom and Oskam 2014). The details (Jarosz et al. 2011)). Using physic-

Fig. 2.1 Schematic

overview of the three types
of interactions between
C. albicans and bacteria
described in this chapter.
This overview is far from
complete. See text for more
16 M.M. Janus et al.

chemical and force microscopic analyses, it was biofilms are the source of recolonization. In the
shown that the strong hydrophobic nature of the oral cavity, environmental challenges are con-
hyphal cell wall attracts cells of P. aeruginosa stantly present; at least twice a day high levels
(Ovchinnikova et al. 2012a, b). By virtue of their of antimicrobial compounds are washed through
hydrophobic nature, surface proteins of the the oral cavity. The mixed-species C. albicans
hyphal cell wall were shown to be mostly respon- bacteria biofilms could therefor be instrumental
sible for the non-specific interactions mediating to healthy recolonization after routine oral
this adhesion. When the bacterium approaches hygiene. Healthy recolonization is of great
the fungus close enough, a specific chitin- importance to maintenance of overall health.
binding protein interacts with chitin in the fungal There is a growing body of evidence suggesting
cell wall (Ovchinnikova et al. 2012a, b). The role that the oral ecology is involved in regulation of
of non-specific interactions and a specific bacte- important physiological functions, such as blood
rial binding protein, but absence of a specific pressure (see Box 1). Consequently, this scaffold
fungal binding protein might illustrate the antag- function of C. albicans in the oral cavity could
onistic nature of this interactions; beneficial for assist in stability of the oral ecology and thus in
P. aeruginosa, but disadvantageous for maintenance of overall health.
C. albicans (Hogan and Kolter 2002; Hogan
et al. 2004). In contrast, the interaction between
Box 1: How oral bacteria impact healthy
S. aureus and C. albicans is probably a benign, if
not mutually advantageous interaction for both
Our blood pressure is regulated by a small
species. Using a similar approach as described
diffusible molecule: NO (nitric oxide).
for P. aeruginosa, interactions between S. aureus
Dietary components with high nitrate con-
and C. albicans were investigated. It was shown
tent, such as spinach and other green
that a specific hyphal wall protein (Als3p) was
vegetables, are able to lower the blood
involved in strong interaction forces (Peters
pressure (Larsen et al. 2006). Nitrate
et al. 2012). A S. aureus counterpart has not yet
(NO3) is converted to nitrite (NO2) and
been identified, but considering the extend of the
subsequently to nitric oxide (NO). We
interaction forces determined using force micros-
humans have enzymes capable of
copy, this will only be a matter of time. Similar
converting NO2 to NO, but we lack
interactions have been observed between Strep-
enzymes that convert NO3 to NO2. Upon
tococcus mutans and C. albicans as well as for
digestion of dietary NO3, our body feeds
C. albicans and a range of other oral bacteria
back the NO3 to the oral cavity through the
(Nobbs et al. 2009; Silverman et al. 2010; Wright
salivary glands. In the oral cavity bacteria
et al. 2013; Bachtiar et al. 2014). The potential
convert the NO3 to NO2 that is taken up by
relevance of such interactions in the oral cavity
the host. Oral disinfection using chlorhexi-
follows from two independent observations.
dine prior to the consumption of dietary
Firstly, upon adhesion to the hyphae of
NO3 prevented the conversion to NO2 and
C. albicans, S. aureus become more tolerant
subsequent lowering of the blood pressure
towards antibiotics (Harriott and Noverr 2010).
(Govoni et al. 2008). Thus, oral bacteria are
Secondly, using a murine gut-model, it was
truly important for a healthy physiology!
shown that when C. albicans was present, bacte-
rial recolonization following antibiotic treatment
was enhanced (Mason et al. 2012b; Mason 2.2 Chemical Interactions
et al. 2012a; Erb Downward et al. 2013). When
these two observations are combined, it is very Chemical interaction within a species is com-
well possible that adhesion to and biofilm forma- monly referred to as intraspecies signaling or
tion on C. albicans protects many bacteria quorum sensing. A small diffusible molecule is
against antibiotic challenges and such surviving synthesized and accumulates in the external
Candida albicans in Multispecies Oral Communities; A Keystone Commensal? 17

medium. Upon reaching a threshold concentra- mechanism by which C. albicans creates an

tion, gene expression is induced and a concerted anaerobic micro-niche is unknown, but could be
response of the population is given. Due to the related to high level of O2 consumption typical
external nature of the quorum sensing molecules for yeasts (Wesolowski et al. 2008). It is known
they are commonly detected by other species that there is enormous niche-diversity in the oral
than the ones producing them (Jarosz et al. cavity (Jenkinson 2011) and conceptually the
2011). Several bacterial QS molecules have observation that C. albicans creates an anaerobic
been shown to affect C. albicans behavior micro-niche explains the niche-diversity that is
(Fig. 2.1), amongst others Homoserine lactone so typical for the oral cavity; aerobic and anaero-
(HSL) produced by P. aeruginosa (Hogan et al. bic niches exist side by side, depending on the
2004), Competence Stimulating Peptide (CSP) microbial species composition.
produced by S. mutans (Jarosz et al. 2009) A metabolic interaction at the substrate level
and autoinducer-2 (AI-2) of Aggregatibacter has been shown for the interaction between
actinomycetemcomitans (Bachtiar et al. 2014). S. mutans and C. albicans. Caries is a long-term
This interaction is bi-directional as C. albicans process in which lactic acid, produced by bacte-
secretes the QS molecule farnesol (Hornby ria, result in demineralization of tooth surfaces.
et al. 2001) which, inhibits Pseudomonas quino- Under healthy conditions, lactic acid is removed
lone signal (PQS) production (Cugini et al. 2007; by saliva flow and microbial metabolism. Health-
Cugini et al. 2010). PQS is needed for the expres- related bacteria such as Veillonella spp. grow on
sion of several virulence factors (Bjarnsholt the lactic acid produced mainly by S. mutans.
et al. 2010) and C. albicans thus decreases the Several studies have described C. albicans as an
virulence of P. aeruginosa using chemical organism capable of synergizing the onset and
interactions. progression of caries induced by S. mutans
(Metwalli et al. 2013; Falsetta et al. 2014; Jarosz
et al. 2009). We have recently shown that the
2.3 Metabolic Interactions presence of C. albicans in a dual-species biofilm
with S. mutans does not synergize the cariogenic
In addition to physical and chemical interactions, capacity in terms of acidity and calcium release,
metabolic interactions probably play a role in the the two main surrogates for caries (Willems
oral cavity. For example, recently it was shown et al., Pathogens and Disease, accepted for publi-
that C. albicans allows growth and biofilm for- cation). In contrast, the presence of C. albicans
mation of anaerobic bacteria under aerobic appears to decrease the cariogenic potential of
conditions (Fox et al. 2014). In the oral cavity the biofilm. Mixed biofilms become less acidic
the presence of anaerobic bacteria is well- over time, and after 72 h of biofilm growth the
established, also in health. For example, the strict bulk pH remains above the critical pH of 5.5
anaerobic Veillonella spp. are related to (Matsui and Cvitkovitch 2010). Since lactic
decreased caries risk because they consume lac- acid production potential was increased in our
tate (the main acid involved in caries develop- mixed biofilms, it is possible that C. albicans
ment). How such strict anaerobes can survive influences the pH in an independent fashion of
in an aerobic niche as the oral cavity is still S. mutans. There are several possible
not entirely known. Recently it was shown that explanations for this observation. A study by
C. albicans allows growth and biofilm formation Ene et al. (2012) showed that C. albicans is
of several strict anaerobic (mainly gut) bacteria able to grow using lactic acid as a carbon source
under aerobic condition (Fox et al. 2014). We (Ene et al. 2012). Furthermore, lactic acid is the
have shown in vitro that in the presence of most preferred source of carbon in hypoxic
C. albicans many strict anaerobic oral bacteria conditions for the fungus and hypoxic conditions
can also grow under aerobic culture conditions are being created by C. albicans (Fox
(Janus et al., submitted for publication). The et al. 2014). Logically, consumption of lactic
18 M.M. Janus et al.

acid by C. albicans would cause the environment et al. 2012)). Briefly, periodontitis is a very com-
to become less acidic. This effect is enlarged as mon oral disease related to chronic inflammation
S. mutans increases in numbers, since sucrose is of the tooth-supporting tissues. While the micro-
then consumed even faster. At an early time point bial biofilms is certainly involved in the onset of
when sucrose is not limited, C. albicans favors the disease, it is the inflammatory response by the
the consumption of sucrose over lactic acid, pro- host that causes irreversible damage to the tooth-
ducing ethanol. When sucrose becomes limited, supporting tissues. One of the bacteria involved
C. albicans is obligated to consume the lactic in periodontitis is P. gingivalis. Important viru-
acid. Alternatively, C. albicans can regulate its lence factors of P. gingivalis are secreted
external pH in response to low pH (Vylkova proteases, the gingipains. These proteases acti-
et al. 2011). This has mainly been studied in vate the complement factor C5, generating C5a
relation to phagocytosis (Vylkova and Lorenz that binds to the C5a-receptor leading to activa-
2014). Production of NH4+ from amino acids tion of inflammation and impairs leukocyte kill-
result in increase of the bulk pH. This phenome- ing. Together this allows other bacteria to thrive.
non would resemble the therapeutic effect So by the mere production of a protease a
described for arginine-containing toothpastes low-level presence of P. gingivalis can orches-
and arginine has been shown to increases trate inflammation.
C. albicans presence in in vitro oral biofilms
(Koopman et al. 2015).
4 Conclusion
3 Keystone Organisms Theory The role of fungi in the complex oral ecosystem
that is dominated by 400+ bacterial species has
Originating from architecture where the keystone remained enigmatic. Recent studies on the role of
represents the top stone of an arch, the keystone C. albicans in the healthy oral cavity has started
paradigm has made its entry into ecology near to shed a light on several key functions of fungi
the end of the last century. When a certain spe- in maintaining a healthy balance. However,
cies has disproportionately large effects on their unmistakably, C. albicans are a minority when
communities, given their abundance, it is thought cell numbers are considered. The overview
to form the keystone of the communitys struc- provided above however indicates that even
ture (Hajishengallis et al. 2012). Traditional when present in low numbers a considerable
infections are based on accumulation of a spe- effect on the total ecology is exerted. For
cific pathogen to levels unacceptable to the host instance by rapid consumption of molecular oxy-
(dominant pathogen). Dysbiosis with the host is a gen, rapid increase of local pH and by providing
result of this accumulation in microbial load. a physical scaffold for oral bacteria to adhere
Keystone pathogens modulate the host and/or to. We therefor present the hypothesis that
the ecology in such a way that is unacceptable C. albicans functions as a keystone commensal
to the host, without increasing its own presence in the healthy oral cavity.

3.1 Keystone Pathogens in Oral
Diseases Bachtiar EW, Bachtiar BM, Jarosz LM, Amir LR,
Sunarto H, Ganin H et al (2014) AI-2 of
In oral ecology, the keystone hypothesis is Aggregatibacter actinomycetemcomitans inhibits
Candida albicans biofilm formation. Front Cell Infect
largely used to describe the role of keystone
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Expert Rev Mol Med 12:e11 aeruginosa quorum-sensing molecule influences Can-
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aeruginosa. Mol Microbiol 65(4):896906 Shoemaker R et al (2001) Quorum sensing in the
Cugini C, Morales DK, Hogan DA (2010) Candida dimorphic fungus Candida albicans is mediated by
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quinolone signal production in LasR-defective Pseu- Jarosz LM, Deng DM, van der Mei HC, Crielaard W,
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(2012a) Force microscopic and thermodynamic analy- extracellular pH. mBio 2(3):e00055-11
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Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2016) 3: 2135
DOI 10.1007/5584_2016_6
# Springer International Publishing Switzerland 2016
Published online: 7 June 2016

The Extracellular Matrix of Fungal Biofilms

Kaitlin F. Mitchell, Robert Zarnowski, and David R. Andes

A key feature of biofilms is their production of an extracellular matrix.
This material covers the biofilm cells, providing a protective barrier to the
surrounding environment. During an infection setting, this can include
such offenses as host cells and products of the immune system as well as
drugs used for treatment. Studies over the past two decades have revealed
the matrix from different biofilm species to be as diverse as the microbes
themselves. This chapter will review the composition and roles of matrix
from fungal biofilms, with primary focus on Candida species, Saccharo-
myces cerevisiae, Aspergillus fumigatus, and Cryptococcus neoformans.
Additional coverage will be provided on the antifungal resistance prof-
fered by the Candida albicans matrix, which has been studied in the most
depth. A brief section on the matrix produced by bacterial biofilms will be
provided for comparison. Current tools for studying the matrix will also be
discussed, as well as suggestions for areas of future study in this field.

Extracellular matrix Biofilm Antifungal resistance Candida albicans
Device-associated infections

1 Introduction a structural scaffold for both adhesion to surfaces

and cohesion between cells (Flemming and
A defining feature of the biofilm lifestyle is pro- Wingender 2010; OToole 2003). Additionally,
duction of an extracellular matrix. The matrix the matrix of certain organisms can contribute to
surrounds the cells within the biofilm, providing the retention of water and nutrients, and in some
cases these nutrients are thought to derive from
matrix materials hydrolyzed by microbial-
K.F. Mitchell, R. Zarnowski, and D.R. Andes (*) produced enzymes (Flemming and Wingender
Department of Medicine, University of Wisconsin-
2010). Perhaps the most medically-relevant func-
Madison, Madison, WI, USA
e-mail:;; tion of the extracellular matrix is its ability to provide a physical barrier between biofilm cells

22 K.F. Mitchell et al.

and the surrounding environment. In a human containing polysaccharides, extracellular DNA

host, this includes cells and products of the (eDNA), lipids (namely rhamnolipids), and
immune system and often drugs used for treat- proteins (Mann and Wozniak 2012).
ment (Costerton et al. 1999; Donlan 2001). Polysaccharides include Psl, Pel, levan, and algi-
The composition of the matrix has been com- nate, whose quantity depends on the strain in
prehensively studied in only a handful of biofilm question, the stage of biofilm development, and
species. Most of these studies have occurred the site of biofilm formation (Ma et al. 2009). For
in bacteria, though the fungi Saccharomyces example, strains designated mucoid or
cerevisiae, Aspergillus fumigatus, Cryptococcus non-mucoid differ in their overexpression of
neoformans and Candida albicans serve as alginate, which occurs during infections of the
exceptions. The diversity of biochemical entities lungs of cystic fibrosis patients (Franklin
discovered, and the observation that matrix et al. 2011; Mann and Wozniak 2012). The
components can be similar to components of other polysaccharides are typically produced by
the fungal cell wall, suggests that the composi- environmental strains or those isolated from
tion of biofilm matrices can be as diverse as the other types of infections. Pel has been exten-
microbes that produce them (Branda et al. 2005; sively studied, and found to lend a protective
Zarnowski et al. 2014). This chapter will review role against aminoglycoside antibiotics (Colvin
the composition and roles of matrix from fungal et al. 2011). DNA in the Pseudomonas matrix
biofilms, with a brief coverage on bacterial also contributes to structural stability, and has
matrices for comparison. Current tools for study- been found to induce antibiotic resistance
ing the matrix will also be discussed, as well as (Yang et al. 2011; Mulcahy et al. 2008).
suggestions for areas of future study in this field. Other molecules have been identified in the
matrices of multiple bacterial biofilms. Namely,
cellulose is a critical component for Escherichia
2 Matrix of Bacterial Biofilms coli, Klebsiella pneumonia, Enterobacter spp.,
Citrobacter spp., and Salmonella typhimurium
Most studies of biofilm extracellular matrix (Hobley et al. 2015; Branda et al. 2005; Hung
components to date have utilized bacterial spe- et al. 2013). PNAG, identified in B. subtilis, is
cies. One well-characterized model system also found in Staphylococcus aureus and Staphy-
involves the gram-positive, spore-forming bacte- lococcus epidermidis, as well as the related mol-
rium Bacillus subtilis, which can form pellicles ecule polysaccharide intercellular adhesin (PIA)
or submerged biofilms depending on the strain (Branda et al. 2005).
and environmental conditions (Vlamakis In addition to the composition and production
et al. 2013). Several polymeric components of of individual matrix components, assembly of
the B. subtilis matrix have been described, such the entire matrix over the course of bacterial
as poly-DL-glutamic acid (PGA) and the proteins biofilm development has been explored in two
TapA, TasA, and BslA. This matrix also contains species. The first of these is Vibrio cholerae,
exopolysaccharide (EPS), which is produced by which produces a matrix of Vibrio polysaccha-
the eps operon (Marvasi et al. 2010). Notably, the ride (VPS) and the proteins RbmA, RbmC, and
EPS component was recently discovered to be at Bap1 (Teschler et al. 2015; Fong et al. 2010;
least partially composed of poly-N-acetyl glu- Reichhardt et al. 2015b). A 2012 study by Berk
cosamine (PNAG), a widely conserved bacterial et al. revealed distinct stages of cellular cluster-
polysaccharide (Roux et al. 2015), and had pre- ing during biofilm formation, with RbmA
viously been shown to act as a positive regulator providing initial adhesion between cells and
of its own synthesis (Elsholz et al. 2014). Bap1 aiding in surface adhesion. Cell clusters
A second model organism for the study of were surrounded by mixtures of VPS, RbmC,
biofilm formation is Pseudomonas aeruginosa, and Bap1 in the later stages of biofilm develop-
which produces an extracellular matrix ment (Berk et al. 2012). Similarly, spatially
The Extracellular Matrix of Fungal Biofilms 23

segregated subpopulations of cells surrounded found in extracellular matrices. To quantify the

by extracellular matrix were observed within gross amount of protein, colorimetric assays such
E. coli pellicles in a 2013 publication by as bicinchoninic acid (BCA), Bradford, Folin-
Hung et al. In addition, this study confirmed Lowry, Kjeldahl or ultraviolet absorption can be
that amyloid fibers formed by curli as a employed. For identification of specific proteins,
major matrix constituent, with cellulose, chromatographic and electrophoretic separation
flagella, and type 1 pili also involved (Hung strategies have been extensively used followed
et al. 2013). Together, these recent findings sup- by various mass spectrometry (MS) analytical
port the notion that biofilm matrix is not simply a techniques (Zarnowski et al. 2014; Reichhardt
disorganized conglomerate, but rather specific et al. 2015a). Recent advances in bottom-up pro-
microenvironments of components that emerge teomics technologies have been especially
in time and space. important to the study of extracellular matrix,
as they allow for in-depth relative and absolute
quantification of proteins in intact or minimally-
3 Fungal Matrix processed biological samples (Zhang et al. 2013).
For example, our group recently utilized shotgun
Multiple types of fungi form biofilms, found both proteomics to achieve global protein identifica-
in the environment and in infection settings. The tion in the C. albicans biofilm matrix (Zarnowski
first investigations of the extracellular matrix of et al. 2014).
fungal biofilm species were performed by the Overall measurement of monosaccharides can
Douglas group, with Candida albicans, nearly be accomplished using the phenol-sulfuric assay
two decades ago (Hawser and Douglas 1994; (DuBois et al. 1951). A specific assay for -1,3
Hawser et al. 1998). Since that point, several glucan quantification is the limulus assay, which
groups have investigated basic aspects of matrix has been used both in vitro and as a diagnostic in
production and its individual components. How- clinical settings (Nett et al. 2007b; Nett
ever, not until the past few years has a larger et al. 2007a). For the most precise quantification
picture of the matrix emerged, using multiple of carbohydrates gas chromatography is the gold
approaches to examine how its various entities standard, and is typically coupled with MS anal-
collectively function to provide structural integ- ysis for qualitative purposes. For structural anal-
rity and protection to the cells of biofilms. ysis, complementary approaches utilizing GC,
MS, and multiple nuclear magnetic resonance
(NMR) procedures provide the granularity
3.1 Tools for Studying the Matrix needed for accurate structural assignments. Sim-
ilar to techniques previously used to analyze the
It has been proposed that successful quantifica- C. albicans cell wall, a combination of several
tion of the entire extracellular matrix is nearly solution-state 1H and 13C NMR techniques have
impossible, as matrix isolation methods could been used to examine the matrix (Shibata
potentially disrupt the cell wall (Flemming and et al. 2007; Zarnowski et al. 2014). Recently,
Wingender 2010). However, recent protocols solid-state 13C, 15N, 31P NMR spectroscopy has
have demonstrated no change in the chemical also proven a powerful ally in generating more
composition of the cell wall following matrix complete descriptions of macromolecular
extraction. These methods have used either assemblies (Reichhardt et al. 2015a; Cegelski
mild detergents (or none at all), combined with 2015). For more detailed information on the
gentle sonication to separate the cells from the molecular size and shape of individual matrix
matrix material (Zarnowski et al. 2014; polysaccharides, small-angle X-ray scattering
Reichhardt et al. 2015a; Lattif et al. 2008). (SAXS) has also been employed.
Biochemical approaches have been used to Lipids in the extracellular matrix have also
analyze the different classes of macromolecules been quantified using gas chromatography. For
24 K.F. Mitchell et al.

crude separation of different lipids, TLC can be et al. 2008; Guo et al. 2000; Reynolds and Fink
used, though the most detailed data can be 2001). Of these, FLO11 is required for
achieved using MS-based shotgun lipidomics. S. cerevisiae biofilm formation (Reynolds and
The system recently employed by our group Fink 2001; Guo et al. 2000; Ishigami et al. 2004).
involved liquid chromatography (LC)/mass S. cerevisiae can produce extracellular matrix
selective detector (MSD) time of flight (TOF) in both the flocculating and biofilm forms, as
with electrospray ionization (Zarnowski well as in structured colonies formed by environ-
et al. 2014). This technique has also been mental isolates. This matrix material has been
employed for analysis of small molecular weight visualized using electron microscopy (Zara
lipophilic molecules, such as ergosterol and other et al. 2009; Kuthan et al. 2003; Vachova
sterols. et al. 2011; Beauvais et al. 2009). The 2009
The final class of macromolecule, nucleic study by Beauvais et al. examined the composi-
acids, can be measured in crude matrix material tion and role of the matrix in FLO1-expressing
spectrophotometrically or with use of specific cells, which exhibit strong flocculation and
dyes (Zarnowski et al. 2014; Martins higher resistance to stress and drugs (Beauvais
et al. 2010). In our groups recent study, the et al. 2009). Flo1p aids in flocculation by
potential presence of coding regions was exam- interacting with sugars on the cell walls of neigh-
ined by creating a clone micro-library of random boring yeast, as its exposed N-terminus possesses
regions of matrix DNA, followed by sequence lectin-like properties (de Groot and Klis 2008;
homology analysis to the Candida Genome Dranginis et al. 2007). Matrix from the floc was
Database. extracted using EDTA and shown to be loosely
For measurement of known matrix entities, attached to the cell surface. The material
monoclonal antibodies have been produced for contained mainly glucose and mannose, with a
both imaging purposes and quantification in negligible amount of protein. GC-MS analysis
microtiter based assays such as ELISA revealed the mannose portion to be a chain of
(Zarnowski et al. 2014; Martinez and Casadevall (16) mannan with (12) and (13) linked
2015). For further characterization of the matrix branches. Beauvais et al. also showed the matrix
material, both SEM and TEM have been used, as from flocculated S. cerevisiae was able to
well as atomic force microscopy (AFM) (Yang exclude high molecular weight molecules such
et al. 2011; Lal et al. 2010). as antibodies or concanavalin A. However,
smaller entities, namely amphotericin B and eth-
anol, were not blocked in this case.
3.2 Saccharomyces cerevisiae Additional studies have described
components of the S. cerevisiae matrix produced
The yeast Saccharomyces cerevisiae has long under different growth conditions. A study with
been used as a model organism for many basic environmental isolates formed fluffy colonies,
aspects of eukaryotic biology, and as a means to as opposed to the smooth surface produced by
investigate fungal biology in a non-pathogenic laboratory strains. Matrix from the environmen-
system. In the last fifteen years, it has also been tal strains contained an unidentified protein unre-
explored as a model for the biofilm lifestyle. lated to flocculins. The matrix material also
Certain strains of S. cerevisiae exhibit floccula- reacted with concanavalin A, indicating the pres-
tion, or clumping of cells, and others can form ence of exposed terminal mannose or glucose
true surface-adherent biofilms (Verstrepen and residues (Kuthan et al. 2003). More recent
Klis 2006; Bojsen et al. 2012). Both flocculation investigations involved cells grown in a three-
and biofilm formation rely on the FLO family of dimensional mat on solid medium, and used mul-
cell surface adhesins, or flocculins, which are tiple analytical techniques to identify protein and
related to the ALS family and the Hwp1p carbohydrate matrix components in both
adhesins in Candida albicans (Nobile S. cerevisiae and C. albicans (Faria-Oliveira
The Extracellular Matrix of Fungal Biofilms 25

et al. 2014; Faria-Oliveira et al. 2015). A number (CatB), and ribotoxin (ASPF1). A group of
of proteins were identified with 2-DE and hydrophobin proteins was also identified, with
MALDI-TOF MS, including the dehydrogenase purported roles in intercellular adhesion during
Tdh3p for both species. For S. cerevisiae, two aerial growth. Additionally, eDNA that matches
different molecular weight polysaccharides were genomic DNA sequence has been identified in
identified, which contained glucose, mannose, the A. fumigatus biofilm matrix (Rajendran
and smaller amounts of galactose. The different et al. 2013). Interestingly, host DNA may also
matrix components identified in each of these play a role in the Aspergillus matrix, as addition
studies, especially the relative abundance of of exogenous DNA resulted in greater structural
proteins between flocculating and mat forms, integrity and matrix carbohydrate production.
highlights the impact of growth conditions on This could reflect the conditions of the cystic
the production and content of extracellular fibrosis (CF) lung, where concentrations of
matrix. This, in addition to the methods used human DNA are relatively high, and provides
for analysis, must be carefully considered when an interesting glimpse into the potential role of
comparing information from different host factors in the matrix of biofilm infections.
investigations. A more recent study grew A. fumigatus
biofilms in RPMI 1640 medium and utilized
solid-state NMR to define matrix composition
3.3 Aspergillus fumigatus (Reichhardt et al. 2015a). Multiple approaches
were combined to examine facets of the entire
Aspergillus biofilms can form during chronic matrix: 13C, 15N, and 31P-NMR, electron micros-
lung infections, such as aspergilloma, and more copy, and protein identification using PAGE and
rarely form on medical devices in humans mass spectrometry. Overall, this investigation
(Ramage et al. 2011; Loussert et al. 2010). Extra- found the matrix to contain roughly 40 % pro-
cellular matrix has been found to surround these tein, 43 % polysaccharides, up to 14 % lipids,
three-dimensional hyphal structures during both and 3 % aromatic-containing components such
in vitro and in vivo in studies with A. fumigatus, as melanin. Combined with existing studies that
the most common species of Aspergillus, with have identified specific matrix components, work
thick material covering the surface of the cells such as this provides powerful quantitative data
(Loussert et al. 2010; Beauvais et al. 2007; to frame future investigations of matrix structure
Beauvais et al. 2014). Extracellular matrix levels and function.
increase during the maturation of Aspergillus In vivo Aspergillus biofilm matrix has also
biofilms, which form after adhesion of conidia been investigated, using both aspergillomas
to a substrate (Beauvais et al. 2007; Kaur and from human patients and a murine model of
Singh 2014). invasive pulmonary aspergillosis (Loussert
The initial study of Aspergillus biofilm matrix et al. 2010). Similarly to in vitro A. fumigatus
content utilized biofilms grown under aerial- biofilms, both biofilm models contained
static conditions in glucose yeast extract (GYE) galactomannan within the cell wall and matrix.
medium (Beauvais et al. 2007). The matrix was One observed difference between the in vivo
found to contain galactomannan, -1,3 glucan, models was the lack of matrix -1,3 glucan in
proteins, polyols, and melanin. This study, using the pulmonary aspergillosis biofilms. However,
mainly immunoassays, revealed the presence of they both differed from the in vitro model in their
galactomannan in both the cell wall and matrix, relatively higher levels of galactosaminogalactan
while -1,3 glucan was primarily located extra- (GAG), a cell wall exopolysaccharide that has
cellularly near the surface of hyphae. The protein been characterized by the Sheppard group (Lee
content, which represented 2 % of the total et al. 2014; Bamford et al. 2015). GAG has been
matrix, included three major secreted antigens: found to mediate adherence, and plays a role in
dipeptidylpeptidase V (DPPV), catalase B the host immune response partially by masking
26 K.F. Mitchell et al.

-1,3 glucan and also through inducing Casadevall 2005). Initial studies of the
interleukin-1 receptor antagonist (IL-1Ra), C. neoformans biofilm matrix sought to identify
which blocks proinflammatory IL-1 signaling GXM using monoclonal antibodies. This con-
(Gravelat et al. 2013; Gresnigt et al. 2014). firmed the presence of GXM surrounding the
Like the C. albicans matrix, the matrix of cells of the biofilm, and GC/MS analysis detected
Aspergillus biofilms is thought to contribute to monosaccharide content consistent with the pres-
antifungal resistance, either through slowing ence of GXM. However, this study also detected
drug diffusion or perhaps through a more specific monosaccharides not found in GXM (glucose,
sequestration mechanism (Beauvais et al. 2007; ribose, and fucose) suggesting the matrix
Bugli et al. 2013; Rajendran et al. 2013). One contains additional types of polysaccharides
matrix material that has been identified to have a (Martinez and Casadevall 2007).
role in A. fumigatus biofilm resistance is eDNA The Cryptococcus matrix is thought to protect
(Rajendran et al. 2013; Shopova et al. 2013). An the biofilm cells from surrounding stressors in
in vitro study by Rajendran et al. showed that the both an infection setting and in the environment,
eDNA, which accumulates over biofilm matura- where the fungus is associated with pigeon
tion, can be degraded by DNase to result in excreta (Martinez and Casadevall 2006b, 2007;
higher biofilm susceptibility to amphotericin B Alvarez et al. 2008). C. neoformans biofilms are
and caspofungin. A possible mechanism by more resistant to changes in temperature, pH, and
which eDNA enters the matrix is autolysis, as UV light compared to planktonic cells, lending
chitinase activity was found to increase DNA evidence to the idea that biofilm growth in nature
release. An additional study yielded increased provides a protective advantage (Martinez and
A. fumigatus biofilm susceptibility to Casadevall 2007). Similar to biofilms formed by
amphotericin B after treatment with alginate other infectious fungi, those of C. neoformans
lyase, which degrades uronic acid-containing display higher levels of antifungal resistance
carbohydrates (Bugli et al. 2013). One explana- compared to planktonically-grown cells
tion for this result is that a currently unknown (Martinez and Casadevall 2006b), and are also
carbohydrate is also contributing to antifungal more resistant to antimicrobial molecules pro-
resistance in the Aspergillus matrix. duced by cells of the innate immune system
(Martinez and Casadevall 2006a). Interestingly,
a study with both C. neoformans and C. gattii
3.4 Cryptococcus neoformans showed the formation of biofilm-like
microcolonies to be associated with successful
Cryptococcus neoformans, which can cause resistance to phagocytosis, as well as escape
meningoencephalitis in humans, has the ability from macrophages. These microcolonies were
to form biofilms on polystyrene plates and on the surrounded by a polysaccharide matrix
surfaces of medical devices such as the shunts containing antibody, suggesting utilization of
used to treat intracranial hypertension (Martinez antibody-mediated agglutination in favor of the
and Casadevall 2015). During biofilm formation, fungus (Alvarez et al. 2008).
extracellular matrix increases over time and
provides structure and complexity to groups of
adherent cells (Martinez et al. 2010; Martinez 3.5 Candida
and Casadevall 2007).
This basidiomycete grows in a yeast form Candida albicans is the most common fungal
covered by a polysaccharide capsule composed pathogen, and frequently forms biofilms on
of glucuronoxylomannan (GXM), a structure implanted medical devices. The first studies
required for virulence. GXM is shed into the describing the C. albicans biofilm matrix were
environment, and the capsule structure is conducted by the Douglas group, and
required for biofilm formation (Martinez and demonstrated the importance of environmental
The Extracellular Matrix of Fungal Biofilms 27

conditions on overall matrix production (Hawser found in much smaller quantities compared to
et al. 1998). Conditions of continuous flow, two other polysaccharides, -1,6 glucan and
which most closely resemble those of in vivo -1,6 mannan with -1,2 linked branches.
biofilm growth, resulted in biofilms with the While these have some similarity to the
most abundant matrix. In support of this, polysaccharides found in the C. albicans cell
in vivo biofilms from both the catheters of wall, several lines of evidence suggest that
patients as well as multiple animal models pro- matrix and cell wall carbohydrates are produced
duce visibly ample matrix (Paulitsch et al. 2009; or assembled in a distinct manner (Chaffin 2008).
Andes et al. 2004; Nett et al. 2010b; Johnson First, the three polysaccharides were found to
et al. 2012). physically interact in a mannan-glucan complex
The content of the C. albicans matrix initially (MGCx), a structure not described in the cell
identifed carbohydrates, hexosamine, phospho- wall. Second, the cell wall and matrix
rus, protein, uronic acid, and eDNA (Baillie and polysaccharides are found in different
Douglas 2000; Al-Fattani and Douglas 2006). proportions to one another and with different
Subsequent studies identified -1,3 glucan as linkages. For example, the length of mannan
one specific carbohydrate component (Nett chains in the matrix can reach up to 12,000
et al. 2007a; Nett et al. 2007b). Several groups mannose residues, whereas those in the cell
have also investigated variations in matrix con- wall have only been reported up to 200 residues.
tent between other Candida species, and have Finally, the cell wall polysaccharide chitin was
noted differences in overall carbohydrate and not detected in the extracellular matrix, further
protein content (Al-Fattani and Douglas 2006; suggesting that cell wall and matrix are distinct.
Silva et al. 2009). Notably, the major matrix Subsequent investigations explored the genetic
component identified in C. tropicalis was basis for each of these matrix polysaccharides by
hexosamine, and biofilms of this species were screening a library of mutant strains lacking
also disrupted by different enzymatic treatments enzymes in each of the carbohydrate production
than those of C. albicans. pathways (Mitchell et al. 2015). A subset of
A comprehensive analysis of the C. albicans mutants was found to have lower levels of total
matrix was recently performed by our group matrix, lower quantities of each polysaccharide,
(Zarnowski et al. 2014). All four classes of as well as increased susceptibility to antifungal
macromolecules were analyzed, and by dry treatment (detailed below). Seven genes were
weight was comprised of 55 % protein, 25 % identified that govern levels of matrix mannan
carbohydrate, 15 % lipid, and 5 % nucleic acid. (ALG11, MNN9, VAN1, MNN4-4, PMR1, and
The relative abundance of protein was much VRG4), while two genes govern levels of matrix
greater than previously reported (Al-Fattani and -1,6 glucan (BIG1 and KRE5). The previously
Douglas 2006), and proteomic analysis indicated studied gene encoding the -1,3 glucan synthase,
458 distinct entities. This revealed many similar FKS1, was included in order to study this third
protein classes to those previously identified, matrix polysaccharide. Strikingly, when mixed
including those implicated in carbohydrate and biofilms containing mutants from the different
amino acid metabolism (Thomas et al. 2006; pathways were grown together, matrix of normal
Faria-Oliveira et al. 2014). Lipids identified in structure and function was restored. This
the C. albicans matrix included both neutral and suggested that the MGCx components assemble
polar glycerolipids, and a small proportion of extracellularly, as the mutants lacking one poly-
sphingolipids. The nucleic acids identified were saccharide could be complemented by neighbor-
mainly non-coding sequences of DNA. ing cells lacking a different polysaccharide. These
Recent study included further detailed analy- findings are represented in Fig. 1. Reflecting a
sis of the matrix polysaccharides. Surprisingly, a community behavior, the assembly of matrix
previously identified carbohydrate with func- materials by the biofilm constitutes an exciting
tional roles in the matrix, -1,3 glucan, was area for further studies.
28 K.F. Mitchell et al.

Fig. 1 Pictured is a
scanning electron
micrograph of a Candida
albicans biofilm. The
extracellular matrix is false
colored in blue, red, and
green, representing its three
major polysaccharides.
Work by Mitchell
et al. reveals that these
components physically
interact, and are each
required for matrix
structure and function
(Mitchell et al. 2015)

3.6 Role of Candida Matrix in Drug from biofilms and added to planktonic cells prior
Resistance to antifungal susceptibility testing. The plank-
tonic cells with matrix added were found to
The early biofilm studies by the Douglas group gain levels of resistance similar to mature
began to explore the role of Candida biofilm biofilms. Further, an additional experiment
matrix in resistance to antifungal therapies. The tracked the penetration of radiolabeled flucona-
growth conditions of continuous flow, associated zole within biofilms, and found the majority to be
with increased matrix, provided the highest level retained within the matrix. This work also first
of protection from drug treatment (Baillie and implicated the matrix carbohydrate -1,3 glucan
Douglas 2000; Al-Fattani and Douglas 2006). It in the resistance phenotype: biofilms treated with
should be noted that antifungal resistance in -1,3 glucanase greatly increased susceptibility
C. albicans is multifactorial, with the matrix to fluconazole treatment both in vitro and in vivo.
emerging as a relevant mechanism during the A 2010 study by the dEnfert group corroborated
mature stage of biofilm growth (Taff the importance of matrix for antifungal protec-
et al. 2013; Ramage et al. 2012). Namely, efflux tion from the polyene antifungal, amphotericin B
pumps reduce intracellular accumulation of (Vediyappan et al. 2010). Further studies have
triazole antifungals during the early phases of demonstrated a role for -1,3 glucan in the resis-
biofilm growth (Mukherjee et al. 2003; Ramage tance against additional classes of antifungals,
et al. 2002). Additional mechanisms thought to and in the species C. glabrata, C. parapsilosis,
impact biofilm drug resistance include the pres- and C. tropicalis (Nett et al. 2010a; Mitchell
ence of drug-tolerant persister cells and changes et al. 2013; Yi et al. 2011; Fernandes et al. 2015).
in the sterol content of the cell membrane Several studies have investigated the produc-
(Mukherjee et al. 2003; Nett et al. 2009; LaFleur tion and regulation of -1,3 glucan in the matrix.
et al. 2006). The -1,3 glucan synthase Fks1p is clearly
Matrix contribution to biofilm resistance was involved directly in production and control of
tested even more directly by Nett et al. (Nett the levels of this matrix material, and the glucan
et al. 2007b). Extracellular matrix was isolated modifier proteins Bgl2p, Phr1p, and Xog1p were
The Extracellular Matrix of Fungal Biofilms 29

found to impact delivery of -1,3 glucan from the (eDNA). Addition of DNase to biofilms increases
cell surface to the extracellular space (Nett the efficacy of some but not all antifungal drug
et al. 2010a, c; Taff et al. 2012). A separate classes (Martins et al. 2010; Martins et al. 2012).
regulatory mechanism for production -1,3 glu- As described above, eDNA is an important struc-
can involves the transcription factor Zap1p, tural component of other biofilm species, and in
which negatively impacts the production of this bacterial species is related to the exchange of
material (Nobile et al. 2009). Nobile et al. found genetic material (Rajendran et al. 2013; Mulcahy
relevant targets of Zap1p to be two et al. 2008). However, in Candida biofilms the
glucoamylases, GCA1 and GCA2, which may exact mechanism of how eDNA contributes to
contribute to the hydrolysis of matrix drug resistance remains unclear, especially in
carbohydrates. Other Zap1p targets include alco- light of these sequences being largely
hol dehydrogenases ADH5, CSH1, and LFD6, non-coding (Zarnowski et al. 2014).
which could perhaps impact the biofilm and
matrix through quorum sensing pathways.
Recent work found that -1,3 glucan, while pres- 4 Conclusions
ent in relatively low quantities, works in con-
junction with the other matrix polysaccharides The studies outlined here have provided insight
to provide the biofilm resistance phenotype into key components of the fungal biofilm extra-
(Mitchell et al. 2015). cellular matrix, as summarized in Table 1. How-
Several other pathways have been implicated ever, continued elucidation of matrix
in the resistance of C. albicans biofilms. The composition, production, and function is critical
transcription factor Bcr1p, a regulator of overall for several reasons. Besides providing funda-
biofilm formation, has been found to influence mental information on the biofilm lifestyle, char-
permeability to dyes and neutrophils, as well as acterization of new matrix materials may identify
sensitivity to fluconazole. This was suggested by novel targets for antifungal therapies. Based on
the finding of different expression levels of this information, if methods of matrix inhibition
Bcr1p in mating competent and mating incompe- are developed, they might prove most useful in
tent biofilms, as well as the finding that Bcr1p combination with existing antifungals that,
was required for abundant matrix levels in a alone, are ineffective against biofilms. As proof
vaginal colonization model (Srikantha of principle, adding -mannosidase or -1,6
et al. 2013; Harriott et al. 2010). The heat shock glucanase to biofilms in combination with flu-
protein Hsp90p has also been found to be a conazole results in a greater reduction of biofilm
regulator of matrix production, and therefore growth in comparison to either treatment alone
drug resistance, as disrupting this pathway results (Mitchell et al. 2015). Additionally, specific
in azole susceptibility and also lower levels of matrix components are upregulated during bio-
-1,3 glucan in the matrix (Robbins et al. 2011). film growth in contrast to planktonically grown
Similar phenotypes were yielded by disrupting cells, with some present only during the biofilm
either SMI1 or RLM1, members of the protein lifestyle. These could serve as diagnostic
kinase C (PKC) pathway (Nett et al. 2011). These markers for biofilm infections of medical
defects were restored by overexpression of FKS1 devices, which are often difficult to differentiate
in the mutants, though no effect was seen when from other types of fungal infection.
upstream components of the PKC pathway were To best advance our existing knowledge of the
disrupted. Thus, the pathway members that extracellular matrix, future studies should focus
impact drug resistance and the integrity of the on several topics. First, the roles of specific
cell wall are thought to overlap in a distinct matrix components should continue to be
manner from the whole PKC pathway. investigated as the components themselves are
An additional matrix component found to identified. While our image of the matrix is
impact antifungal resistance is extracellular DNA becoming that of a complex structure, with
30 K.F. Mitchell et al.

Table 1 Extracellular matrix components of fungal biofilms

Biofilm species Carbohydrate Protein Lipid Nucleic acid Other
Saccharomyces Glucose, mannose in Relatively low
cerevisiae -1,6 chains with -1,2 protein levels
and -1,3 branches (Beauvais
(Beauvais et al. 2009); et al. 2009);
two different molecular multiple proteins
weight polysaccharides identified
containing glucose, including Tdh3,
mannose, and galactose Hsp26, and Sod2
(Faria-Oliveira (Faria-Oliveira
et al. 2015) et al. 2014)
Aspergillus Galactomannan, -1,3 Proteins, Comprises up eDNA Melanin,
fumigatus glucan, including to 14 % of (Rajendran polyols
monosaccharides hydrophobins total matrix et al. 2013) (Beauvais
(Beauvais et al. 2007); (Beauvais (Reichhardt et al. 2007)
cell wall et al. 2007); et al. 2015a)
exopolysaccharide 40 % of total
galactosaminogalactan matrix
(Gravelat et al. 2013); (Reichhardt
43 % of total matrix et al. 2015a)
et al. 2015a)
Cryptococcus Glucurunoxylomannan,
neoformans xylose, mannose,
(Martinez and
Casadevall 2007)
Candida 25 % of total matrix, 55 % of total 15 % of total 5 % of total Phosphorus,
albicans includes -1,6 mannan matrix, with matrix, matrix, primarily uronic acid,
with -1,2 branches, 458 distinct includes non-coding hexosamine
-1,6 glucan, and -1,3 entities neutral and sequences (Al-Fattani
glucan (Zarnowski (Zarnowski polar (Zarnowski and Douglas
et al. 2014) et al. 2014) glycerolipids, et al. 2014); 2006)
sphingolipids eDNA
(Zarnowski (Al-Fattani and
et al. 2014) Douglas 2006)
Candida Higher concentration of carbohydrate and
glabrata protein than C. parapsilosis matrix (Silva
et al. 2009)
Candida Higher levels of carbohydrate than protein
parapsilosis (Silva et al. 2009)
Candida Higher levels of carbohydrate than protein Phosphorus,
tropicalis (Fernandes et al. 2015); lower levels of uronic acid,
protein than C. albicans matrix (Al-Fattani hexosamine
and Douglas 2006) (Al-Fattani
and Douglas

individual components acting in concert, some of interacting matrix components? Do these inhibit
these components may be more critical than or promote the overall integrity of that combined
others in conferring certain physical properties. matrix, or perhaps lead to the alteration of cellu-
These queries will expand as studies involving lar processes? Further, what is the role of host
mixed species biofilms become more prevalent: components that interact with the fungal matrix
do biofilms with multiple microbes contain during biofilm infections?
The Extracellular Matrix of Fungal Biofilms 31

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Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2016) 3: 3747
DOI 10.1007/5584_2016_7
# Springer International Publishing Switzerland 2016
Published online: 27 April 2016

Fungal Biofilms: Update on Resistance

Elisa Borghi, Francesca Borgo, and Giulia Morace

Over the past decade, the emergence of biofilm-related invasive fungal
diseases has been the subject of numerous studies focused on antifungal
resistance and its impact on antifungal therapy in severely ill patients. The
majority of the studies investigated the molecular mechanisms involved in
antifungal resistance and pathogenicity of biofilm production by Candida
albicans and Aspergillus fumigatus, the most common etiologic agents of
yeast and mold invasive infections. The main mechanism characterizing
biofilm-related antifungal resistance is the production of extracellular
matrix, a physical barrier preventing the drugs from entering and
expressing their activity. However, over-expression of efflux pumps,
genetic changes of drug targets, persister cells, biofilm-host immune
system interaction, proteins leading to filamentation, all together contrib-
ute to the onset of biofilm antifungal resistance. Some of these
mechanisms are shared with planktonic cells and are often related to
developmental phases of biofilm formation. All physical and genetic
factors leading to biofilm-related antifungal resistance have been briefly

1 Introduction process of invasive fungal infections and there-

fore it is considered an important virulence fac-
Fungal biofilms are complex communities of tor. However, the most intriguing feature of
cells and hyphae attached to a substrate and fungal biofilms is their marked resistance to
protected by an extracellular polymeric matrix. both host immune cells and antifungal drugs
Biofilm formation is involved in the pathogenic commonly used to treat invasive fungal diseases.
Fungi are able to form biofilms on both abiotic
surfaces and animal tissues, having broad
E. Borghi (*), F. Borgo, and G. Morace implications for healthcare. The resistance
Department of Health Sciences, Universita` degli Studi di
Milano, Milan, Italy displayed by biofilms to antifungals represents a
e-mail: considerable therapeutic problem as it may

38 E. Borghi et al.

involve all the main classes of antifungal agents As Ramage et al. (2012) pointed out, fungal
currently available. biofilm resistance involves both physical barriers
Over the past decade, the emergence of and regulatory processes, some of the latter
biofilm-related invasive fungal diseases has already shown for planktonic cells. Thus, anti-
been the subject of numerous studies with fungal resistance of biofilms is multifactorial and
emphasis on antifungal resistance and its impact heterogeneous, often related to developmental
on the treatment of severely ill patients (Pierce phases of biofilm formation (Taff et al. 2013;
et al. 2013, 2015; Cowen et al. 2014; Nett 2014; Lattif et al. 2011) (Fig. 1).
Mathe and Van Dijck 2013; Mitchell et al. 2013; In the following sections the different physi-
Rajendran et al. 2013; Srikantha et al. 2013; Taff cal and genetic factors leading to antifungal
et al. 2013; Ramage et al. 2012; Shapiro resistance of biofilms will be briefly discussed.
et al. 2012; Nett et al. 2007, 2010a, b, 2011;
Watamoto et al. 2009; Bizerra et al. 2008;
Al-Fattani and Douglas 2006).
2 The Extracellular Matrix
Candida albicans and Aspergillus fumigatus,
the major etiologic agents of invasive fungal
Microbial biofilms are characterized by the pres-
diseases among yeasts and molds respectively,
ence of an extracellular matrix (ECM) that
have been the fungal species more extensively
embeds the cells contributing to the complex
studied for investigating the mechanisms
tridimensional architecture of the community.
involved in pathogenicity and biofilm-related
ECM presence is considered a defining feature
antifungal resistance, two aspects closely
of biofilms and its composition varies among
correlated (Fan et al. 2015; Nobile et al. 2014;
different species of microorganisms (Flemming
Sherry et al. 2014; Mathe and Van Dijck 2013;
and Wingender 2010; Al-Fattani and Douglas
Rajendran et al. 2011, 2013; Srikantha
2006; Reichhardt et al. 2015). Despite several
et al. 2012, 2013; Taff et al. 2013; Bink
differences, carbohydrates, proteins and nucleic
et al. 2012; Shapiro et al. 2012; Lattif
acids are the main constituents in both bacterial
et al. 2011; Robbins et al. 2011; Ferreira
and fungal biofilms (Van Acker et al. 2014).
et al. 2010; Nett et al. 2010a, 2010b; Vediyappan
The ECM is alternatively named extracellular
et al. 2010).
polymeric substance (EPS) and confers to sessile

Fig. 1 Schematic overview of fungal resistance intrinsic resistance of fungal biofilms. The number of
mechanisms involved in various biofilm phases. The arrows for each factor indicates the greater (two arrows)
arrows represent the different factors that contribute to or less impact (one arrow) in each developmental phase
Fungal Biofilms: Update on Resistance 39

cells a broad range of advantages such as adhe- polyols, melanin, proteins, and DNA (Beauvais
sion and cohesion properties, mechanical et al 2014; Loussert et al. 2010).
properties, nutritional sources, enzymatic
activities, and protection (Flemming and
Wingender 2010).
2.1 Matrix-Related Antifugal
Biofilm forming-ability and subsequent
matrix production has been demonstrated for
almost all pathogenic fungi (Ramage
Despite differences in the composition, ECM
et al. 2012). However, extensive studies have
actively participates in the drug resistance phe-
been made mostly for Candida albicans, by
notype of biofilm-embedded cells. The confirma-
Andes group (Zarnowski et al. 2014; Nett and
tion of its role has been clearly demonstrated by
Andes 2006; Mitchell et al 2015), and to a lesser
Nett et al. that added biofilm matrix to planktonic
extent for other Candida spp. (Fernandes
cells obtaining a drug-resistant phenotype (Nett
et al. 2015; Silva et al. 2009) and Aspergillus
et al. 2007).
fumigatus (Wuren et al. 2014; Loussert
The most intuitive mechanism, although not
et al. 2010; Reichhardt et al. 2015).
the most relevant, is the capability of reducing
The chemical composition of ECM produced
penetrance of drugs throughout biofilm layers.
has been recently reported for some fungal spe-
Despite biofilm spatial heterogeneity and the
cies. Four macromolecular classes were found to
presence of water channels that facilitate nutrient
be the major constituents of C. albicans EPS:
influx and waste efflux (Chandra et al. 2001;
proteins and glycoproteins (55 % [wt/wt]),
Ramage et al. 2005), the penetration of antimi-
carbohydrates (~25 % [wt/wt]), lipids (~15 %
crobial drugs could be not sufficient to achieve
[wt/wt]), and nucleic acids (~5 % [wt/wt])
an appropriate distribution in all the biofilm areas
(Zarnowski et al. 2014). Concerning the
(Van Acker et al. 2014). Although some authors
polysaccharides, the main cell wall constituents,
(Al-Fattani and Douglas 2004) observed, by
-1,3-glucans, were found to be poorly
using a filter disk assay, a good biofilm penetra-
represented in the matrix, whereas the most
tion by azoles, amphotericin B and flucytosine,
abundant were -1,2-brached, and
the used technique as well as the lower presence
of matrix in the in vitro growth of Candida
C. tropicalis matrix contains mainly
biofilms could have overestimated the distribu-
hexosamine (27.4 %), with smaller amounts of
tion into small niches present into the biofilm
carbohydrates (3.3 %, including 0.5 % glucose),
(Van Acker et al. 2014). Many studies
and proteins (3.3 %) (Al-Fattani and Douglas
demonstrated that static growth of biofilms
resulted in a lower matrix production in compar-
Less is known about matrix composition in
ison to flow-through or in vivo models (Baillie
other Candida species, but according to Silva
and Douglas 2000; Nett and Andes 2006;
et al. (2012) C. parapsilosis biofilms consist of
Uppuluri et al. 2009).
high amounts of carbohydrates and small
Poor or delayed drug diffusion could in turn
amounts of proteins, whereas C. glabrata display
promote a secondary mechanism of resistance.
high amounts of both proteins and carbohydrates.
Cells exposed to low and inadequate
In Cryptococcus neoformans, the capsular
concentrations of drugs, in fact, could develop
component glucurunoxylomannan is the predom-
or put in place defensive strategies to survive the
inant component, together with xylose, mannose,
action of the compound.
and glucose (Martinez and Casadevall 2007).
However, recent studies suggested that spe-
The hydrophobic matrix of A. fumigatus is
cific constituents of ECM play a crucial role in
composed of galactomannan, galactosamino-
drug resistance.
galactan, -1,3 glucans, monosaccharides,
40 E. Borghi et al.

2.1.1 Candida albicans and other the amphotericin B resistance (Fernandes

Candida spp. et al. 2015). The authors observed that
C. albicans biofilm resistance is multifactorial amphotericin B administration to preformed bio-
and different mechanisms participate in a stage- film dramatically increased the carbohydrate and
dependent manner (Ramage et al. 2012). How- protein contents of ECM, resulting in a thicker
ever, a matrix-dependent mechanism has been and drug-proof biofilm.
fully elucidated by Andes group (Nett
et al. 2007 and 2010b; Taff et al. 2012).
2.1.2 Aspergillus fumigatus
-1,3-glucans have been shown to be able to
In vivo, in both aspergilloma and invasive asper-
sequester azoles, echinocandins and polyenes,
gillosis, A. fumigatus has been observed to pro-
resulting in a multidrug resistant mechanism
duce ECM (Loussert et al. 2010; Seidler
(Nett et al. 2010a). It relies on the expression of
et al. 2008). The in vivo ECM composition has
FKS1, encoding a -1,3 glucan synthase, and has
been demonstrated to be highly similar to those
been demonstrated to be biofilm-specific, as
obtained in vitro (Loussert et al. 2010), and its
overexpression or heterozygous disruption of
production is stimulated by the presence of
FKS1 does not alter susceptibility of planktonic
serum or its proteins (Shopova et al. 2013;
cells (Nett et al. 2010b). Moreover, the crucial
Wuren et al. 2014).
role of -1,3 glucans has been corroborated by
Galactomannan and galactosaminogalactan
studies from mutants of three genes controlling
(GAG) are the major components of ECM and
-1,3 glucans delivery into the matrix, two glu-
constitute its scaffold; -1,3 glucan, found in
can transferases and an exo-glucanase (BGL2
lower proportion, mediate hyphae cohesion
and PHR1, and XOG1, respectively). These
(Loussert et al. 2010).
mutants, with reduced glucans content, displayed
Rajendran et al. recently demonstrated that
enhanced susceptibility to the above-mentioned
eDNA plays a crucial role in A. fumigatus biofilm
antifungals (Taff et al. 2012).
production (2013). eDNA matrix content
Recently, another C. albicans matrix compo-
increases with the maturation of the biofilm,
nent, the extracellular DNA (eDNA), has been
released by autolytic processes catalyzed by
observed to mediate, at least partially and with
chitinases. This increase goes in parallel with
unknown mechanism, the resistance to
the increase in antifungal resistance. Moreover,
echinocandins and polyenes, but not to azoles
the authors, by adding exogenous DNA to early-
(Martins et al. 2012). Accordingly, DNAse treat-
phase biofilms, observed an increase of biofilm
ment was shown to enhance drug susceptibility.
biomass elucidating a role for eDNA in promot-
Extracellular DNA (eDNA) and its role in
ing and maintaining biofilm architectural integ-
antimicrobial resistance have been first identified
rity. Similarly, DNase I treatment drastically
in bacterial biofilms (Whitchurch et al. 2002;
altered the structural integrity of mature biofilms.
Tetz et al. 2009). Bacterial eDNA exerts other
The eDNA role in antifungal resistance was
roles, such as nutrient source, genetic informa-
demonstrated by the same group by treating
tion exchange, and biofilm increased stability.
preformed A. fumigatus biofilms with
C. albicans eDNA has been shown improve
amphotericin B, caspofungin and azoles in com-
the stability of mature biofilms, without affecting
bination with DNase. An increase in susceptibil-
it establishment (Martins et al. 2010).
ity to echinocandins and polyenes was observed.
eDNA has been demonstrated also in other
This behavior was not shown analyzing azoles
Candida species (Al-Fattani and Douglas 2006;
(Rajendran et al. 2013), probably due to the lack
Sapaar et al. 2014).
of active cell proliferation, and thus of ergosterol
Furthermore, a recent study on C. tropicalis
biosynthesis, in mature biofilm.
biofilm demonstrated a matrix involvement in
Fungal Biofilms: Update on Resistance 41

3 Efflux-Pumps Activity A. fumigatus in vivo biofilms. Due to the high

in Fungal Biofilm number of efflux pumps in A. fumigatus, most of
and Expression of Drugs them yet to be characterized, it is possible that
Targets other genes could compensate the lack of
AfMDR4 overexpression resulting in an equal
Genes encoding for efflux pumps have been resistant phenotype. Indeed, da Silva Ferreira
identified and extensively studied in and colleagues (2006) by performing a global
C. albicans, and several authors reported an transcriptomic analysis reported mdr1 gene to
increasing expression during sessile growth be overexpressed in mycelial growth during
(Sanglard and Odds 2002; Ramage et al. 2012). voriconazole exposition.
In C. albicans planktonic cells, resistant Thus, efflux pumps-mediated resistance is rel-
phenotypes are due to altered expression of three evant in the first phases of fungal biofilm whereas
genes: CDR1 and CDR2, encoding for in mature biofilms other mechanisms should
ATP-binding cassette (ABC) transporters, and prevail.
MDR1, encoding for major facilitator superfamily The ergosterol biosynthetic pathway is the
(MFS) transporters (Sanglard and Odds 2002). target of both polyenes and azoles. An overall
By using single, double and triple mutants of decreased content in ergosterol has been
C. albicans for these genes, Mukherjee observed in C. albicans sessile cells in compari-
et al. (2003) demonstrated that efflux pumps- son to the planktonic counterpart (Mukherjee
mediated fluconazole resistance is dependent on et al. 2003), being more pronounced in mature
the phase of biofilm formation, being evident in biofilms.
early phases of the process but not in mature Recent in vitro and in vivo biofilm models
biofilms. Accordingly, Ramage group showed (Khot et al. 2006; Nett et al. 2009) showed an
that 24 h- and 48 h-old Candida biofilms do not overexpression in C. albicans biofilms of
display altered efflux pumps activity (Ramage ERG25, a C4 methyl sterol oxidase with a role
et al. 2002). in C4- demethylation of ergosterol biosynthesis
CDR1/2 and MDR1 homologs have been intermediates. ERG25 promotes the conversion
identified also in other pathogenic Candida spe- of lanosterol to non-ergosterol intermediates,
cies and in C. neoformans (Morschhauser 2010). reducing the ergosterol content in biofilm
C. tropicalis CtMDR overexpression (Bizerra membranes.
et al. 2008) has been shown in 24 h grown Hence, it is possible to hypothesize that a
biofilms compared with their planktonic modulation of ergosterol could contribute to
counterparts. Similar results were obtained in C. resistance in later phases of biofilm formation.
glabrata for CgCDR1 and CgCDR2, with a max-
imum of overexpression in early or intermediate
phases of biofilm formation (Song et al. 2009). 4 Other Mechanisms Promoting
Genomic sequence analysis showed in Biofilm Resistance
A. fumigatus the presence of putative 278 differ-
ent MFS and 49 ABC transporters (Nierman Adhesion to abiotic surfaces, cell to cell interac-
et al. 2005). Rajendran et al. (2011) tion as well as hyphal development are crucial
demonstrated that A. fumigatus voriconazole events for biofilm formation. Therefore, cell wall
resistance dramatically increases as germlings proteins involved in the processes of adhesion
proliferate to mycelia. Moreover, by assessing and/or filamentation, and transcription factors
mRNA expression of efflux pump AfMDR4, the regulating their expression have been deeply
authors showed a phase-dependent increase, investigated as potential players in biofilm anti-
higher at 24 h, and strain-dependent increase, fungal resistance. Although many studies are
further enhanced by voriconazole administration. focused on C. albicans (Blankenship and
AfMDR4 overexpression was also confirmed in Mitchell 2006), in a recent work, Fan and
42 E. Borghi et al.

colleagues (2015) demonstrated that similar pro- et al. 2014) have been recently shown to enhance
cesses could be present also in A. fumigatus. By polyene and azole susceptibility, respectively. In
deleting cspA gene of A. fumigatus, encoding for particular, Oligo G was demonstrated to modu-
the cell surface protein A, the authors achieved a late per se both fungal growth, by inhibiting
reduction in biofilm formation that led to filamentation, and biofilm formation, by altering
enhanced sensitivity to amphotericin B and biofilm morphology.
itraconazole. Mucolytic agents are also under investigation.
In patients with invasive fungal infection, dis- Ambroxol (2-amino-3,5-dibromo-N-[trans-
persion of biofilm cells and interaction of sessile 4-hydroxycyclohexyl] benzylamine), an expec-
cells with the ECM play an important role in torant used in many lung diseases, showed a
persistence and antifungal treatment failure. good activity against C. parapsilosis biofilms
Hsp90 is an important protein involved in (Pulcrano et al. 2012) improving fungal suscep-
fungal morphogenesis and virulence, and in tibility to voriconalole. As for alginate lyase, this
development of biofilm and drug resistance effect was biofilm-specific, as drug combination
(Shapiro et al 2012). Robbins and colleagues lacked to show a synergistic effect on planktonic
(2011) found that genetic depletion of Hsp90 cells.
impaired dispersion of C. albicans biofilm Many natural compounds have also been
cells in vitro, while compromised function of demonstrated to exert an anti-biofilm effect,
Hsp90 abrogated azole resistance in such as chitosan (Cobrado et al. 2013),
C. albicans biofilm, probably by reducing the polyphenols (Sardi et al. 2013), and fulvic acid
levels of ECM glucans. The same authors (Sherry et al. 2012). In particular, a carbohydrate
demonstrated that inhibition of Hsp90 with derived fulvic acid (CHD-FA), already known to
geldanamycin reduced echinocandins resistance be safe for topic and oral administration (Gandy
in A. fumigatus biofilm. et al. 2012), was shown to exert a rapid activity
on both planktonic and sessile cells of
C. albicans without differences in the minimal
5 Unconventional Compounds inhibitory concentrations (MICs).
to Implement Fungal Biofilm
6 Drug Tolerance
Studies addressing the multifaceted resistance of
fungal biofilms make clear the need to develop Besides multifactorial biofilm-mediated resis-
new strategies to treat such infections. tance, drug tolerant cells, named persisters,
Due to the crucial role of ECM in biofilm have been shown to characterize Candida biofilm
resistance, behaving as a drug sponge for communities (Lewis 2010; Li et al. 2015). As in
many of the available antifungals (Nett bacterial biofilms, these cells represent a
et al. 2010a and 2010b), new compounds sub-population that spontaneously and stochasti-
targeting matrix production as well as promoting cally arise and are genetically identical to the
its disaggregation could help in eradicating ses- clonal population, representing thus a phenotypic
sile pathogens. variant (Lewis 2010; LaFleur et al. 2006).
DNase treatment was shown to be effective in Chronic carriage of Candida, an important
enhancing the activity of polyenes and risk factor for subsequent infection, has been
echinocandins (Martins et al. 2012; Rajendran shown to increase the number of persister cells
et al. 2013) that, whilst recognized as the most in the microbial population (LaFleur
active classes against biofilm, are unable to et al. 2010). Periodic application of antifungals
completely kill biofilms (Ramage et al. 2014). may in fact select for strains with increased
Alginate lyase (Bugli et al. 2013) and the levels of persister cells. This aspect has broader
alginate oligomer, OligoG, (Tndervik implications in the clinical setting of susceptible
Fungal Biofilms: Update on Resistance 43

patients, as prolonged drug treatments and pro- Furthermore, persisters show a different regu-
phylaxis could favor drug tolerance and infec- lation of genes involved in both ergosterol and
tion recurrence. -1,6 glucan pathways (Khot et al. 2006).
Biofilm communities are enriched in Modifications in membrane and cell wall
persisters, although their percentage is strain- compositions could represent a complementary
dependent (Li et al. 2015). Studies performed pathway leading to drug tolerance.
on several strains mutated in their capability to
form biofilm, e.g., strains lacking the transcrip-
tion factors, led to the conclusion that attachment 7 Conclusions
to substrates, rather than complex biofilm
architectures, is the crucial step promoting The resistance to antifungal drugs is a well-
persisters selection (LaFleur et al. 2006). established health problem and the mechanisms
Persisters are dormant cells that exhibit toler- underlying this phenomenon are well known,
ance to multiple drug classes, including mainly through in vitro studies on planktonic
amphotericin B and azoles (LaFleur et al. 2006; cells (Perlin et al 2015; Cowen et al 2014; Perlin
Bink et al. 2012; Li et al. 2015). 2014; Bowyer et al 2011; Marie and White
The presence of persister cells results in a 2009). The antifungal resistance, which often
biphasic killing curve, with the majority of cells results in a therapeutic failure, has been progres-
dying rapidly at concentration of antimicrobial sively more associated to the ability of fungi to
close to the MIC, and a small fraction surviving develop biofilms. Several studies have shown
even at the highest concentrations (LaFleur that the physical barrier of biofilm extracellular
et al. 2006). matrix is the major mechanism allowing embed-
The mechanism responsible of antimicrobial ded cells to prevent drugs from entering and
tolerance is still not completely understood. expressing their activity. However, biofilm anti-
Transcriptional and proteomic analysis of fungal resistance is complex, involving different
persisters showed a differential regulation of genetic mechanisms some of them in common
genes that could explain the dormant status of with planktonic cells. Over-expression of efflux
these cells. In particular, glycolysis, tricarboxylic pumps, genetic changes of drug targets, persister
acid cycle (TCA) and protein synthesis have cells, biofilm-host immune system interaction,
been demonstrated to be down-regulated, proteins leading to filamentation, all together
whereas the alternative glyoxylate shunt contribute to the onset of biofilm antifungal resis-
enzymes were over-activated (Li et al. 2015). tance at early, intermediate or mature phases of
This switch resulted in a lower production of biofilm development.
reactive oxygen species (ROS), avoiding Overcoming biofilm-related resistance should
ROS-enhanced killing. Moreover, expression of represent an important progress in antifungal
proteins involved in stress response, as heat- therapy and would have broad implications for
shock proteins HSP90, HSP120, and HSP70, healthcare applications. Different approaches,
were also induced (Li et al. 2015). Accordingly, such as targeting stress responses, matrix produc-
superoxide dismutase mutants biofilms (sod4 tion, or other biofilm-specific mechanisms of
sod5) were shown to form less miconazole- resistance, are currently under investigation.
tolerant persisters (Bink et al. 2011). Up to date,
miconazole, echinocandins and liposomal
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DOI 10.1007/5584_2016_8
# Springer International Publishing Switzerland 2016
Published online: 12 May 2016

Fungi, Water Supply and Biofilms

Catherine KauffmannLacroix, Damien Costa,

and Christine Imbert

Even though it has been studied for many years, water-related infectious
risk still exists in both care and community environments due to the possible
presence of numerous microorganisms such as bacteria, fungi and protists.
People can be exposed directly to these microorganisms either through
aerosols and water, after ingestion, inhalation, skin contact and entry through
mucosal membranes, or indirectly usually due to pre-treatment of some
medical devices. Species belonging to genera such as Aspergillus, Penicil-
lium, Pseudallesheria, Fusarium, Cuninghamella, Mucor and in some
particular cases Candida have been isolated in water from health facilities
and their presence is particularly related to the unavoidable formation of a
polymicrobial biofilm in waterlines. Fungi isolation methods are based on
water filtration combined with conventional microbiology cultures and/or
molecular approaches; unfortunately, these are still poorly standardized.
Moreover, due to inappropriate culture media and inadequate sampling
volumes, the current standardized methods used for bacterial research are
not suitable for fungal search. In order to prevent water-related fungal risk,
health facilities have implemented measures such as ultraviolet radiation to
treat the input network, continuous chemical treatment, chemical or thermal
shock treatments, or microfiltration at points of use. This article aims to
provide an overview of fungal colonization of water (especially in hospitals),
involvement of biofilms that develop in waterlines and application of
preventive strategies.

D. Costa and C. Imbert

Laboratoire de Parasitologie et Mycologie Medicale,
CHU de Poitiers, 2 rue de la Miletrie, UBM, BP577,
Poitiers 86021, France
C. KauffmannLacroix (*) Laboratoire Ecologie Biologie des Interactions (EBI),
Laboratoire de Parasitologie et Mycologie Medicale, Universite de Poitiers, UMR CNRS 7267, equipe
CHU de Poitiers, 2 rue de la Miletrie, UBM, BP577, Microbiologie de lEau, UFR Medecine-Pharmacie, Bat
Poitiers 86021, France D1, 6 rue de la Miletrie, TSA 51115, Poitiers 86073,
e-mail: France

50 C. KauffmannLacroix et al.

1 Introduction well documented, especially regarding the

Aspergillus-related risk (Ao et al. 2014; Warris
Numerous studies have shown that water could et al. 2002). However, it has been shown that air
be contaminated by many microorganisms, both control may not be sufficient to ensure the pre-
in hospital and in community environments: vention of aspergillosis in units receiving immu-
virus, bacteria, protists and also microscopic nocompromised patients (VandenBergh
fungi, such as single-celled yeasts and multi- et al. 1999; Warris et al. 2001b). Actually, this
cellular filamentous fungi. People can be contamination may occasionally originate from
exposed to microorganisms present in water the aerosolisation of a mouldy aquatic niche
after ingestion (drinking), inhalation (Anaissie et al. 2003). Rigorous control of both
(showering), skin contact (showering or bathing) air and water supply is essential to control the
and entry through mucosal membranes related infectious risk. Studies reported in this
(showering and bathing). The fungal risk related article focused on the fungal presence in water
to water has been highlighted in some nosoco- and were generally conducted in the context of
mial outbreaks due to Fusarium sp, Aspergillus microbial investigations in health facility
sp or Exophiala janselmei sp and also in networks subsequent to an outbreak or were
drowning-related infections due to designed to search for specific fungi such as
Pseudallescheria sp (Ao et al. 2014; Figel Pseudallesheria sp or black yeasts. Available
et al. 2013; Goncalves et al. 2006; Heinrichs data concerning investigations on hospital water
et al. 2013; Nucci et al. 2002; Pires-Goncalves networks further to bacterial outbreaks (Pseudo-
et al. 2008; Warris et al. 2001a; Warris monas aeruginosa, Legionella pneumophilla)
et al. 2002). Historically, hydric fungal contami- contributed to the development of an analytical
nation has been considered as a minor health method applied to fungi (Kauffmann-Lacroix
issue in health facilities because of the rather et al. 2008). Current knowledge on water as a
opportunistic behavior of related infections. fungal biotope and on interactions established
However, fungi can also be responsible for between fungi and other microorganisms poten-
severe systemic infections and given an increas- tially present in water still remain incompletely
ing number of fragile or immunocompromised understood. This article deals with the fungal
patients, effective control of the microbiological human infectious risk related to water exposure.
water quality, including fungi, has become of
increasing interest. Fungi of environmental ori-
gin can contaminate neutropenic patients in 2 Biofilms in Water
hematology units, patients receiving a solid-
organ transplant and patients from intensive One of the major issues related to the presence of
care units receiving high-dose corticosteroids fungi in water is their possible growth, together
who should benefit from prophylactic measures with other microorganisms such as bacteria and
against bio-aero contamination-related risk. protists, in complex structures called biofilms.
Fungi can cause candidemia or, more frequently Indeed, water networks particularly favor biofilm
and more worrisome in this context, fatal formation. Flemming has reported that 95 % of
diseases due to filamentous fungi infection. Air- the bacterial biomass occurred as biofilms on the
borne fungi responsible for nosocomial inner surface of the drinking water distribution
infections usually belong to the Aspergillus system whereas less than 5 % existed in the
genus. Aspergillus conidia potentially respon- planktonic form (Flemming 2002). If current
sible for nosocomial aspergillosis can also be knowledge on drinking water biofilms has been
airborne from a water source, thereby obtained mainly from studies on bacterial
establishing a potential link between aero- and biofilms, it has been also shown that Penicillium
water-contamination (Anaissie and Costa 2001). expansum was able to develop a mature biofilm
Mold aerocontamination in health facilities is in around 48 h in a model mimicking drinking
Fungi, Water Supply and Biofilms 51

water distribution system conditions (Simoes at the level of shower heads and siphons. This is
et al. 2015). The development of a biofilm in also the case in endoscopy reprocessing units,
drinking water typically involves seven succes- dialysis units and dental units.
sive phases: initial adhesion (4 h), germlings
(8 h), initial monolayers (12 h), a monolayer of
intertwined hyphae (24 h), mycelial develop- 3 Methods Dedicated to Fungi
ment, hyphal layering and bundling, and finally Isolation from Water
the development of the mature biofilm (48 h)
(Simoes et al. 2015). In the context of dental unit The filtration method applied to bacteria in
waterlines, it has been shown that biofilms start health facilities has been helpful for developing
to develop in the early hours following water a specific method adapted to fungi: samplings are
supplying for a newly installed dental unit performed on cold water and filtered volumes are
(Williams et al. 1995). Actually, by developing larger than the ones recommended for bacterial
within a biofilm, sessile microorganisms become controls (Kauffmann-Lacroix et al. 2008).
less susceptible or sometimes totally resistant to Indeed, fungi are usually inactivated in hot
different antimicrobial treatments, compared to water samples (>55  C), highlighting the fact
microorganisms growing planktonically. This that Legionella and fungi tests cannot be
resistance is partially explained by the presence performed using the same water sample.
of persisters which are highly tolerant to According to the literature, sampled volumes
treatments, especially in the deeper layers of the usually range between 50 mL and 500 mL. How-
biofilms. Other factors can contribute to suscep- ever, this volume varies considerably according
tibility loss: the matrix produced by sessile to the different studies: 50 mL (Arvanitidou
microorganisms and consisting of exopolymers et al. 2000), 100 mL (Schiavano et al. 2014),
containing open water channels limits the pene- 250 mL (Kadaifciler et al. 2013), 500 mL
tration of antimicrobial agents, thereby (Sammon et al. 2010; Warris et al. 2001a; Warris
constituting a physical barrier to antimicrobial et al. 2010) or 1 L (Goncalves et al. 2006;
agent efficacy and strongly limiting the diffusion Mesquita-Rocha et al. 2013). Such major
of oxygen and nutrients inside the biofilm differences in sampling volumes are probably
(Brown et al. 1988; Costerton et al. 1995). In explained by the variable fungal contamination
addition, the process of biofilm formation is level in the water studied but also by differing
regulated by specific genes of which expression laboratory customs and means. For example, in
is triggered by adhesion; regulation of the genes France, the water delivered to the point of use is
encoding efflux pumps can significantly limit the only lightly contaminated by fungi because of
activity of some antifungal agents (Costerton preliminary treatments to make it drinkable. In
et al. 1995; Ramage et al. 2012). Even though such cases, the recommended sampling volume
recent years have been marked by many for fungal analysis could be 1 L (Kauffmann-
advances, comprehension of the mechanisms Lacroix et al. 2008). The lack of standardized
that govern the resistance of fungal cells within methods of search for fungal contaminations tak-
biofilms has remained incomplete. That much ing into consideration volume, culture medium
said, biofilms can be considered as reservoirs of and incubation temperature could explain some
microbial contamination of water systems by failures to conclusively prove the water origin of
periodic release of fungal cells, isolated or in healthcare associated infections: due to fail in
clusters with matrix and other microorganisms. fungal isolation or misidentification of
Biofilms grow at an interface such as the one co-existing different phenotypes. In addition,
existing between air and water, and are promoted the fact that some fungi cannot be isolated from
by flux, especially in cases of slow flux some samples does not prove that these species
alternating with stagnation periods. These were not previously present (Anaissie
conditions are found in water systems, in com- et al. 2003). Regarding conditions for experimen-
munity and hospital environments, for example tal cultivation, use of a culture medium
52 C. KauffmannLacroix et al.

supplemented with an antibiotic is studies: 25  C (Goncalves et al. 2006; Sammon

recommended. However, various culture media et al. 2010) ; 25  C and 37  C (Mesquita-Rocha
(Figs. 1 and 2) are used to isolate fungi from et al. 2013); 23.5  1.5  C (Schiavano
water samples and the fact that they vary from et al. 2014) ; 35  C and 42  C (Warris
one laboratory to another may be an issue. For et al. 2010), 35  C (Warris et al. 2001a). Impor-
example, depending on studies, authors used tantly, an incubation temperature close to 30  C
Sabouraud dextrose agar supplemented with should be preferred as it would support biodiver-
chloramphenicol (Arvanitidou et al. 2000; sity, whereas a warmer temperature, such as
Mesquita-Rocha et al. 2013; Schiavano 37  C or 40  C could promote some specific
et al. 2014) or sometimes with penicillin (Warris species such as A. fumigatus complex (Oliveira
et al. 2001a; Warris et al. 2010) ; tap water agar, et al. 2013). Finally, most of the time, incubation
half-strength corn meal agar, neopeptone- was performed for one week (Goncalves
glucose rose Bengal aureomycin and oomycete et al. 2006; Sammon et al. 2010; Schiavano
selective medium were also used (Goncalves et al. 2014; Warris et al. 2001a; Warris
et al. 2006) as well as malt extract agar with et al. 2010) but it could be extended to 15 days
chloramphenicol (Sammon et al. 2010). Incuba- (Mesquita-Rocha et al. 2013); some authors
tion temperature also varies according to the incubated water samples for three or four weeks

Fig. 1 A. fumigatus on Czapek, Sabouraud-Gentamicin and Sabouraud medium respectively (up to down)

Fig. 2 A. fumigatus, A. flavus and A. nidulans respectively (up to down) on Czapek medium
Fungi, Water Supply and Biofilms 53

at room temperature (Arvanitidou et al. 2000). fungal species identification (Schelenz

Only a few laboratories currently implement the et al. 2015). They are currently based on the use
new molecular methods for routine analyses; in of DNA sequences variation designed to distin-
the future, however, the fungal ecology of water guish species or strains. Multiple genes ranging
will be certainly more and more extensively from the universal ribosomal DNA region ITS and
explored thanks to these methodological the large ribosomal subunit D1D2 to protein
advances. encoding genes such as the -tubulin and calmod-
ulin gene regions are now used to delimit species
within aspergilli.
4 Fungi Isolated from Water Numerous genera of filamenteous fungi with a
number of different species, (Acremonium,
Fungi are eukaryotic and aerobic microorganisms, Alternaria, Aspergillus, Cladosporium,
nonchlorophyllous heterotrophs, and they are Exophiala, Fusarium, Penicillium,
characterized by two different forms: the asexual Trichoderma. . .) and yeasts (Candida) can be
or anamorph one, which is observed in culture (for isolated from water, notwithstanding the hydro-
example A. fumigatus) and the sexual one, which phobic nature of certain structures (spores,
is not always known (for example Neosartorya blastospores). Fungi infect many organic
fumigata) (Kwon-Chung and Sugui 2009). Less substrates and are responsible for their degrada-
than 10 % of an estimated 1.5 million of fungal tion or transformation. Their growth is promoted
species have been described to date; consequently, by heat and moisture. As regards filamentous
the identification of fungi up to the species is quite fungi that are pathogenic for humans, we do not
difficult and lacking in most of the studies. Asper- currently know in which cases water is the origi-
gillus is a genus bringing together different spe- nal biotope or else represents a habitat that could
cies based on morphological, physiological and be described as transitional. The recent identifica-
phylogenetic characteristics (Balajee et al. 2007). tion of certain fungi to clarify the species by
Aspergillus has been associated with nine means of molecular biology appears to support
teleomorph genera, but phylogenetic data suggest the idea that water could be the preferred habitat
that it is linked together with other genera. After of certain species. Quantitative evaluation of the
the recent changes in the International Code of different fungi isolated from water was shown to
Nomenclature for algae, fungi and plants, the depend on its origin: surface water presents a
Aspergillus genus now contains 339 species (Sam- higher risk of colonization compared to deeper
son et al. 2014). Recent molecular approaches samples such as groundwater (Gottlich
have offered prominent alternative methods com- et al. 2002). A recent study (Oliveira et al. 2013)
pared to conventional approaches based on mac- identified different species in waters with different
roscopic and microscopic characteristics, and origins from surface source and groundwater
multiple recent studies have demonstrated the in Portugal. These authors underscored the great
limited utility of morphological methods used sin- biodiversity of fungi by isolating 27 different
gly for identification of clinically relevant species. genera. Several pathogenic species were found,
It is increasingly recognized that comparative including Aspergillus species, A. fumigatus,
sequence-based methods used in conjunction A. calidoustus (Hageskal et al. 2007) and
with traditional phenotype-based methods can A. viridinutans, Fusarium sp, and Zygomycetes,
offer better resolution of species within this Mucor racemosus, Cuninghamella bertholletiae.
genus. Various diagnostic tools have been devel- Interestingly, the presence of Penicillium
oped for medically important groups of fungi but brevicompactum was reported in water in another
are still lacking in standardization (Roe study, even if Penicillium sp are only exception-
et al. 2010); however very recent publications of ally responsible for human infections in Europe
standardized methods for DNA extraction, ampli- (only one case of infection associated with
fication and sequence analysis can be applied to P. brevicompactum) (De la Camara et al. 1996).
54 C. KauffmannLacroix et al.

However, A. calidoustus (group A. usti) is pioneer French study in 1985, the fungal micro-
emerging as a pathogen reported in immunocom- flora was investigated in 38 samples of
promised patients. In a recent study of the section chlorinated tap drinking water. Interestingly, fil-
A. usti, strains isolated in immunocompromised amentous fungi and yeasts were isolated in 81 %
patients were investigated showing the emergence and 50 % of the water samples respectively; as
of A. calidoustus among lung-transplanted regards yeasts, Candida genus was the most
patients with pulmonary aspergillosis resistant to isolated, and half of the filamentous fungi
azole antifungals of the third generation (Egli belonged to three genera: Penicillium, Aspergil-
et al. 2012). Furthermore, frequent isolation of lus and Rhizopus (Hinzelina and Block 1985).
fungi belonging to the genus Pseudallesheria From that time, other studies devoted to air and
(Scedoporium) in patients having escaped drown- water analyses have highlighted the presence of
ing was reported in the 2000s (Tintelnot fungi in drinking water and reported the almost
et al. 2008) and is another example of exposure systematic presence of Penicillium and Aspergil-
to water-related risk of fungal infection. This lus genera in positive samples. Their presence
observation was confirmed by reported cases in may be related to their high frequency in air
patients who had been injured during a tsunami in (Gangneux et al. 2002; Siqueira et al. 2011).
Thailand or Japan. Finally, black yeasts were Aside from Penicillium and Aspergillus,
studied because of their general availability in European studies have often reported the pres-
wetlands; they are characterized by their resis- ence of Acremonium, Cladosporium, Epicoccum,
tance in very hostile environments such as hyper- Exophiala, Fusarium, Phoma, Trichoderma and
saline water, sea or ice from icebergs (Heinrichs Phialophora in drinking water (Arvanitidou
et al. 2013). et al. 1999; Arvanitidou et al. 2000; Goncalves
et al. 2006; Gottlich et al. 2002; Hageskal
et al. 2006; Hageskal et al. 2007). More recently,
5 The Fungi Isolated from Water black yeasts and filamenteous fungi were
Supply in Health Facilities reported as coming from water in hemodialysis
units (Arvanitidou et al. 2000; Figel et al. 2013;
According to some studies, filamentous fungi can Pires-Goncalves et al. 2008; Rao et al. 2009;
be isolated in water from both hospital and com- Schiavano et al. 2014; Varo et al. 2007). Patients
munity environments, and yeasts would resul- with chronic renal insufficiency undergoing
tantly be less frequently found (Fig. 3). In a haemodialysis or peritoneal dialysis may be

Fig. 3 Cladosporium sp isolated from an endoscopic rinsing solution filtration. (a) macroscopic aspect on malt agar
(b) microscopic aspect (conidiophores and conidia)
Fungi, Water Supply and Biofilms 55

susceptible to a number of primary or opportu- Netherlands (Warris et al. 2003) or Greece

nistic pathogens including fungi from water and (Panagopoulou et al. 2007). The observed
dialysate in haemodialyse units (Arvanitidou differences are correspondingly related to geog-
et al. 2000; Unal et al. 2011); at times the link raphy, climate, and culture procedures but also,
between the causal agent and water remains as previously mentioned, to non-standardized
missing (Proia et al. 2004). In hemodiafiltration methods leading to the use of different culture
procedures, dialysis water is used in the form of media, sampling volumes. . . Even if the results
dialysate and infusate up to 34006800 L directly of these studies are difficult to compare, the
following ultrafiltration through two or three fungal composition of water seems to vary
dialysis water monitor ultrafilters (Locatelli between hospitals according to water supply
et al. 2010). Microbiological water quality is conditions, and probably seasons and network
consequently an important issue in dialysis and features (material, age. . .). Overall, available
the water supply used to prepare the dialysate can studies have evaluated the fungal colonization
be one of the sources of contamination (Rao of water but have only exceptionally shown a
et al. 2009). In Brazil, legislation regulating direct link between water colonization level and
water microbiological quality for dialysis does infections. Finally, in hospitals, water must be
not cover waterborne microbes such as fungi considered as an environmental source of fungi
(Figel et al. 2013). Likewise in Brazil, the fungal that is airborne, and transmitted through card-
contamination in water distribution systems of a board and food.
tertiary care hospital devoted to medical assis-
tance of children with cancer was recently
investigated over a period of 12 months 6 Transmission of Fungi
(Mesquita-Rocha et al. 2013). These authors Responsible for Water-related
showed that the highest concentrations of fungi Infections in Health Facilities
in the water system occurred in autumn and sum-
mer. Generally, along with authors from other In France and in many other countries, there exist
countries, they have isolated a wide variety of numerous standards and rules to oblige hospitals
filamentous fungi from the water studied, but the to monitor and control the large water volumes
isolated strains were hardly in any way related to used for food, health care (hygiene, toilets. . .),
invasive infection. hemodialysis, medical device disinfection, air
Health facilities do not react in the same way conditioning systems, etc. The control of
to water fungal contamination, which has many microbiological water quality consequently
sources and causes: surface water or groundwa- requires the implementation of organizational,
ter, storage tank, hot water tank, nature and age technical and practical actions, including pro-
of the pipes in the network. . . It has been shown phylactic approaches. Nevertheless, water can
that the differing conditions depending on the remain a reservoir for hospital-acquired
differing origins of the water may lead to quali- infections.
tative and quantitative differences of fungal col- Regarding fungal risk, patients are potentially
onization, which is extensively increased in exposed to infections, especially lung infections,
water originating from superficial water or hav- following aerosol inhalation (humidifier, shower,
ing previously stagnated in a storage tank dental care) and also direct contact with medical
(Anaissie et al. 2002; Warris et al. 2001a). Stud- devices that were previously washed with water.
ies performed in hospital environments have par- Direct contact with water or aerosols may also be
ticularly targeted A. fumigatus of which the responsible for wounds or mucosa membrane
contamination level tended to differ according infections. Finally, water especially for perito-
to geographical location: the United States neal dialysis, drinking water and ice may be
(Anaissie et al. 2002; Squier et al. 2000), Norway sources of local infections, even if reported
(Hageskal et al. 2006; Hageskal et al. 2007), the cases nowadays remain the exception. When
56 C. KauffmannLacroix et al.

water-related fungal infections are suspected, fungi of environmental origin such as Aspergillus
microbiologists must prove, in order to establish sp were among the most frequent species. How-
the environmental source of infection, that the ever, Candida sp. yeasts were also present; they
pathogenic fungi isolated from a patient and most probably originated from the saliva reflux
those isolated from water are the same. Unfortu- occurring due to negative pressure when the
nately, the genotyping approaches needed to rotary dental instruments stop. Moreover, the
associate them are only rarely performed variety of fungal species was two times higher
(Anaissie et al. 2002; Squier et al. 2000). In a in both biofilm samples and in the output water
study reporting an outbreak of A. fumigatus in a sampled from rotary dental instruments as com-
neonatal intensive care unit, the authors used pared to water sampled from tanks. These data
microsatellite strain typing to prove that suggest that biofilm dispersal could contribute to
A. fumigatus strains isolated from neonates and the output water fungal contamination. In addi-
those isolated from environment were genotypi- tion, other studies have shown the presence of
cally related. The source of infection may have fungi in the same context. For example, in Brazil,
been the humidity chambers of neonate filamentous fungi were isolated in 70 % of the
incubators (Etienne et al. 2011). overall water sampled from either three-water
syringe, handpieces, water reservoir or the
water supply of dental units (Lisboa
7 Specific Case of Dental Unit et al. 2014). Overall, 16 filamentous fungi genera
Waterlines were isolated and among them the most
represented were: Acremonium (46.7 %),
Conditions encountered in dental unit waterlines Exophiala (14.7 %), Penicillium (9.4 %) and
strongly favor biofilm formation: significant Aspergillus (8.9 %) (Lisboa et al. 2014). In
water stagnation periods between patients, or Istanbul, Turkey, similar investigations revealed
during nights and weekends, together with the the presence of Penicillium sp, Cladosporium sp,
high surface ratio water volume, water circula- Candida sp, Cryptococcus sp and Aspergillus sp
tion in laminar flow (maximum flow at the center in dental unit waterlines (Kadaifciler et al. 2013).
and minimum at the periphery). The presence of All in all, many studies have highlighted fungal
multiple and complex flora in these waterlines, of presence in dental unit waterlines, but the fungal
both environmental and human origins, is related community seemed variable, probably
to a potential health risk for both patients and depending on the sampling location (i.
dental staff exposed to the water drops and e handpieces, air/water syringe, water tanks,
aerosols generated during dental care (Coleman incoming water). Development of specific
et al. 2014; Szymanska 2003). Nowadays, the biofilms in each sampled condition could explain
microbial contamination of dental unit waterlines the variability of the fungal community.
is well documented especially as regards bacte- Polymicrobial biofilms are able to grow in rather
rial identification (Barbot et al. 2012; Kumar poor nutritional conditions and to promote the
et al. 2010; ODonnell et al. 2011; Szymanska survival of numerous microbial species such as
2003; Szymanska 2005b). The presence of bac- Candida sp yeasts, which normally exist under
teria in biofilms formed in dental unit waterlines more nutritional conditions. Laboratory
is likewise well-documented but fungi are also experiments have also shown that human micro-
involved. In 2015 a Polish study investigated bial species such as Streptococcus gordonii,
25 dental units and independent water tanks and C. albicans, C. glabrata and C. parapsilosis
revealed the presence of fungi in 12 units: 11 of have been able to survive and sometimes to pro-
the 25 collected biofilm samples were liferate in water as soon as it contained a small
contaminated by fungi, as well as 16 of the out- concentration of saliva (1 or 2 % v/v) (Barbot
put water samples from rotary instruments et al. 2011; Costa et al. 2013). Furthermore, it has
(Szymanska 2005a). Interestingly, in this study, been shown that some species of free-living
Fungi, Water Supply and Biofilms 57

amoebae have been able to help the survival or applied to tap and showerhead allow a sterilizing
even promote the proliferation of Candida filtration at the point of use, which is considered
albicans, C. glabrata and C. parapsilosis in efficient to obtain microbiologically controlled
water (Vanessa et al. 2012). Free-living amoebae water (Fig. 4). Warris and collaborators recently
are commonly found in water systems and can evaluated the efficacy of point of use filter
contribute to the fungal risk associated with devices on taps and showerheads in view of
water by the biofilm. reducing patient exposure to filamentous fungi
These considerations once again raise the by developing a laboratory test (simulated test
issue of the prevention of infectious risk related rig) and monitoring filter efficiency in real
to water and at this time, the one radical action conditions in a pediatric bone marrow transplan-
able to eliminate a preformed biofilm requires the tation unit (Warris et al. 2010). Their results
replacement of contaminated surfaces; in the -obtained in laboratory conditions showed
case of dental unit waterlines, chemical agents that filters were highly effective in reducing the
(based on chlorine or hydrogen peroxide. . .) and number of colony-forming units of A. fumigatus
physical measures (pipe flushing) are only par- alone or in combination with Fusarium solani
tially active on biofilms, even when combined. from contaminated water for a period of at least
Up to now none of the cases of severe fungal 15 days. Finally, the use of sterile water in
infection have been definitively linked to dental humidifiers and in the rinsing of critical medical
unit waterline exposure, but the presence of such devices represents a significant improvement. In
fungi, at times including opportunistic a bone marrow transplantation unit, authors have
pathogens, may expose patients and dental staff shown that point of use filters completely retain
to diseases as asthma, rhinitis, other respiratory mould contamination on the first day; however
problems, etc. Epidemiological research should high concentration of particles in the water
be planned to evaluate this potential infectious occluded filters, thereby putting a premature
risk. Once again, all these data reinforce the need end to the study (Warris et al. 2010). That
for better monitoring and control of the microbial shows why point of use filters must be changed
quality of dental unit waterlines in order to limit regularly, according to supplier
the infectious risk related to dental care. recommendations.
Numerous factors are involved in the degra-
dation of network water-quality, including water
8 Prophylactic and Curative stagnation, corrosion, furring up, retrograde con-
Actions Developed in Hospitals tamination and biofilm formation. Measures for
the technical monitoring of plumbing and of
Hospital water networks are a potential source of some other critical points, combined with regular
hospital-acquired infections. Currently infectious microbiological tests, contribute to insure the
risk control is mainly related to bacterial risk and water quality. Health facilities can now archive
is based on the implementation of a program of all of these data. Bacterial monitoring is a very
both technical improvement and monitoring of sensitive indicator of microbiological pollution;
water quality. In many countries, health facilities however due to inappropriate culture media and
have implemented some preventive measures inadequate sampling volumes, the current
such as ultraviolet radiation to treat the input standardized methods used in bacterial research
network or continuous chemical treatment of are not suitable for fungal research.
cold water, usually using chlorine (hypochlorite Curative methods are based on the different
or chlorine dioxide), or at the point of use with types of maintenance applied to non-operating
microfiltration. Hot water can be treated by simi- networks; shock chlorination as well as thermal
lar processes (Ministe`res des solidarites and shocks can be carried out. Treatments using
DGS/DHOS 2005). In care units with immuno- peracetic acid or hydrogen peroxide can also be
compromised patients, end-filters (0.2 m) performed and are allowed by some legislations
58 C. KauffmannLacroix et al.

Fig. 4 End-filters (a) Tap, (b) Showerhead

(for example in France: Ministe`res des solidarites better evaluation of the risk of developing inva-
and DGS/DHOS 2005). Hydrogen peroxide sive water-related fungal infection, especially for
would be more efficient against bacteria than elderly, dialyzed, transplanted and more gener-
against yeasts and viruses (Ministe`res des ally for all immunocompromised patients. Cur-
solidarites and DGS/DHOS 2005). In some rently, in numerous health facilities, for example
cases, maintenance on the network has to be in French hospitals, the prevention of water-
planned, at times requiring partial replacement related risks is more focused on bacteriological
of pipes. Regarding the technological strategies risk. There are many types of water in health
deployed for the prevention of dialysis water facilities, including drinking water, dialysis
pollution, including fungal contamination, daily water, sanitary water and water supply related
overnight thermal disinfection procedures have to health care. Through current regulations, bac-
proved at times to be more effective than fre- terial environmental risk is well-controlled. Hot
quent chemical disinfection (Bolasco water is not usually a vector of the molds respon-
et al. 2012). In addition, these authors showed sible for fungal infections; on the other hand,
the interest of two-stage reverse osmosis for dial- cold water is a potential vector which must be
ysis procedures. taken into account. Although the fungal risk has
not been systematically evaluated, and even
though it is quite low, at least in Europe (low
9 Conclusion number of colony forming units reported by
liter), it remains a possibility that should not be
A number of studies clearly show the frequent neglected. Despite the measures for air quality
presence of fungi in hospital and community control in hospitals, control of invasive fungal
water. In the future, identification by sequencing infection risk in immunocompromised patients
isolated species may provide us with valuable can at times be insufficient and we must remain
epidemiological information and will allow attentive to other potential sources of fungal
Fungi, Water Supply and Biofilms 59

contamination, such as water, whose impact hospitals and healthcare. Woodhead Publishing Lim-
needs to be monitored. Finally, the development ited/University of Leeds, Leeds, pp 166207
Costa D, Barbot V, Latappy C et al (2013) The role of
of new anti-biofilm strategies may also help to saliva in the persistence of Streptococcus gordonii in
limit the presence of fungi in water and, conse- water. Eur J Water Qual 44:113121
quently, water-related fungal risk. Costerton JW, Lewandowski Z, Caldwell DE et al (1995)
Microbial biofilms. Annu Rev Microbiol 49:711745
De la Camara R, Pinilla I, Munoz E et al (1996) Penicil-
Acknowledgments The authors would like to acknowl- lium brevicompactum as the cause of a necrotic lung
edge Mr Arsham Jeffrey for revising the English text. ball in an allogeneic bone marrow transplant recipient.
Bone Marrow Transplant 18(6):11891193
Egli A, Fuller J, Humar A et al (2012) Emergence of
Aspergillus calidoustus infection in the era of
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Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2016) 3: 6382
DOI 10.1007/5584_2016_9
# Springer International Publishing Switzerland 2016
Published online: 15 June 2016

Diagnostic of Fungal Infections Related

to Biofilms

Maurizio Sanguinetti and Brunella Posteraro

Fungal biofilm-related infections, most notably those caused by the Can-
dida and Aspergillus genera, need to be diagnosed accurately and rapidly
to avoid often unfavorable outcomes. Despite diagnosis of these infections
is still based on the traditional histopathology and culture, the use of
newer, rapid methods has enormously enhanced the diagnostic capability
of a modern clinical mycology laboratory. Thus, while accurate species-
level identification of fungal isolates can be achieved with turnaround
times considerably shortened, nucleic acid-based or antigen-based detec-
tion methods can be considered useful adjuncts for the diagnosis of
invasive forms of candidiasis and aspergillosis. Furthermore, simple,
reproducible, and fast methods have been developed to quantify biofilm
production by fungal isolates in vitro. In this end, isolates can be
categorized as low, moderate, or high biofilm-forming, and this categori-
zation may reflect their differential response to the conventional antifun-
gal therapy. By means of drug susceptibility testing performed on fungal
biofilm-growing isolates, it is now possible to evaluate not only the
activity of conventional antifungal agents, but also of novel anti-biofilm
agents. Despite this, future diagnostic methods need to target specific
biofilm components/molecules, in order to provide a direct proof of the
presence of this growth phenotype on the site of infection. In the mean-
time, our knowledge of the processes underlying the adaptive drug resis-
tance within the biofilm has put into evidence biofilm-specific molecules
that could be potentially helpful as therapeutic targets. Surely, the suc-
cessful management of clinically relevant fungal biofilms will rely upon
the advancement and/or refinement of these approaches.

M. Sanguinetti (*) B. Posteraro

Institute of Microbiology, Universita` Cattolica del Sacro Institute of Public Health (Section of Hygiene),
Cuore, Rome, Italy Universita` Cattolica del Sacro Cuore, Rome, Italy
e-mail: e-mail:

64 M. Sanguinetti and B. Posteraro

1 Introduction this goal has been primarily achieved with the

pathogenic fungi Candida albicans (and, at
Rapid and accurate diagnosis of fungal lesser extent, non-albicans Candida species)
infections, especially those invading blood- and Aspergillus fumigatus (Herwald and
stream and deep tissues (i.e., lung, abdomen, Kumamoto 2014; M uller et al. 2011).
peritoneum, meninges, endocardium, sinus, This chapter is, therefore, aimed at reviewing
orbit, etc.) and thus termed invasive, is para- the current state of the clinical laboratory diag-
mount to improve the clinical and therapeutic nosis of IFIs, with particular focus on the IFIs
management of infected patients in many hospi- caused by species of Candida and Aspergillus.
tal settings (Perfect 2013). For example, how Note that the two genera were found to cause
much early is the diagnosis of candidemia/inva- approximately 85 % of IFIs in large prospective
sive candidiasis (IC) or invasive aspergillosis surveillance studies that were conducted recently
(IA), and the consequent initiation of appropriate in North America (Neofytos et al. 2009; Azie
antifungal treatment (Garey et al. 2006; Parkins et al. 2012). Not surprisingly, major progress in
et al. 2007; Upton et al. 2007), so much favorable the IFI diagnosis has regarded the development
will be the outcome of these infections especially of new laboratory assays for rapid detection of
when they are associated with biofilm formation five most common Candida species
(Tumbarello et al. 2007; Rajendran et al. 2015). C. albicans, C. glabrata, C. tropicalis,
In the last years, the clinical mycology labo- C. parapsilosis, and C. krusei account for 92 %
ratory has enhanced its diagnostic capability of cases of candidemia globally (Guinea 2014)
thanks to the acquisition of new methods, which as well as of A. fumigatus that alone accounts for
have been progressively introduced in the routine 90 % of cases of IAa severe invasive fungal
analysis of clinical specimens so as to replace disease (IFD) characterized by high mortality
conventional methods (Arvanitis et al. 2014). In rate (Abad et al. 2010). The role of newly devel-
one case, use of new methods has changed the oped assays for the diagnosis of candidiasis and
mycological diagnostic workflow; in another aspergillosis, including medical device-
case, they are adjunct diagnostics for invasive associated infections, will also be discussed.
fungal infections (IFIs). In this context, a grow- Above all, it is important to note that recently
ing number of pathogenic Candida or Aspergil- published international guidelines underscore
lus species can be identified successfully, thus basic aspects to be taken into consideration
encompassing species that differ with respect to when managing patients with the suspect of IFI
the virulence potential and the antifungal suscep- (Arendrup et al. 2012a; Cuenca-Estrella
tibility profile (Hadrich et al. 2012; Ziccardi et al. 2012; Cornely et al. 2014; Tortorano
et al. 2015). et al. 2014; Chowdhary et al. 2014; Arendrup
Biofilm formation is a common feature to et al. 2014a). Thus, it is desirable that clinical
many clinically encountered fungi, including microbiology laboratories serving hospitals with
not only Candida and other yeasts (Cryptococcus patients at risk of fungal infection are complying
neoformans, Cryptococcus gattii, Rhodotorula with these guidelines. For brevity, only some of
species, Malassezia pachydermatis, Saccharo- these aspects will be addressed in this chapter.
myces cerevisiae, and Trichosporon asahii) but
also fungi such as Aspergillus fumigatus, Fusar-
ium species, Mucorales, Histoplasma 2 Diagnostics for Candida
capsulatum, Coccidioides immitis, and Infections
Pneumocystis species (Sardi et al. 2014). Never-
theless, while there is growing interest in 2.1 Culture Based Diagnostics
unveiling the true involvement of fungal biofilms
in human disease (Williams and Ramage 2015), Three entities need to be considered when
diagnosing IC: (i) candidemia in the absence of
Diagnostic of Fungal Infections Related to Biofilms 65

deep-seated candidiasis (DSC; i.e., infections of simultaneously 24 microbial pathogens (includ-

tissue sites beneath mucosal surfaces), ing 5 Candida species, already mentioned in
(ii) candidemia associated with DSC, and (iii) Sect. 1), proved to be accurate not only with
DSC in the absence of candidemia (Clancy and monomicrobial BCs (Altun et al. 2013). Using a
Nguyen 2013). Approximately one-third of combined testing with both the methods, approx-
patients with IC fall into each of above categories imately 98 % of bloodstream infections were
(Leroy et al. 2009). Blood cultures (BCs) remain correctly diagnosed during a 17-month period
the gold standard for the diagnosis of invasive of clinical routine in our lab. Specifically,
Candida infections, and should be the initial 65 (5.3 %) of 1223 monomicrobial BCs, that
diagnostic test when candidemia is suspected provided unreliable results with the MALDI-
(Cuenca-Estrella et al. 2012). Conversely, the TOF MS system, were accurately detected with
gold standard tests for DSC are sterilely collected the FilmArray BCID panel; 9 of
cultures of infected fluids or tissues, but the sen- 65 microorganisms were Candida yeasts. Of
sitivity of these cultures is low, which may reflect note, 153 (89.5 %) of 171 polymicrobial BCs
difficulties in identifying optimal sampling sites had complete identification results with
or uneven distributions/low burdens of viable FilmArray BCID, including all but 1 of 29 BCs
organisms (Clancy and Nguyen 2013). Further- that grew Candida species together with bacteria
more, collecting samples from deep-seated sites (unpublished data). Also, by our approach, the
through surgery or other invasive procedures is median time to identification was shortened
often precluded by underlying medical (19.5 h vs 41.7 h with the culture-based reference
conditions. In keeping with these observations, method; P <0.001), whereas a minimized use of
it was estimated that around 3050 % of patients FilmArray BCID led to significant cost savings
with IC are actually not diagnosed by BCs (unpublished data).
(Clancy and Nguyen 2013). Similarly, the Luminex xTAG fungal assay
Cultures take 25 days to complete13 a multiplex-PCR Luminex xMAP bead probe
days to grow and an additional 12 days for fluid array using xTAG analyte-specific
identification of the organism (Arvanitis reagentsthat was developed to detect clinically
et al. 2014). However, the accurate species iden- significant Candida species (C. albicans,
tification is pivotal for initiating targeted (though C. glabrata, C. parapsilosis, C. tropicalis,
delayed) antifungal treatment (Chandrasekar C. krusei, C. lusitaniae, C. guilliermondii),
2010; Ostrosky-Zeichner 2012), especially C. neoformans, H. capsulatum, and Blastomyces
when antifungal susceptibility testing (AFST) dermatitidis from BCs, was shown to have sensi-
results are not promptly available (Sanguinetti tivity and specificity of 100 % and 99 %, respec-
et al. 2015). It is considerable that the time to tively (Balada-Llasat et al. 2012). As expected,
identification can now be accelerated through the turnaround times with the assay were consider-
use of new technologies directly on positive BCs. ably faster than conventional culture-based
These include pathogen-specific real-time PCR, identifications, as well as the assay was able to
peptide nucleic acid fluorescence in situ detect mixed Candida infections that were
hybridization (PNA FISH), and matrix-assisted observed in 5.8 % of the cases (Balada-Llasat
laser desorption ionization time-of-flight mass et al. 2012). However, as in its current format
spectrometry (MALDI-TOF MS) (Borman and the assay takes 5 h to complete, as well as a
Johnson 2013; Arvanitis et al. 2014). meticulous technique to prevent cross-
Despite its well-documented good perfor- contamination in the open system, it is expected
mance (Lagace-Wiens et al. 2012; Spanu that the DNA extraction protocol will be
et al. 2012), MALDI-TOF MS often fails to optimized to enhance the usability of the
identify polymicrobial BCs in their entirety. Luminex xTAG fungal assay in clinical practice.
Alternatively, the multiplex PCR-based However, both the PNA FISH and multiplex
FilmArray BCID panel, that detects approaches are currently limited by the relatively
66 M. Sanguinetti and B. Posteraro

low number of fungal species that the systems are indisputable value so as to uniquely compete
designed to detect, with the consequence that with other molecular, usually genomic, methods
infections with uncommon species will be not (Borman and Johnson 2013). It is important to
diagnosed, as well by the inability of the systems note that very few false identifications occur with
to discriminate between cryptic Candida species. MALDI-TOF MS systems, as the absence of a
For example, the inclusion of fungal probes for match with a suitable spectrum in the reference
detecting Candida dubliniensis, Candida database leads to log(score) values that are too
orthopsilosis/C. metapsilosis, and Candida low to be accepted (<1.7). Thus, a continuous
nivariensis/C. bracarensis would be beneficial updating of the fungal (yeast) database, by
since these species can be recovered from BCs adding the spectra from those same isolates that
and should be distinguished from C. albicans, have been ultimately identified by other methods,
C. parapsilosis, and C. glabrata, respectively greatly enhance the identification capability of
(Cornet et al. 2011). MALDI-TOF MS. In this way, recurring to lon-
The growing diversity of infecting species/ ger and labor-intensive molecular techniques in
strain makes the identification of clinical yeasts the clinical routine practice will be restricted to
increasingly challenging. Results from a recent very rare occasions (Posteraro et al. 2013).
meta-analysis of published articles on the Vitek
2 and other two major systems, namely the
AuxaColor and the API ID32C, showed that the 2.2 Nonculture Based Diagnostics
accuracy of identification of clinically important
yeasts using conventional commercial systems is Beside to classical techniques, nonculture
currently not optimal, particularly for less com- approaches are noninvasive and promise more
mon species (Posteraro et al. 2015). So, clinical rapid diagnosis than conventional approaches
microbiologists should reconsider the clinical (Perfect 2013). Newer nucleic acid (NA)-based
utility of these systems, particularly in view of or antigen-based diagnostics are able to identify
new diagnostic tools such as MALDI-TOF MS cases of IC, particularly DSC, by detecting Can-
(Posteraro et al. 2013). dida NA and cellular components (i.e., -D-glu-
With the advent of MALDI-TOF MS in the can [BDG], mannan [MN]) that persist in the
mycological field (Santos et al. 2011), it is now blood or that are released from deep fluid or
possible to identify all the closely related species tissue sites. As a major cell wall component of
within the aforementioned Candida species most fungal pathogenstwo unique, but notable
complexes, that, except for C. dubliniensis, are exceptions are Mucor and Cryptococcusthe
predictably not identifiable by any conventional presence of BDG in blood serves as pan-fungal
method that is commercially available for routine marker for most IFIs, including IC (Marty and
clinical use. Following the progressive introduc- Koo 2009). In clinical settings, BDG tests have a
tion of MALDI-TOF MS in the diagnostic sensitivity and specificity of 6490 % and
workflow in our laboratory and other European 73100 % for detection of candidemia, with
laboratories (Ling et al. 2014), automated very high negative predictive (7397 %), and
systems such as the Vitek 2 are almost exclu- there multiple factors that contributed to false-
sively used to perform antimicrobial susceptibil- positive results, such as surgical gauze or
ity testing of clinical isolates. On the other hand, sponges, administration of immunoglobulin or
the PNA FISH assay showed a 100 % agreement albumin, hemodialysis, and serious bacterial
with the result of culture-based MALDI-TOF infections or mucositis, in addition to those
MS identifications on 35 blood samples positive recognized for the Aspergillus galactomannan
for yeasts (Calderaro et al. 2014). (GM) test (e.g., amoxicillin-clavulanate,
Apart from the limitation of the growth of piperacillin-tazobactam, platelet transfusion,
organism in culture, the MALDI-TOF MS sys- etc.) (Arendrup et al. 2015). In C. albicans, bio-
tem, when applied on the isolated yeast, has an film (pathogenic) cells differ from commensal
Diagnostic of Fungal Infections Related to Biofilms 67

cells in that they are surrounded by an the intra-abdominal candidiasis diagnosis

exopolymeric matrix, also termed extracellular (5 days) and antifungal therapy (6 days) (Tissot
matrix (ECM), that is mostly composed of et al. 2013). Two populations are shown to con-
-mannan, -1,6 glucan, and -1,3 glucan (i.e., sistently benefit from BDG testing, namely
BDG) (Taff et al. 2013). Compared to patients with hematological malignancies and
commensals, cells in biofilms produce higher those who have undergone allogeneic
levels of soluble BDG, which can be detected in hematopoietic stem cell transplants, particularly
culture supernatants and in the serum of rats during episodes of neutropenia (Theel and Doern
carrying a C. albicans-infected central venous 2013). Nonetheless, careful dissection of
catheter (CVC) (Nett et al. 2007). In addition to published data reveals certain scenarios (e.g.,
promote biofilm resistance to multiple the ICU) where BDG testing is relevant and
antifungals (Taff et al. 2013), glucan modifica- may lead to improved patient outcome (Posteraro
tion enzymes (encoded by BGL2, PHR1, and et al. 2011). With regard to serial testing of high-
XOG1 genes) may play a biofilm-specific role risk patients, neither the ECIL-3the 3rd
in mediating the delivery and organization of European Conference on Infections in Leukemia
mature ECM (Taff et al. 2012b). Thus, these guidelines categorized BDG testing as B II,
enzymes may represent potentially attractive indicating that there is moderate evidence to
targets for detection of, and for therapeutic support recommendation for use in patients
interventions against candidiasis (Herwald and with leukemia (Marchetti et al. 2012)nor the
Kumamoto 2014). EORTC/MSG provides BDG timing or interval
In a recent study, Nguyen et al. (2012) found testing guidelines. Intriguingly, a recent meta-
that the sensitivity of a real-time quantitative analysis of cohort studies from the ECIL-3
PCR assay (ViraCor-IBT) was superior to BDG reported a diagnostic odds ratio of 111.8 vs
in diagnosing candidemia and DSC. In addition, 16.3 for the presence of IFDs in hematological
both PCR and BDG tests were significantly more patients following two consecutively positive
sensitive than traditional BCs (the respective BDG results compared to a single positive BDG
sensitivities were 88 %, 62 %, and 17 %; result (Lamoth et al. 2012). This meta-analysis
P 0.005 and 0.003 for BCs vs BDG and also reported a pooled sensitivity of 49.6 %,
PCR, respectively), whereas the combination of alongside a PPV and NPV of 83.5 % and
BC and BDG or PCR had sensitivity of 79 % and 94.6 %, respectively. Nevertheless, despite the
98 %, respectively. According to data from a strong NPV, and the consistently low sensitivity
recent meta-analysissensitivity and specificity reported among studies, a negative BDG test
of BDG for the diagnosis of IC were shown to be cannot exclude the diagnosis of IFD, as well the
5797 % and 5693 %, respectively (Karageor- value of BDG as a screening test is questionable
gopoulos et al. 2011)the serum BDG assay is (Theel and Doern 2013). In the last context,
currently recommended in guidelines from the findings from a very recent meta-analysis of pro-
European Organization for Research and Treat- spective cohort studies suggest that the BDG
ment of Cancer/Invasive Fungal Infections assay has high sensitivity and specificity for dis-
Cooperative Group and the National Institute of criminating between patients with and without
Allergy and Infectious Diseases Mycoses Study IFD, but that, in clinical practice, BDG assay
Group (EORTC/MSG) (De Pauw et al. 2008) and results should be evaluated together with clinical
from the European Society of Clinical Microbi- and microbiological (e.g., other surrogate marker
ology and Infectious Diseases (ESCMID) (s)) findings (Hou et al. 2015).
(Cuenca-Estrella et al. 2012). In another study, While NA amplification (i.e., PCR)
the diagnostic accuracy of BDG not only was techniques are still lacking standardization
higher than that of Candida score and coloniza- (Alanio and Bretagne 2014), commercial tests
tion index (both predictive rules of IC; Leon are also available for measurement of MN,
et al. 2014), but also BG positivity anticipated anti-mannan antibody (AMN), and Cand-Tec
68 M. Sanguinetti and B. Posteraro

Candida antigen. Of the four (including BDG) in the therapy decision-making process and the
tests compared in one study, BDG and the discontinuation of empirical antifungal therapy
combinations of BDG plus MN and of MN plus (Martnez-Jimenez et al. 2015b).
AMN showed the highest sensitivity (Held The primary testing goal should be that of
et al. 2013). Finally, the C. albicans germ tube identifying patients early in the course of
antibody (CAGTA) assay, that detects antibodies IC. To this regard, a novel technology, based on
against the surfaces of C. albicans germ tubes by the application of T2 magnetic resonance
indirect immunofluorescence, was shown to be (T2MR), was exploited to develop an automated
helpful in diagnosing candidemia associated instrument platform, T2Dx, which provides a
with deep-seated infection (Martnez-Jimenez patient sample-to-answer clinical diagnostic
et al. 2014). In an effort to establish the place test, namely the T2Candida (Pfaller et al. 2015).
of CAGTA test in routine clinical practice, a new In this FDA-cleared test, whole blood is
diagnostic tool for IC that was based on positive subjected to PCR amplification of Candida
CAGTA and BDG assays was introduced by sequences, followed by hybridization to
Leon et al. (2012). Later, the same group nanoparticles that elicit a T2MR signal to detect
evaluated the performance of BDG and Candida speciesthis without the need for cul-
CAGTA for the diagnosis of IC in a prospective ture or nucleic acid extraction steps. Exciting
cohort of 107 unselected, non-neutropenic ICU results from a preliminary study of the T2MR
patients, and showed that two consecutive BDG method showed that it is able to reduce the time
levels 80 pg/mL allowed discrimination to result to an average of 2 h, in contrast to the
among IC and high-grade colonization (Martn- 48-h average of BCs (Neely et al. 2013). Next, in
Mazuelos et al. 2015). Furthermore, the best a clinical trial, the T2MR method was found to
combinations found to diagnose candidemia in have an overall sensitivity per patient of 91.0 %,
31 patients were CAGTA and BDG using cutoffs with a mean time to species identification of only
of 1/80 and 80 pg/mL, respectively (sensitivity 4.4 h, compared to the 25 days typically needed
96.8 % and specificity 84 %), and CAGTA and by the automated BC systems (Mylonakis
MN using cutoffs of 1/80 and 75 pg/mL, respec- et al. 2015). Across all studies to date, T2Can-
tively (sensitivity 93.5 % and specificity dida has an overall specificity of >99.4 % from
86.0 %) (Martnez-Jimenez et al. 2015a). An >1560 patients, and 12 proven cases of IC have
approach to improve the performance of antigen been detected with T2Candida in patients with
assays is the combination with other diagnostic negative BCs (Pfaller et al. 2015). As the 5 target
tools. One example is a study by us, who used Candida species of the T2MR method account
BDG measurement together with the Candida for the majority of cases of candidemia (Guinea
score in 14 IC patients and increased the sensi- 2014), and have distinct antifungal susceptibility
tivity from 92.9 to 100 % (Posteraro et al. 2011). profiles (Miceli et al. 2011), the rapid speciation
However, this gain in sensitivity came at the allowed by this method may drive the appropri-
price of a loss of specificity from 93.7 to ate choice of antifungal therapy. In 5000-patient
83.5 %. Similarly, Levesque et al. (2015) Monte Carlo simulations, the average time to
showed that, on the day the clinical diagnosis initiation of antifungal therapy was
of IC was made after liver transplantation, the 0.6  0.2 days for T2Candida (used for detec-
combination of two sequential BG-positive tion and species-level identification directly from
samples (>146 pg/mL) and a colonization whole blood) vs 2.6  1.3 days and
index of 0.5 resulted in sensitivity, specificity, 2.5  1.4 days, respectively, when either PNA
PPV, and NPV of 83 %, 89 %, 50 %, and FISH or MALDI-TOF MS methods were used to
97.6 %, respectively. Interestingly, the BDG identify Candida species from positive BCs
assay, used in combination with the CAGTA (Aitken et al. 2014).
test, provided NPV as high as to justify the use
Diagnostic of Fungal Infections Related to Biofilms 69

3 Diagnostics for Aspergillus the yield of cultures may improve under labora-
Infections tory conditions that mimic the physiological tem-
perature (Tarrand et al. 2005). In agreement with
3.1 Culture Based Diagnostics these observations, it was recently argued that
the fungal yield from sterile or non-sterile
As with IC, proven diagnosis of IA is usually sitesspecimens usually analyzed include spu-
made by demonstrating fungal hyphae in tissue tum, bronchoalveolar lavage (BAL) fluid,
biopsy specimens (De Pauw et al. 2008). How- aspirates from lesions, cerebrospinal fluid
ever, one of major problems to the regard is (CSF), and other tissuesmay at least in part
obtaining tissue for standard histopathological depend on the suboptimal sampling or the
analysis, because of the contraindication to per- unstandardized specimen processing in the rou-
form biopsy in severely ill patients, who could be tine clinical microbiology laboratory (Arendrup
at risk of bleeding due to thrombocytopenia et al. 2015). As specified in the aforementioned
(Chandrasekar 2010; Ostrosky-Zeichner 2012). guidelines (see Sect. 1), factors argued to
Even when tissue is available, several filamen- improve respiratory fungal diagnostics include
tous fungi appear morphologically identical at (i) rapid, direct microscopy with optical
the microscopic observation (Perfect 2013), so brighteners such as Calcofluor white and
that uniform, septate, narrow-angle-branching Blankophor, (ii) increasing the volume of clinical
hyphae of Aspergillus are undistinguishable specimen plated, (iii) use of both selective and
from those of the pathogenic Fusarium or nonselective media, and (iv) prolonged incuba-
Scedosporium (Cuenca-Estrella et al. 2011). tion of primary culture plates (Hamer et al. 2006;
Also with the pathogenic Mucoralesunique Fraczek et al. 2014; Arendrup et al. 2015). Most
features to these fungi are broad, sparsely sep- stains are inexpensive and can be performed eas-
tate, ribbon-like hyphae, with open angle (90  ) ily in various specimens, as well as microscopy
branchesmorphology of fungi in tissue is not performed in conjunction with cultures improves
definitive criterion because it can be difficult for their positive predictive value by confirming pos-
even experienced pathologists (Lanternier itive culture results (Arvanitis et al. 2014).
et al. 2012). Nevertheless, histopathology allows Although contamination of clinical specimens
for both the tissue invasion by fungi and the host by Aspergillus may occur (Burco et al. 2012;
response or tissue necrosis to be detected, as well Kanamori et al. 2015), positive culture results,
as for clarifying if a positive culture is the result especially when repetitive and associated with
of infection, colonization, or contamination positive smear results, are strongly suggestive
(Arvanitis et al. 2014). of aspergillosis (Arvanitis et al. 2014).
In general, the sensitivity of culture for the Despite being still essential, cultures provide
diagnosis of aspergillosis is low, and Aspergillus results after too long a delay for optimal IFI
species are almost never recovered in BCs even management (Ostrosky-Zeichner 2012). Species
in disseminated disease (Chandrasekar 2010; identification may take many days, especially
Ostrosky-Zeichner 2012). In two large multicen- with fungi that are slow to sporulate, which
ter surveillance studies, among transplant may delay the selection of the appropriate anti-
recipients with a positive molecular test for IA, fungal (Van Der Linden et al. 2011). However,
only 2550 % had a positive culture result while accurate identification of the infecting
(Neofytos et al. 2009; Kontoyiannis pathogen remains a fundamental step in the
et al. 2010). However, one study, published in mycological diagnostic process also for aspergil-
2005, showed that incubation of cultures from losis, the differentiation to the species level
Aspergillus-infected tissue specimens at 35  C within the Aspergillus species complexes is
(in room air) led to a 31 % increase in sensitivity very problematic, at least with traditional macro-
compared to incubation at 25  C, suggesting that scopic and microscopic analyses (Balajee
70 M. Sanguinetti and B. Posteraro

et al. 2007). Although a polyphasic approach of molds (Gautier et al. 2014). In our study
(Samson and Varga 2009)it combined pheno- (De Carolis et al. 2012), using water suspensions
typic (morphology and extrolite profiles) and of fungal mycelia and/or conidia and a home-
molecular (e.g., ITS, calmodulin, -tubulin, made reference database encompassing spectra
actin) charactersshould be used, a robust from Aspergillus (33 species), Fusarium (12 spe-
Aspergillus species delineation is possible sim- cies), and Mucorales (10 species), MALDI-TOF
ply using molecular methods (Balajee MS accurately identified 91 (96.8 %) of 94 fungal
et al. 2009b). In this end, partial -tubulin or isolates tested. Interestingly, the log(score)
calmodulin gene sequences are currently utilized values of the 91 isolates with correct results
to identify a species within a given Aspergillus were all higher than 2.0; the remaining 3 isolates
species complex (Balajee et al. 2009a), because (1 Emericella nidulans, 1 Aspergillus niger, and
conventional locithe nuclear ribosomal ITS 1 Aspergillus versicolor) could be identified only
regions (ITS1, 5.8S rRNA, and ITS2)are not to the genus level, as their log(score) value was
variable enough to allow resolution of closely lesser than 2.0 (1.817, 1.874, and 1.796, respec-
related species of fungi (Borman and Johnson tively), but had concordant species designations
2013). So, using these additional loci, a number as compared with the multilocus sequence anal-
of cryptic (or sibling) speciesmorphologically ysis results. In another study (Alanio et al. 2011),
indistinguishable from each other but separable using a chemical (acid formic and acetonitrile)
with only DNA-based molecular methods extraction of the fungal colonies and a database
(Hawksworth 2006)are nowadays identified built with the reference spectra from 146 mold
within Aspergillus section Fumigati, and it is strains, mold isolates obtained from sequential
relevant in view of that some species have clinical samples were prospectively analyzed.
in vitro antifungal susceptibility profiles which Eighty-seven percent (154/177) of isolates,
differ significantly from that of A. fumigatus including 86 A. fumigatus, 38 other aspergilli
sensu stricto (Van Der Linden et al. 2011). (9 species), and 53 other molds (23 species),
As with Candida species, the time to identifi- were identified to the species level, whereas the
cation for colony-growing aspergilli from clini- MALDI-TOF MS-based approach failed in 12 %
cal specimens can be considerably shortened (21/177) of isolates belonging to species not
using MALDI-TOF MS. However, unlike yeasts, represented in the reference library. Although
MALDI-TOF MS-based identification of fila- the methods employed by investigators at each
mentous fungi, including aspergilli, fusaria, single center proved to work well in their own
Mucorales, and dermatophytes is more challeng- evaluations, multicenter studies are necessary for
ing (Posteraro et al. 2013). Inconclusive the standardization of MALDI-TOF MS use into
MALDI-TOF MS results often occur due to routine mold identification.
discrepancies between the methods used for rou-
tine testing and for spectral database construc-
tion, and several research groups have worked to 3.2 Nonculture Based Diagnostics
develop and validate sample preparation
protocols and fungus-specific databases The development and validation of newer diag-
(Sanguinetti and Posteraro 2014). Since early nostic approaches are currently in the spotlight of
efforts by us (De Carolis et al. 2012) and others scientific research, but the lack of a reliable gold
(Alanio et al. 2011) to date, two clinically com- standard to be used as a comparator makes very
prehensive mold databases have been described, difficult to estimate the performance of a new
one comprising 294 reference spectrawhich diagnostic test for IA (Arvanitis and Mylonakis
correspond to 294 strains from 152 species (Lau 2015). For most trials and guidelines, this prob-
et al. 2013)and the other one comprising 2832 lem has been in part circumvented by using the
reference spectra (four replicas for each strain) aforementioned EORTC/MSG criteria (De Pauw
which correspond to 708 strains from 347 species et al. 2008), which divide the patients suspected
Diagnostic of Fungal Infections Related to Biofilms 71

to have IA into four categoriesproven, proba- the aforementioned meta-analysis by Karageor-

ble, possible, and unlikely IAbased on risk gopoulos et al. (2011), the pooled sensitivity
factors, radiological, and microbiological (75.6 %) and specificity (85.3 %) among patients
criteria. However, an unavoidable number of with IFIs (including IA) did not differ signifi-
patient cases classified as possible IA, due to cantly in the subgroup of studies that evaluated
the absence of one of the microbiological criteria the performance of the assay in
that define a probable IA case, will in fact have IA. Unfortunately, unlike GM, the BDG test can-
the disease, and their misclassification will in not differentiate between IFIs due to Aspergillus
turn lead to erroneous calculation of the perfor- spp. and IFIs due to other fungi (Marty and Koo
mance estimates of a diagnostic test in the devel- 2009).
opment (Arvanitis and Mylonakis 2015). As a potential alternative to the Platelia GM
Among the newer molecular and serologic assay (Arvanitis and Mylonakis 2015), a lateral
tests for IA, some of them such as GM and flow device (LFD) that uses the JF5 monoclonal
BDG are now widely used in clinical practice, antibody was shown to detect a glycoprotein
while others such as the Aspergillus PCR have antigen in the serum and BAL of patients with
yet to be implemented in clinical laboratories IA, holding promise to be used as rapid point-of-
(Arvanitis et al. 2014). As a component of the care test for IA (Thornton 2008). In an early trial
cell wall of Aspergillus spp. (Leeflang (White et al. 2013), the performance of this LFD
et al. 2008), the GM antigen can be detected in was compared to real-time PCR (targeting the
clinical specimens, and the serum and BAL GM 28S rRNA gene) and GM detection on serum
tests, both using the Platelia GM enzyme-linked from an EORTC/MSG-defined hematological
immunoassay, were approved by the FDA, and population. In a proven/probable-IA population
are incorporated into clinical guidelines (see versus a no-IA population, the LFD performance
Sect. 1) and routinely used for discriminating parameters were comparable to those of both
between patients with possible or probable IA PCR and GM, with a sensitivity and specificity
(De Pauw et al. 2008). False-positive results of 81.8 % and 98 %, respectively (White
have been reported for patients receiving et al. 2013). In combination with PCR, the LFD
piperacillin-tazobactam antibacterial agents provided both 100 % sensitivity and 100 % spec-
(Viscoli et al. 2004), although this association ificity, besides to being easy to use and to
seems to be now confuted (Mikulska allowing for rapid testing of easily obtainable
et al. 2012). While the sensitivity and specificity specimens (White et al. 2013). Likewise, a
of GM on serumusing 0.5 optical density small pilot study conducted at two university
(OD) index as the cutoff for positivitywas hospitals in Austria and Germany, on BAL fluid
estimated to be 79 % and 8186 %, respectively, samples from 78 patients at risk for invasive
in two large meta-analyses (Leeflang et al. 2008; pulmonary aspergillosis (IPA), showed that the
Pfeiffer et al. 2006), GM performed on BAL was combination of the GM assay and PCR or, if PCR
shown to have a pooled sensitivity and specificity is not available, the LFD test, allows for sensitive
of 85 % and 94 %, respectively, in an earlier and specific diagnosis of IPA (Hoenigl
meta-analysis of studies that used an OD index et al. 2012). Furthermore, in a recent prospective
of 1 (Guo et al. 2010), and, impressively, of 92 % study of 221 patients with underlying respiratory
and 98 %, respectively, in a more recent meta- diseases (and without hematological malignancy
analysis of studies that used an OD index of 1.5 or previous solid organ transplantation), BAL
(Heng et al. 2015). In that study, comparing LFD for IA provided sensitivity and specificity
serum GM and Aspergillus PCR testing on BAL (77 % and 92 %, respectively) that differed
fluid, BAL-GM conferred greater sensitivity, but markedly from those of GM (OD index 0.5,
lower specificity than the serum GM test, and 97 % and 81 %, respectively; OD index 1.0,
similar specificity as the PCR assay (Heng 97 % and 93 %, respectively; OD index 3.0,
et al. 2015). With regards to the BDG assay, in 61 % and 99 %, respectively), BDG (cutoff
72 M. Sanguinetti and B. Posteraro

80 pg/mL, 90 % and 42 %, respectively; cutoff was conducted with the aim to evaluate if previ-
200 pg/mL, 70 % and 61 %, respectively), and ous recommendations for serum are also applica-
mycological culture (29 % and 97 %, respec- ble to plasma PCR and to compare the analytical
tively) (Prattes et al. 2014). These findings were performance of plasma PCR with that of serum
confirmed by a more recent semiprospective PCR (Loeffler et al. 2015). This study showed
multicenter study that evaluated the performance that, despite the absence of major differences in
of the LFD test for BAL IPA detection in 133 crit- the molecular processing of serum and plasma,
ically ill patients. Sensitivity and specificity were the formation of clot material potentially reduces
80 % and 81 %, respectively, that contrasted available DNA in serum. Interestingly, in a com-
with a sensitivity of 50 % and a specificity of panion study by the EAPCRI (White et al. 2015),
85 % exhibited by the fungal BAL culture, clinical performance and clinical utility (time to
thereby suggesting that LFD may be a reliable positivity) of the standardized Aspergillus PCR
alternative for IPA diagnosis in ICU patients if were calculated for both plasma and serum
GM results are not rapidly available (Eigl specimens. These specimens were concomitantly
et al. 2015). obtained from hematological patients in a multi-
Primarily due to the lack of standardization, center retrospective anonymous case-control
PCR for the diagnosis of IA has shown in indi- study, with cases diagnosed according to the
vidual studies sensitivity and specificity which EORTC/MSG criteria. While confirming the
ranged from 43 to 100 % and 64 to 100 %, analytical finding that the sensitivity of Aspergil-
respectively, and from 36 to 100 % and 70 to lus PCR using plasma (94.7 %) is superior to that
100 %, respectively, depending on whether using serum (68.4 %), it was shown that PCR
serum and whole blood or BAL fluid were tested positivity occurred earlier when testing plasma,
(Arvanitis et al. 2014). According to this, the providing sufficient sensitivity for the screening
results from two comprehensive meta-analyses of IA (White et al. 2015).
of studies where PCR tests were performed on
blood specimens showed a pooled sensitivity and
specificity of 8488 % and 7576 %, respec- 4 Biofilm Production in Fungal
tively, which led to conclude that PCR is unable Isolates
on its own to confirm or exclude IA (Mengoli
et al. 2009; Arvanitis et al. 2014). A multicenter It is estimated that 8090 % of hospital-acquired
effort was recently completed by the European bloodstream and deep tissue infections are
Aspergillus PCR Initiative (EAPCRI) to provide associated with the use of indwelling medical
guidelines and specific recommendations, that devices such as CVC and prosthesis (Chandra
should improve the in vitro performance of et al. 2014). Most cases of central line-associated
Aspergillus spp. PCR using whole blood and BSI (CLABSI)that occur, in the United States,
serum specimens (White et al. 2010; White at a rate of 1.5 per 1000 CVC days and a mortal-
et al. 2011). Likewise, when PCR was used to ity rate of 1225 %involve microbial biofilms
detect the Aspergillus spp. DNA in BAL on the catheter surfaces (Yousif et al. 2015).
specimens, this test exhibited a pooled sensitivity Once a mature biofilm is formed within the
and specificity of >90 % in two recent meta- human host through a medical device, the infec-
analyses (Sun et al. 2011; Avni et al. 2012). tion becomes recalcitrant to antimicrobial treat-
Excluding BAL, it is clear that, if considering ment (Ramage et al. 2012) and can develop into a
whole blood and serum PCR equivalent in terms chronic condition (Yousif et al. 2015). Although
of performance, the serum PCR might be used catheter removal is the conventional manage-
preferentially, as serum is easier to process and ment of catheter-associated fungemia (Walraven
allows for other serological tests to be performed and Lee 2013), salvaging the catheter can often
at the same time (Arvanitis and Mylonakis 2015). help to minimize mechanical complications that
However, a very recent study by the EAPCRI the procedure itself carries; so, many techniques
Diagnostic of Fungal Infections Related to Biofilms 73

have been used to prevent the biofilm formation whereas MBF and HBF strains had dense
by targeting different stages of biofilm matura- structures which covered the whole surface of
tion (Yousif et al. 2015). the assay discs. When the procedures were com-
A plenty of studies reproducing a fungal ses- pared, species-specific biofilm production
sile community in vitro has allowed not only to patterns were noted among the isolates, with an
study the development of a biofilm, its interac- overall categorical agreement between the
tion with other microorganisms and the environ- procedures of only 44 %. Interestingly, all the
ment, and its susceptibility to available four Candida speciesC. albicans,
antifungal agents, but also to search for new C. parapsilosis, C. glabrata, and C. tropicalis
therapies (Williams and Ramage 2015). In the known to be the most common causes of
meantime, these studies have revealed biofilm catheter-related BSI, produced biofilms with
features which led to the development of several high biomass or high metabolic activity,
methodologies strategies (Williams and Ramage suggesting that both the features may play an
2015). For example, published work indicates important role in colonization (Marcos-
that biofilms grown in microtiter dishes do Zambrano et al. 2014). Finally, the authors
develop some properties of mature biofilms, showed that XTT and CV can be used as com-
such as antibiotic tolerance and resistance to plementary procedures for studying biofilm pro-
immune system effectors (Ramage et al. 2012). duction, and they claimed the necessity of studies
Two methods are mostly used to quantify that investigate the outcome of patients infected
biofilm production by fungal isolates in vitro by HBF or HMA isolates of pathogenic yeasts
(Costa et al. 2013). One method quantifies bio- (Marcos-Zambrano et al. 2014).
mass production using crystal violet stain With regards to Aspergillus, the well-
(CV) (OToole 2011) and the other one, the met- established behavior of the fungus to form bio-
abolic activity of viable embedded biofilm cells film in vitro (Mowat et al. 2007, 2008; Seidler
based on reduction of tetrazolium salt XTT to et al. 2008) was shown to correlate with localized
formazan dye XTT (Pierce et al. 2008). How- and invasive forms of aspergillosis, which are
ever, poor agreement between the both methods both characterized by the mycelial development
was observed in earlier studies, which were of A. fumigatus that arises from inhaled conidia
performed exclusively on C. albicans isolates (Loussert et al. 2010; Muller et al. 2011). In
and with a small number of isolates (Alnuaimi addition, Aspergillus species have been reported
et al. 2013; Taff et al. 2012a). Furthermore, as to cause biomaterial-related biofilm infections,
variability in biofilm formation can be observed which involve catheters, cardiac pace makers,
among C. albicans isolates, the categorization of heart valves, and joint replacements (Williams
isolates based on this can be used to predict how and Ramage 2015). Like in C. albicans biofilms,
pathogenic the isolate will behave clinically the ECM that surrounds the intricate hyphal net-
(Sherry et al. 2014). By measuring the biomass work of A. fumigatus (Beauvais et al. 2007) in
production by CV and the metabolic activity by either IA or aspergilloma may account for the
XTT, Marcos-Zambrano et al. (2014) studied resistance of the fungus against phagocytic and
577 yeast isolates causing fungemia and antimicrobial attacks (Ramage et al. 2012).
established tentative cutoff values to classify Clinical biofilm-associated infections are dif-
the isolates as being low (<0.097), moderate ficult to diagnose (and treat), but the differences
(0.0970.2), or high (>0.2) biofilm-forming that exist between planktonic and sessile micro-
LBF, MBF, or HBF, respectivelyand as having bial cells could be exploited into the search for
low (<0.44), moderate (0.441.17), or high new diagnostic targets. While advanced molecu-
(>1.17) metabolic activityLMA, MMA, or lar in situ or imaging techniques may be effective
HMA, respectively. Consistent with the findings to demonstrate biofilms in vivo, older immuno-
by scanning electron microscopy analysis, LBF histochemical or immunofluorescent techniques,
strains had less tick and compact structures, that utilize specific polyclonal or monoclonal
74 M. Sanguinetti and B. Posteraro

sera, represent a still useful tool to targeting distributions of wild-type fungal populations pro-
pathogens in host tissues (Hall-Stoodley vide a measure of the epidemiological cutoff
et al. 2012). However, use of these antibodies is values (ECVs). These values, in the absence of
often limited by that they are thought to bind to specific CBPs, may be very useful in antifungal
the ECM non-specifically and by almost overall resistance surveillance to monitor the emergence
lack of commercially available antibodies spe- of resistant isolates (i.e., those with gene
cific for fungal pathogens (Loussert et al. 2010; mutations associated with reduced therapeutic
Hall-Stoodley et al. 2012). In a study (Bugli responses) (Pfaller 2012). In particular, while
et al. 2014), we generated an anti-gliotoxin breakpoints are not yet available for the CLSI,
mouse polyclonal antibody that was used, in an the EUCAST CBPs have been established for
immunofluorescent assay, for detecting the A. fumigatus (and for some Aspergillus species)
mycotoxin in A. fumigatus biofilms. This assay for the majority of the licensed antifungal agents
proved to be able to demonstrate A. fumigatus (Arendrup et al. 2012c; Hope et al. 2013). There-
gliotoxin not only on the hyphae cultured onto fore, EUCAST testing is somewhat more appeal-
the human lung epithelial cell line A549, but ing for guiding antifungal therapy (Arendrup
also, directly, on the hyphae into respiratory et al. 2015), whereas both the EUCAST and
tract specimens or lung tissues from patients CLSI reference methods are equivalent for epi-
with IA. demiological surveillance purposes (Arendrup
et al. 2013; Pfaller et al. 2009).
However, commercial techniques such as the
5 Antifungal Susceptibility Vitek 2 yeast susceptibility test, Etest, and
Testing Assays Sensititre YeastOne are currently approved by
the US FDA for clinical testing (Posteraro and
5.1 Planktonically-Growing Isolate Sanguinetti 2014). These techniques, all easy-to-
Testing use modifications from the CLSI/EUCAST ref-
erence methods, show a good essential agree-
Two standardized microdilution-based ment (defined as MICs within 2 dilutions) with
procedures by the Clinical and Laboratory the reference methods, but the categorical agree-
Standards Institute (CLSI 2008a, 2008b) and ment (i.e., agreement in categorizing an isolate as
the European Committee on Antibiotic Suscepti- susceptible, intermediate, or resistant) may be
bility Testing (Arendrup et al. 2012b, 2014b) are lower, especially for the echinocandin class of
universally accepted for performing AFST, but antifungal agents (Pfaller et al. 2012; Arendrup
these procedures are complex, time-consuming, and Pfaller 2012; Astvad et al. 2013). Thus, Can-
and not intended for routine use (Alastruey- dida isolates with MIC values around or above
Izquierdo and Cuenca-Estrella 2012). the breakpoints should be referred for reference
Following a multistep process based on the testing, particularly for isolates with possible
analysis of MIC distribution curves for wild- echinocandin resistance which is emerging
type populations and the clinical relationship (Arendrup and Perlin 2014).
between MIC values and efficacy, CLSI/ As MIC determination is strongly
EUCAST MIC breakpoints (i.e., obtained with recommended for the patient management
the aforementioned reference methods in (Cuenca-Estrella 2014), epidemiological surveys
specialized mycology laboratories) are, to date, of deep, blood, and mucosal infections should be
available to interpret the AFST results of done to monitor antifungal susceptibilities of
amphotericin B, azoles, and echinocandins for Candida and Aspergillus isolates. Therefore, it
Candida, and amphotericin B and azoles for is mandatory that the laboratories that serve
Aspergillus (Cuenca-Estrella 2014). Therefore, patients at risk of IFIs are able to perform
besides to be an important step in establishing AFST for Candida and to screen for azole resis-
fungal clinical breakpoints (CBPs), the MIC tance in A. fumigatus. In this end, a multi-dish
Diagnostic of Fungal Infections Related to Biofilms 75

azole-agar dilution plate (itraconazole 4 g/mL, MIC. Whether these endpoints held the promise
posaconazole 0.5 g/mL, voriconazole 1 g/mL) to potentiate the practicability and the clinical
was developed as a screening test for identifying utility of AFST, it needs to be determined in
potentially resistant A. fumigatus isolates future studies that are specifically aimed at better
(Howard and Arendrup 2011). While this test defining reproducibility and standardization of
can be performed directly from primary plates these advanced AFST assays.
in parallel with the subculturing of the fungus, in
order to provide an early indication of possible
azole resistance (Astvad et al. 2014), it is impor- 5.2 Sessile/Biofilm-Growing Isolate
tant that more than one colony are eventually Testing
tested to take into account the coexistence of
susceptible and resistant isolates (Arendrup As aforementioned, cells in biofilms display phe-
et al. 2015). As up to 10 % of the A. fumigatus notypic properties that are radically different
isolates will be positive at the azole-plate screen- from their planktonic counterparts, including
ing (Escribano et al. 2012), isolates need to be their resistance to antimicrobial agents (Ramage
confirmed as resistant by means of AFST refer- et al. 2012). Many catheter-associated infections
ence method and also by cyp51A gene sequenc- are related to intraluminal biofilms, which are
ing (Astvad et al. 2014). notoriously difficult to treat, as biofilm-related
Finally, through the AFST methods coupled (sessile) MICs (SMICs) are often dramatically
with detection of molecular fungal alterations elevated than MICs in planktonic (non-biofilm)
conferring reduced antifungal drug susceptibility form (Tobudic et al. 2012). Several methods for
(Perlin 2009), often directly from clinical fungal susceptibility testing under biofilm
specimens (Denning et al. 2011; Zhao growth conditions have been developed (Ramage
et al. 2013) it is now possible to ensure a close and Lopez-Ribot 2005; Pierce et al. 2010;
antifungal resistance surveillance in many clini- Sternberg et al. 2014; El-Azizi et al. 2015) and
cal settings. In most IA cases, the detection of validated in a variety of studies published
cyp51A gene mutations in primary clinical recently (Tobudic et al. 2012). In general, these
specimens may be the sole strategy for detecting methods rely upon the XTT-based approach that
Aspergillus resistance to (tri)azoles due to the provides the most reproducible, accurate, and
absence of culture growth, which makes an easy modality for the antifungal drug testing of
MIC determination impossible (Verweij fungal biofilms (Pierce et al. 2008; Nett
et al. 2009; Howard and Arendrup 2011). How- et al. 2011). In the static model, fungal biofilms
ever, NA-based assays, though allowing for formed into 96-well microtiter plates can be
quicker detection of azole-resistance in culture directly used for the biofilm AFST assay. The
positive samples, are to date not standardized or readout of this assay is colorimetric, based on the
practical for most clinical laboratories (Cuenca- reduction of XTT by metabolically active fungal
Estrella 2014), besides to be unable to reveal the biofilm cells. A typical experiment takes approx-
influence from other resistance mechanisms imately 48 h, of which 24 for biofilm formation
(Verweij et al. 2009; Howard and Arendrup and an additional 24 for AFST (Pierce
2011). et al. 2010). In a catheter continuous flow
Novel assays, such those based on flow model, C. albicans biofilm can be eradicated by
cytometry or MALDI-TOF MS are upcoming liposomal amphotericin B at the MIC, as
tools for AFST (Posteraro and Sanguinetti documented by a 20 % reduction in the growth
2014). Based on short-time antifungal drug expo- of hyphal network that was observed after 24-h
sure of fungal isolates, these assays could pro- biofilm formation and a 24-h treatment with the
vide a reliable means for quicker and sensitive drug, in comparison with the untreated control
assessment of AFST, through the determination (Seidler et al. 2010). In our study (Fiori
of new endpoints in alternative to the classical et al. 2011), in vitro activities of anidulafungin
76 M. Sanguinetti and B. Posteraro

and caspofungin against biofilms of Candida and methods that targeting specifically biofilm
Aspergillus clinical isolates were evaluated and molecules can provide a direct proof of the real
compared with those of the older antifungals presence of a biofilm on the site of infection. In
amphotericin B, fluconazole (only for Candida the meantime, removal or replacement of medi-
isolates), and voriconazole. The MICs and MECs cal devices, where appropriate, represents the
were recorded after incubation for 2448 h, while first line in clinical management, followed by
the SMICs were determined by the 96-well antifungal management, of biofilm infections.
microtiter assay. We found that Candida species While the treatment with conventional antifungal
exhibited high SMIC90s against fluconazole, agents, such as liposomal amphotericin B and
voriconazole, and amphotericin B, whereas the echinocandins, is fundamental, experimental
anidulafungin SMIC90s were very low, as were evidences show that it is possible to enhance
those for caspofungin. In comparison, Aspergil- antifungal activity through ECM degrading
lus species exhibited higher SMIC90 not only molecules, natural compounds, and microbially
against amphotericin B and voriconazole but derived substances. In addition, our understand-
also against the echinocandins. ing of the processes underlying the adaptive drug
In a more recent study (Rajendran et al. 2015), resistance within the biofilm has shed light on
C. albicans LBF and HBF were tested for their specific molecules that could be potentially use-
response to voriconazole, amphotericin B, and ful as therapeutic targets. Further progress and
caspofungin at low (clinically relevant; 2 mg/L) refinement of diagnostic and therapeutic
concentration and high (potentially useful in approaches will be expected, in the next future,
antifungal lock therapy; 200 mg/L) concentra- in order to provide a reliable means of success-
tion. The results show that LBF and HBF isolates fully managing fungal biofilms of clinical
were significantly different in susceptibility to importance.
voriconazole and caspofungin at both 2 mg/L
(P <0.05) and 200 mg/L (P <0.001), whereas
amphotericin B was shown to be equally effec-
tive against LBF and HBF isolates, although a
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200 mg/L (HBF, P <0.0005; LBF, P <0.005). What makes Aspergillus fumigatus a successful path-
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DOI 10.1007/5584_2016_10
# Springer International Publishing Switzerland 2016
Published online: 7 June 2016

Disinfectants to Fight Oral Candida


nia Silva
M. Elisa Rodrigues, Mariana Henriques, and So

Oral biofilms, especially those caused by oral mycobiota, which include
Candida species, are very difficult to eradicate, due to their complex
structure and recalcitrance. Moreover, the mouth is prone to be colonized
since it presents different types of surfaces, especially biomaterials and
dental implants, often associated with a high rate of infections. Therefore,
although disinfection of the oral cavity is of major importance, the number
of commercially available disinfectants is not high. However, new
solutions, as silver nanoparticles are being developed to help oral
biofilms eradication.

1 Introduction infections, specially due to its heterogeneous

composition (from ceramics to metals) and struc-
The oral cavity is one of the most complex ture (from very smooth, as the crown surface, to
cavities of the human body, comprising hundreds very rough, as the implant). These infections can
of different bacterial, viral, and fungal species. be less complex as caries, periodontitis and
The complexity of the oral cavity is in fact thrush or can evolve to severe diseases, as endo-
increased by the high number of different carditis or glomerulonephritis.
surfaces that can co-exist there, namely teeth, Several microorganisms can be found in the
gingival sulcus, tongue, cheeks, hard and soft oral cavity, including the well-known bacteria,
palates and medical devices (prosthetic devices such as streptococci, lactobacilli, staphylococci
or implants). In addition to the oral specific and corynebacteria, as well as fungi. Candida
environment (variations on pH and sugar concen- species belong to the latter and are the most
tration, etc.), biomaterials are very prone to frequent (isolated from 75 % of participants),
followed by Cladosporium (65 %),
Aureobasidium, Saccharomycetales (50 % for
M.E. Rodrigues, M. Henriques (*), and S. Silva both), Aspergillus (35 %), Fusarium (30 %) and
CEB, Centre of Biological Engineering, LIBRO Cryptococcus (20 %). Moreover, four of these
Laboratorio de Investigacao em Biofilmes Rosario
Oliveira, University of Minho, 4710-057 Braga, Portugal predominant genera are known to be pathogenic
e-mail: in humans (Ghannoum et al. 2010). So, the

84 M.E. Rodrigues et al.

mycobiota is a medically important component aggregates of cells called biofilms (Silva

of the oral microbiota, since opportunistic fungal et al. 2012). Candida biofilms can play a signifi-
infections commonly afflict the oral mucosa, cant role in clinical oral infections because of
being of special importance on immunocompro- their resilience and resistance to normal host
mised hosts. removal mechanisms and also antifungal ther-
apy. In general, for Candida species, biofilm
formation occurs as a result of a sequence of
2 Oral Candida Species Biofilms events: (i) adherence/colonization to the surface;
(ii) cell proliferation/invasion and well organized
The genus Candida contains over 200 different colony formation; (iii) matrix production with
species that are ubiquitously distributed. How- differentiation into a mature three-dimensional
ever, only a few of these have been implicated in structure consisting of yeast, pseudohyphae,
human oral infection (Calderone 2002). Oral and/or hyphae embedded within extracellular
candidoses have been recognized throughout matrix and finally (iv) detachment of biofilm
human history and are often described as being cells to promote colonization and infection of
the disease of the diseased, reflecting the oppor- distal sites (Seneviratne et al. 2008; Silva
tunistic pathogenic nature of Candida. Whilst et al. 2012). Involvement of Candida biofilms
Candida species are generally regarded as harm- in human oral infections is well recognized, par-
less members of humans, infection can arise if a ticularly when occurring on biomaterials, such as
colonized individual becomes immunocompro- acrylic and metals, used for implanted medical
mised (Silva et al. 2012). The most prevalent devices. Silva et al. (2009) confirmed that NCAC
Candida species recovered from the human clinical isolates, as it is described for C. albicans
mouth, in both commensal state and cases of (Chandra et al. 2001), are also able to produce
oral candidoses, is Candida albicans (Pfaller biofilms, although these were less extensive for
and Diekema 2007; Samaranayake et al. 2009). C. parapsilosis comparatively to C. tropicalis
However, the socalled non-Candida albicans and C. glabrata (Fig. 1a) (Silva et al. 2009).
Candida (NCAC) species are increasingly As it is well known, biofilms formation on
recognized as important agents of oral infections denture surfaces is promoted by poor oral
(Pfaller and Diekema 2007). In terms of oral hygiene and practices, such as failure to remove
prevalence, C. albicans is followed by the denture whilst sleeping and reduced denture
C. glabrata, C. krusei, C. tropicalis and cleaning. In the event of host debilitation, which
C. parapsilosis (Weems 1992; Williams causes an ecological shift in favour of Candida
et al. 2011). The apparently increased involve- growth, candidal biofilms may develop also on
ment of NCAC species in oral candidoses may the mucosa itself, as it is possible to observe in
partly be related to improvements in diagnostic Fig. 1b (Silva et al. 2011).
methods, such as the use of primary agars with As described previously, the presence of can-
ability to differentiate species, and the introduc- didal biofilms reduce the likelihood of removal
tion of molecular techniques in the routine diag- of organisms by the host defense mechanism and
nosis of fungaemia (Liguori et al. 2009). antifungal agents. From the clinical perspective,
However, the increased prevalence of NCAC the most important feature of Candida biofilms
species in disease could also be a reflection of is, in fact, their role in resistance to conventional
the inherently higher level of antifungal drug antifungal therapy (Hawser and Douglas 1995).
resistance in some NCAC species (Gonzalez Several groups have demonstrated that Candida
et al. 2008) compared to C. albicans, as this cells present in biofilms show a dramatic increase
would promote their persistence in mixed- in the levels of resistance to the most commonly
species infections treated with traditional anti- used antifungal agents (Ramage et al. 2001).
fungal agents. Candida species virulence has Although the mechanisms of biofilm resistance
been attributed to their ability to form structured to antifungal agents are not fully understood, the
Disinfectants to Fight Oral Candida Biofilms 85

Fig. 1 (a) Scanning electron microscopy images of epithelium after 24 h. Candida cells are shown in red,
Candida species colonizing acrylic surfaces and and the nucleic of the epithelial cells appear in blue in
(b) Confocal laser scanning microscopy images of confocal microscopy images; 3000 X corresponds to orig-
Candida species colonizing and invading human oral inal magnification of scanner electron microscopy images

current consensus is that biofilm resistance to 3.1 Chlorhexidine

drugs is a complex multifactorial phenomenon
involving different molecular mechanisms of Chlorhexidine is a highly cationic polybiguanide
resistance, compared to those displayed by (bisbiguanide) with a broad spectrum of antimi-
planktonic cells (Silva et al. 2012). So, it is of crobial activity, including C. albicans and other
major importance to know which oral common NCAC species (Suci and Tyler 2002;
disinfectants are available to fight these biofilms. Ellepola and Samaranayake 2001). It is consid-
ered one of the most important medication in a
basic health system (WHO 2013).
Additionally, chlorohexidine acts both as fun-
3 Oral Disinfectants
gicide and fungistatic agent, but the specific
mode of action is not yet fully understood. Nev-
As described, Candida biofilms are very complex
ertheless, several studies have assessed the anti-
structures that can be formed on both biotic
microbial effects of chlorohexidine identifying
and abiotic surfaces, and are associated with
effects on Candida viability and structural integ-
important and recalcitrant infections. Therefore,
rity such as: (i) disruption of the attachment to
disinfection of oral biomaterials is of major
the surface substrate; (ii) cell wall changes as
retraction of the plasmalemma from the cell
As well as the antifungal agents available, the
wall, and condensation and fragmentation of the
number of disinfectants used for elimination of
cell wall (indicating possible lysis and escape of
fungal colonization is very low. The most com-
cytoplasmic components through the plasma-
mon are chlorhexidine, benzalkonium chloride
lemma); (iii) increased vacuolation, aggregation
and nystatin, among others. However, as their
and clumping of the cytoplasm contents; and
effect on biofilms is still reduced, other
(iv) inhibition of budding (MacNeill et al. 1997;
alternatives, as silver nanoparticles, are arising.
86 M.E. Rodrigues et al.

Bobichon and Bouchet 1987; Ellepola and 2000; Budtz-Jorgensen 1990). Chlorhexidine is
Samaranayake 2001). also used as prophylaxis for both chemotherapy-
Chlorhexidine has also been shown to bind and radiotherapy-induced mucositis (Kowanko
avidly to negatively charged surfaces such as et al. 1998). It has been suggested that chlorhexi-
epithelial cells (Audus et al. 1992), and to adsorb dine may be used as a pre-treatment on denture
to enamel and salivary proteins (Hjeljord acrylic to reduce subsequent adherence of
et al. 1973). Because of this, it has been C. albicans (Spiechowicz et al. 1990; McCourtie
suggested that a crucial feature of this compound et al. 1985; McCourtie et al. 1986).
is its high substantivity in the oral cavity, i.e. the However, there are some problems that lead to
capacity to be retained in the mouth (Bonesvoll therapeutic failure of chlorhexidine: both the dil-
et al. 1974a, b). The slow and continuous release uent effect of saliva and the cleansing action of
of chlorhexidine from pellicle-covered oral the oral musculature reduce the availability of
surfaces seems to be a relevant pharmacody- the compound below the effective therapeutic
namic feature, prolonging the therapeutic effect concentration (Ellepola and Samaranayake
in the oral environment (Ellepola and 2001); and the reduced susceptibility to
Samaranayake 2001). However, this characteris- antifungals of Candida biofilms (Suci and Tyler
tic has also been associated with the destruction 2002; Baillie and Douglas 1999; Hawser and
of the normal ultrastructure of epithelial cells and Douglas 1995).
their receptors for microbes (Vaahtoniemi 1997),
as well as with denaturation and precipitation of
salivary mucinous proteins (Gjermo 1989). This 3.2 Benzalkonium Chloride
may lead to side effects such as interference with
taste sensations, soreness of the oral mucosa, and Benzalkonium chloride is a quaternary ammo-
discoloration of the teeth (Gjermo 1989). nium compound consisting of a mixture of
This antimicrobial agent is widely prescribed alkylbenzyldimethylammonium chlorides
as an adjunctive therapeutic supplement due to whose alkyl group has different even-numbered
its antimicrobial activity against a broad spec- alkyl chain lengths. This compound has been
trum of organisms that includes Candida used as a biocide, a cationic surfactant and a
(Epstein 1990; Zegarelli 1993; Suci and Tyler phage transfer agent.
2002; Ellepola and Samaranayake 2001; Torres The amphiphilicity of benzalkonium chloride
et al. 2007). In particular, it is used in mouth rinse is essential for its antimicrobial activity. The
formulations to reduce microbial burden in the action of this compound begins with the interac-
oral cavity, related to biofilms, having an tion of the hydrophilic cationic region with the
improved action compared to washes with anti- negatively charged components of the surface of
fungal agents as nystatin and clotrimazole the pathogen, establishing electrostatic
(Treister and Woo 2010). However, the effec- interactions. These outcompete the divalent
tiveness of chlorhexidine as an antimicrobial cations that commonly stabilize the structure of
agent is influenced by its concentration and expo- the pathogens surface. After this contact, the
sure time, and no firm conclusions have been hydrophobic region of benzalkonium chloride
taken regarding the best method to prevent infec- penetrates the hydrophobic bilayer, compromis-
tion in the oral cavity (Fathilah et al. 2012). Nev- ing cellular permeability controls and causing
ertheless, a 0.2 % chlorhexidine gluconate cell leakage and lysis (Coughlin et al. 1983;
solution is typically used in clinical practice Fazlara and Ekhtelat 2012; Mangalannalli-
(Salem et al. 1987) against Candida-associated Illathu and Korber 2006; McDonnell and Russell
denture stomatitis and in acute 1999).
pseudomembranous candidosis; and a 2 % chlor- The main antimicrobial activity of
hexidine gluconate is used as an overnight den- benzalkonium chloride membrane disruption,
ture disinfectant (Ellepola and Samaranayake is most effective against Gram-positive bacteria,
Disinfectants to Fight Oral Candida Biofilms 87

having an effect also on Gram-negative bacteria, et al. 2009). In fact, the incorporation of nystatin
some enveloped virus, fungi, yeasts and protozoa into the thin-film polymers was able to reduce
(Fazlara and Ekhtelat 2012). biofilm formation between 70 % and 80 %
It is an active ingredient of biocides used in (Redding et al. 2009). In studies in which the
different applications, from clinical, food line to concentration of nystatin was evaluated, the
domestic household (Mangalannalli-Illathu and higher concentration tested had the highest
Korber 2006). Its main use is as a preservative effect, being time dependent, and decreasing
in ophthalmic solutions (as eye drops), in and/or disappearing after 1 week, 15 days or
concentrations ranging from 0.004 to 0.01 % 1 month depending on the concentrations
since higher values can be caustic and cause (Skupien et al. 2013).
irreversible damage to the cornea (Nelson and Despite polyenes being very poorly absorbed
Goldfrank 2011; Baudouin et al. 2001). Addi- through the gut and their use being relatively
tionally, it has also been tested against Candida limited, in view of their efficacy on the control
in plastic surfaces of medical devices of Candida growth and biofilm development it is
(e.g. denture materials), achieving a pathogen possible to consider that nystatin can be used as
reduction when used at least at a concentration an alternative disinfectant to fight Candida
of 5000 mg/L. In opposition, benzalkonium chlo- species.
ride was shown to be unable to prevent fungal
adherence to extracellular matrix proteins
(Imbert et al. 2003b).
3.4 Silver Nanoparticles

3.3 Nystatin Silver nanoparticles (SN), due to their unique

properties, are starting to be used in many day-
Nystatin belongs to the polyene class, fungicidal to-day applications, including medical
due to the ability to interact with the ergosterol applications. Although there are various theories
component within the fungal cell membranes about the microbicidal action of SN, the exact
to generate pores causing cell leakage and loss mechanism is not clearly known and is yet a
of cytoplasmatic content. This agent is generally debate topic. SN has the ability to anchor to the
regarded to have the broadest spectrum of microorganisms cell wall and subsequently pen-
antifungal activity (Silva et al. 2001). Nystatin etrate it, thereby causing structural changes in the
is frequently used topically and can be cell membrane integrity. It has also been pro-
administrated in a variety of oral formulations posed that the microbicidal activity can be due
in the treatment of oral candidoses including to the release of silver ions by nanoparticles,
suspensions and pastilles (Williams et al. 2011). which can interact with important enzymes and
Silva et al. (2013) revealed that on single inactivate them. It has also been postulated that
pre-formed biofilms, nystatin was able to signifi- nanoparticles can even modulate the signal trans-
cantly inhibit C. glabrata and C. albicans duction in microorganisms (Prabhu and Poulose
biofilms. Thus, one strategy that has been the 2012).
focus of recent research is to actually modify The effect of SN against Candida biofilms
the medical surfaces so they are less prone to was first reported by Monteiro et al. (2011).
biofilm formation by microorganisms, including In this study, SNs were synthesized by silver
Candida species (Price et al. 2002; Price nitrate reduction with sodium citrate and
et al. 2005). In addition, thin-film polymer stabilized with ammonia. Then, these
formulations with incorporated antifungal nanoparticles were applied (during 24 h) to
agents, such as chlorhexidine and nystatin have adherent cells (2 h) or biofilms (48 h) of
also recently been shown to inhibit C. albicans C. albicans and C. glabrata previously devel-
biofilms growth on denture materials (Redding oped (Fig. 2).
88 M.E. Rodrigues et al.

Fig. 2 Scanning electron microscopy images showing conditions: (a) control and (b) biofilms treated with SN
the structure of Candida albicans (i) and Candida (Note agglomerated SN in an enlarged view of part of
glabrata (ii) mature biofilms under different experimental biofilms (Monteiro et al. 2013))

The results for adhered cells showed a signifi- this species is known to be resistant to conven-
cant reduction in the total biomass and in the tional antifungal drugs, making it very difficult to
number of cultivable cells for SN concentration eliminate. Furthermore, the effective SN concen-
at/or higher than 3.3 g/mL for both species. So, tration against C. glabrata biofilms can be con-
SNs showed the ability to inhibit biofilm forma- sidered low when compared with the
tion when applied to adherent cells. Importantly, concentrations of conventional antifungal used.
SNs were more effective in the reduction of the For instance, fluconazole at concentrations rang-
number of cells and total biomass when applied ing from 50 to 1250 g/mL was ineffective
on adhered cells than pre-formed biofilms against C. glabrata biofilms (Fonseca
(Monteiro et al. 2011). Regarding pre-formed et al. 2014).
Candida biofilms, the highest SN concentration In the view of the SN application as disinfec-
tested (54 g/mL) significantly reduced biofilms tant of medical devices against Candida biofilms
of C. glabrata. In general, C. glabrata biofilms proliferation, and considering that the loss of the
have reduced thickness and are devoid of hyphae chemical stability might reduce their effective-
compared with C. albicans biofilms (Silva ness, a study was conducted to verify whether
et al. 2009; Samaranayake et al. 2005). Conse- heating or changing pH of a SN stock solution, as
quently, all these features can help explain the well as the treatment period, would affect the
better effect of SN against C. glabrata biofilms, anti-biofilm activity (Monteiro et al. 2014). Sev-
which is still of major clinical importance since eral parameters were evaluated regarding SN
Disinfectants to Fight Oral Candida Biofilms 89

(54 g/mL) stability, namely temperature (50  C, Recently, Longhi et al. (2015) revealed that the
70  C, and 100  C) and pH (5.0 and 9.0) in combination of SN and fluconazole reduced the
mature biofilms grown on acrylic resin concentration of the latter around 1664 times
specimens. Surprisingly, SN colloidal against planktonic cells of C. albicans and a
suspensions heated at 50  C and 70  C for significant dose-dependent decrease in the viabil-
30 min did not display modifications in their ity of both initial and mature C. albicans biofilm
absorption spectra as compared with SN suspen- (Longhi et al. 2015). In a clinical context, these
sion that was not heated. In addition, heating SN combinations are very important to decrease the
at 50  C and 70  C did not compromise the concentrations used on traditional antifungal
effectiveness of SN against Candida biofilms therapy and reduce the probability of their
(Monteiro et al. 2014). Regarding pH variations increased resistance.
and despite the appearance of dark aggregates, In view of all these attributes it is possible to
evidencing instability of SN at both acid and consider that SN can be used as an alternative
basic pH, the results also showed that the disinfectant to fight Candida biofilms; however,
variations in pH did not impair the effectiveness further research is needed to elucidate the mode
of SN in reducing Candida species biofilms. One of action, cytotoxicity and the longevity of SN
of the factors that may contribute to Candida formulations and microbial properties of SN
biofilms antifungal resistance is the presence of generated by the incorporation of nanoparticles
extracellular matrix (Silva et al. 2001). The for example into denture acrylic.
matrix of Candida biofilms is constituted by sev-
eral proteins, carbohydrates (Silva et al. 2009)
and DNA (Martins et al. 2010). Antifungal drugs 3.5 Other Alternative Disinfectants
may bind to constituents of the extracellular
matrix, preventing the drugs from reaching the Besides the previously described disinfectants,
cells in the deeper layers of the biofilm the literature suggests other alternatives, as the
(Vediyappan et al. 2010). Therefore, strategies use of 0.5 % sodium hypoclorite concentration
focusing on eradication of extracellular matrix that can help disinfect tissue conditioners and
may collaborate to fight Candida infections denture lines (Skupien et al. 2013).
associated with biofilms. Monteiro et al. (2013) In addition, povidone iodine is recognized as
applied SNs on C. glabrata mature biofilms and an effective broad spectrum biocidal agent,
it was possible to observe a significant reduction whose in vitro biocidal activity has been studied
in matrix protein and DNA contents (Monteiro for years against bacteria, yeast, moulds, viruses,
et al. 2013). Moreover, an in vitro study com- fungi, protozoa, actinomycetes and rickettsia
pared the antifungal effects of nystatin and SN on (Duarte and Hamdan 2006; Zamora 1986;
pre-formed single and dual species (C. albicans Amend and Pietsch 1972). Typical iodine
and C. glabrata) biofilms on acrylic resin solutions present significant oral toxicity, but
surfaces under conditions that attempted to this complex exhibits markedly lower toxicity,
mimic the oral environment (Silva et al. 2013). being less hazardous in case of accidental inges-
This work revealed that C. albicans and tion (Kumar et al. 2009). Povidone iodine has
C. glabrata colonized acrylic surfaces in the been mostly used for surgical scrubbing and as a
presence of artificial saliva and co-existed in prophylactic irrigation solution against surgical
dual species biofilms without antagonism and site infection (Chundamala and Wright 2007). A
that both nystatin and SN were able to inhibit study has also demonstrated that when used at
single and dual species biofilms. SNs were also 2.5 %, povidone iodine is able to completely
tested in combination with chlorhexidine and a inhibit yeast adherence, suggesting that it could
synergetic activity was observed for C. glabrata be a good candidate for prevention of candidoses
biofilms, achieving a reduction in biofilm bio- related to medical devices (Imbert et al 2003a).
mass of more than 84 % (Monteiro et al. 2013). However, it has only been applied: in
90 M.E. Rodrigues et al.

gynaecology for vaginitis associated with Can- infections, with special relevance in immuno-
dida (Dugal and Chaudhary 2013); as an antisep- compromised patients.
tic for infected inflammatory conditions of the
mouth and pharynx caused by C. albicans (Silva
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Adv Exp Med Biol - Advances in Microbiology, Infectious Diseases and Public Health (2016) 3: 95103
DOI 10.1007/5584_2016_11
# Springer International Publishing Switzerland 2016
Published online: 11 June 2016

Updates on Therapeutic Strategies Against

Candida (and Aspergillus) Biofilm Related

Fuad Kamel Muakkassa and Mahmoud Ghannoum

Fungal biofilm related infections are commonly associated with medical
devices with biofilms contributing to the virulence of the involved fungal
species. If infection does occur, removal of medical device is often
warranted. However, this is not always possible. Moreover, biofilm
associated infections are often resistant to antifungals and host immunity.
Therefore, a need for new agents and strategies to combat these
devastating infections is needed. Although no randomized clinical trials
have been conducted or are likely to be conducted in the future, the
Infectious Disease Society of America (IDSA) and the European Society
of Clinical Microbiology and Infectious Diseases (ESCMID) utilized
available published data and clinical experience of the infectious disease
community to propose strategies to treat biofilm associated devise
infections. In this chapter we describe the emerging therapies for biofilm
related infections.

ranging from lesser superficial skin and mucosal

1 Introduction
infections to life-threatening systemic infections.
Candidas growth and virulence arises from mul-
The Candida genus, a eukaryotic organism, is
tiple factors including dimorphism (converting
one of the relatively few fungi that can cause
between budding yeast and branching septal
severe human infection and disease. Despite its
hyphal forms), ability to adhere via adhesion
known role as a human commensal organism, it
proteins (agglutinin-like sequence (ALS) fam-
has the ability to inflict a spectrum of infections
ily), binding proteins (invasins), secretion of
hydrolases (e.g. phospholipases and aspartic
F.K. Muakkassa and M. Ghannoum (*) proteinases), pH regulation mechanisms, meta-
Department of Dermatology, Center for Medical bolic adaptations, environmental stress reactions,
Mycology, Case Western Reserve University, University
heat shock proteins, trace metal and nutrient
Hospitals Case Medical Center, Cleveland, OH 44106,
USA acquisition, and biofilm formation (Mayer
e-mail: et al. 2013). Medically relevant fungi that

96 F.K. Muakkassa and M. Ghannoum

produce biofilms include Candida, Aspergillus, and more complex biofilms than other fungal
Cryptococcus, Trichosporon, Coccidioides, and species (Kuhn et al. 2002a). This may contribute
Pneumocystis (Fanning and Mitchell 2012). Bio- to C. albicans common pathogenicity in humans,
film infections in general, including both fungal especially when introduced through implantable
and bacterial, can affect any of the organ systems devices.
in humans, and the Center for Disease Control In the past decades there has been an increase
and Prevention estimates over 60 % of chronic in candidiasis, paralleling the increased use of
infections are related to biofilms (Ehrlich medical implants, which are common substrates
et al. 2004). for biofilm formation (Lopez-Ribot 2014). In
As of 2004, Candida spp. are the fourth most contrast to the planktonic form (free floating
common cause of nosocomial blood stream cells), the organized structure of biofilms
infection (BSI), with Candida albicans, specifi- combines metabolic plasticity, increased concen-
cally, being the most common cause of invasive tration of drug efflux pumps, and architectural
candidiasis (IC), followed by Candida glabrata formation of the biofilm matrix. The biofilm
(Pfaller and Diekema 2007). Dispersion of yeast, matrix increases the organisms resistance to
by detachment from infected indwelling devices, chemical, immunological, and mechanical
can result in hematogenous spread of yeast from forces. Contact sensing through cell wall
biofilms surfaces, resulting in disseminated molecules and thigmotropism (directional hyphal
infections. Candida is the third leading cause of growth on uneven surfaces) help the fungus grow
infection related to catheters, and if and adhere to surfaces touching their biofilm, and
disseminated, it can cause up to 40 % mortality invade into substratum layers (Mayer
(Vila et al. 2015). Biofilms may form on a variety et al. 2013). Thigmotropism takes place second-
of surfaces with catheter, dental, and mucosal ary to complex signaling pathways that recruit
surfaces being the most common (Mayer actin and microtubules at the apex of filamentous
et al. 2013). The most commonly inserted medi- fungi. Calcium channels, GTPases, and protein
cal devices in the United States are urinary and complexes all play a role in signaling. This gives
intravascular catheters, which are also the two the fungus an advantage, orienting and guiding
most common causes of nosocomial bloodstream the organism towards the most suitable environ-
infections (Trautner and Darouiche 2004). This ment (Brand and Gow 2009). Due to a transcrip-
poses a significant problem because there has tional activator (required for biofilm formation),
been an increase in the use of indwelling GCN4, many metabolism processes and
catheters in medicine. More than 200 million substrates are at higher levels in biofilms as
catheters of all types are used each year in the well. Biofilm cells have an increased turnover
United States (Thomas et al. 2004). The biofilm, of protein due to an overexpression of protein
composed mainly of the polysaccharide -1,3 synthesis genes and higher concentrations of
glucan, forms in sequential stepsadhesion of translation factors, ribosomal proteins. If nutrient
initial yeast cells, proliferation, formation of deficient, this may play a role in recycling of
hyphal cells, accumulation of extracellular nutrients in the biofilm matrix furthering their
matrix, and finally dispersion of yeast cells persistence to survive (Fanning and Mitchell
(Mayer et al. 2013). The biofilm matrix is 2012). For example, the gene MET3, which
comprised of biopolymers, mainly encodes for assimilation of sulfur metabolism,
polysaccharides and proteins, however lipids is up-regulated in biofilm cells, and does not
and nucleic acid are also commonly found. This show such high concentrations in planktonic
matrix is often used in a combined term, cells (Murillo et al. 2005). One goal of the scien-
exopolymeric substances (EPS). The EPS is a tific community is to further understand how
slimy layer that protects the fungus from outside biofilms function in order to properly target
forces (Purevdorj-Gage and Stoodley 2004). infectious fungi, outsmarting their mechanisms
Candida albicans has shown to produce larger of resistance.
Updates on Therapeutic Strategies Against Candida (and Aspergillus). . . 97

2 Biology and Resistance adhesion of fungal cells to the substrate in the

of Biofilms first 11 h. The mature phase is between hour
31 and 72, at which the cells are encased in a
As mentioned above, biofilms contribute to a thick ECM. These studies were done using wild-
variety of virulence factors for the organism. type and knock-out strains of the efflux pump
They also contribute to microbial resistance to genes: CDR and MDR1. The knock-out strains
antimicrobials. The network of EPS can hinder a were more susceptible to fluconazole (Mukherjee
drugs ability to penetrate the biofilm (Lopez- et al. 2003). One study has shown that a family of
Ribot 2014). The EPS adhesive and cohesive genes called the quinidine drug resistance (QDR)
forces cause strong cell-cell and cell-surface family, in fact, changes functionality of the bio-
interactions, enforcing this protective barrier, film regulation, and thus, inhibits the virulence of
not solely against drugs, but also environmental C. albicans (Shah et al. 2014). This is a great
factors, such as radiation, oxidation, desiccation, example of how the role of biofilms, resistance
predators and host-immune functions (Lopez- mechanisms, and overall virulence of the fungus
Ribot 2014). In fact, nuclear magnetic resonance are intertwined and all play a role in how we may
(NMR) imaging analysis has shown aggregate target and prevent infection.
interaction between the biofilm matrix and the
antifungal fluconazole (Zarnowski et al. 2014).
Biofilm production is regulated by genes and 3 Approach to Discovery
several transcription factors. BGL2 and PHR1 and Novel Treatment
are glucan transferases that are positive
regulators of -1,3 glucan production (Mayer In the previous section we discussed the role of
et al. 2013). Fluconazole was more effective biofilms, and how the structure, metabolism, and
against biofilms produced by fungal mutants gene expression of biofilms increase Candidas
lacking BGL2, PHR1, and XOG1 (a gene virulence. Here we will discuss how we came to
encoding exo-gluconase) in vivo and in vitro, establish certain treatment guidelines, and new
during biofilm growth (Taff et al. 2012). treatments on the horizon. In the next section we
Although fluconazole is not commonly used to present tables summarizing the Clinical Practice
treat biofilm related infections, this demonstrates Guidelines for the Management of Candidiasis:
the role enzymes involved in the fungal biofilm 2009 Update by the Infectious Disease Society of
play in their reaction to medications. C. albicans America and the European Society of Clinical
has multidrug resistant transporter genes (MDR1, Microbiology and Infectious Diseases Guideline
CDR2, and PDR16) which are overexpressed in for the Diagnosis and Management of Candida
in vivo biofilms (Fanning and Mitchell 2012). Diseases 2012: Non-neutropenic Adult Patients.
Biofilms demonstrate up-regulation of other vir- These are guidelines based on clinical practice
ulence factors as well. ALS-1 is the most and research. Physicians are to use their judg-
up-regulated adhesion protein in candidal ment in following these guidelines case by case.
biofilms (Garcia-Sanchez et al. 2004). Sterol bio- Each patient has a unique medical history and
synthesis genes are also up-regulated in presentation of illness, for which these guidelines
C. albicans and contribute to resistance to azole may do just as the name suggests and guide the
drugs as well (Nett et al. 2009). Mukherjee physician in his or her therapeutic choices. It is
et al. showed that the mechanism underlying important to become familiar with these current
fluconazole resistance depends on the biofilm treatment recommendations. It is equally impor-
phase. Efflux pumps were shown to be responsi- tant to understand the upcoming practices and
ble for resistance at early phase; while sterol emerging therapeutics in candidal infection con-
synthesis is responsible for resistance at the trol. First and foremost, efforts must be made to
mature phase. The early phase is defined as the prevent invasive candidiasis from occurring.
Steps to insure candidal biofilms from emerging
98 F.K. Muakkassa and M. Ghannoum

and subsequent dissemination are crucial. required higher concentrations to reach suscepti-
Nonpharmacologic methods of prevention bility in four antifungals: amphotericin B, nysta-
include hand hygiene, judicious use of antibiotics tin, chlorhexidine, and fluconazole (Chandra
in the community (to prevent resistance), and et al. 2001). In a follow up study, new
proper use of catheters and other implanted formulations of drugs were shown to have activ-
devices (Pfaller and Diekema 2007). It is com- ity against candidal biofilms. These included
mon practice to remove or replace any foreign lipid formulations of amphotericin B and
device from the patient if it were to harbor bio- echinocandins (caspofungin and micafungin)
film producing fungi or cause infection. Data (Kuhn et al. 2002b).
from 1915 patients from seven trials Models for bench-work research on biofilms
demonstrated that two factors correlated with have come a long way, with the use of high-
greater clinical success in patients with throughput screening (a method of testing high
candidemia: use of an echinocandin and the numbers of compounds with machinery and data
removal of central venous catheters (CVC) processing software). One example of this is a
(Andes et al. 2012). Interestingly, Nucci recent study using a microarray platform of
et al. showed in an analysis of 842 patients, that nano-scale biofilms encapsulated in different
early CVC removal did not show any clinical matrices. This research design puts in perspec-
benefit in adults who developed candidemia tive how biofilm growth is affected by drugs
(Nucci et al. 2010), potentially pointing out that when grown in different mediums. This can
time of removal of catheter may be irrelevant. At potentially be translated from in vitro results to
times, however, patients require the use of such patient care, if we were to take into consideration
devices. For example, in the intensive care unit, a all the different materials that are used in pros-
hypotensive patient whose venous access is poor thetics and other implantable medical devices
may only have one source (a central venous (Srinivasan et al. 2014). A previous in vivo
catheter) for crucial fluid replacement and study tested the medium of the drug rather than
vasopressors (medicines that raise blood pres- the fungus. The authors implanted a material
sure). In such cases, we must rely on anti-fungal called amphogel (a dextran based hydro-gel),
medications. A 2004, in vivo, study inserted which absorbs amphotericin B, in mice. The gel
catheters in rabbits and subsequently infected was inoculated with C. albicans and with time
the, with C. albicans. This showed that showed decreased surviving fungal cells and less
amphotericin B almost cleared fungal growth biofilm formation. This matrix for drug may be
and fungal biofilms when assessed with cultures used in the future to coat implantable devices in
and scanning electron microscopy. These results humans (Zumbuehl et al. 2007).
were significant when compared to the control There have been recent advances in achieving
arm (untreated rabbits) and fluconazole treated such mechanistic methods of biofilm inhibition.
arm of the study. This may give insight into the One study found that in combination with stan-
salvation of infected catheters in humans dard antifungals (fluconazole and capsofungin), a
(Schinabeck et al. 2004). As of yet we have no non-steroidal anti-inflammatory drug, flufenamic
Food and Drug Administration (FDA) approved acid (FFA), had up to 99 % prevention of
medicines that are biofilm specific. We must rely C. albicans biofilm formation. FFA alone had
on ways to inhibit growth of or disruption of up to 8085 % reduction of biofilm formation,
biofilms to render the microbe susceptible to dependent upon concentration of FFA (Chavez-
current antifungal medications (Nobile and John- Dozal et al. 2014).
son 2015). Further proof that biofilms are more Miltefosine, an alkylphospholipid, has shown
resistant to anti-fungals then their planktonic efficacy against C. albicans biofilm formation
structural counterpart, is demonstrated in an and against preformed biofilms, in azole-resistant
in vitro study by Chandra et al. using denture pathogens. The efficacy was demonstrated
model biofilms; C. albicans with biofilm in vitro (planktonic forms) and topically, in vivo
Updates on Therapeutic Strategies Against Candida (and Aspergillus). . . 99

(murine oropharangeal models) (Vila Microbiology and Infectious Diseases Guideline

et al. 2015). Furthermore, immunomodulatory for the Diagnosis and Management of Candida
properties of statins (medication for high choles- Diseases 2012: Non-neutropenic Adult Patients.
terol in humans) has been shown to slow Candi- In this section we present summary tables
dal growth. A follow up multicenter study has (Tables 1 and 2) of treatment recommendations
demonstrated a potential benefit of use of statins for device related candidal infections. These
in hospital patients who developed candidemia infections are more likely to have been biofilm
(Cuervo et al. 2013). New tools to defend against related. The treatment of biofilms specifically,
biofilm fungal infections are yet to be discov- however, is trickier and more complicated.
ered; however, some tools may be right under There are no standard set of guidelines for these
our nose, such as these statins. treatments. Research has suggested that removal
Certain biofilms may be polymicrobial, mean- of catheter, when warranted, improves survival
ing different species of fungi are involved, or in C. albicans biofilm related infections. When
perhaps fungi mixed with bacterial biofilm. This removal is not possible, antifungal lock therapy
poses a treatment challenge, because of the large may be beneficial. Antifungal lock therapy uses
evolutionary difference between prokaryotic high dose concentrations in medical devices
bacteria and eukaryotic fungi. One study has (mainly catheters) to sterilize the equipment. To
found that Guanylated polymethacrylates, a enforce what has been discovered and discussed
copolymers of 2-guanidinoethyl methacrylate in the previous section, the drugs that most often
(2-GEMA) and methyl methacrylate (MMA), work for biofilm related infections are liposomal
which are structural imitators of the amino amphotericin B and echinocandins, while azoles
acids arginine and alanine, respectively, has are less reliable.
strong activity against C. albicans and Staphylo-
coccus aureus (a Gram positive bacteria)
polymicrobial biofilms (Qu et al. 2015). This 5 Moulds: Apergillus
presents a great opportunity for the future of and Fusarium
medicine as monotherapy is usually preferred,
to avoid further side effects and other dangers Lastly, we will briefly review other clinically
of polypharmacy. important fungal species, besides Candida.
Lastly, another study has identified 19 other Aspergillus also forms biofilms on either biotic
compounds that inhibit over 30 % metabolic or abiotic surfaces. The processes are similar to
function of biofilms versus clotrimazole alone other fungi. Initial cells (condia) adhere to
(LaFleur et al. 2011). Understanding that this substrates, multiplying and forming hyphal
study is conducted in vitro, we have a long way forms (mycelia). Extracellular matrix binds the
to go before such molecules can be formed into biofilm together (Fanning and Mitchell 2012).
drugs to be used for management of biofilm- The in vivo biofilm, which invasive Apergillus
associated infections in humans, but the prospect fumigatus forms, is composed of hyphae with an
for the future is exciting. extracellular matrix of polysaccharides,
galactomannan, and galactosaminogalactan.
There is a growing need for new therapies against
4 American and European Aspergillus, due to recent resistant forms
Guidelines to Therapy (Beauvais and Latge 2015). A recent prospective
multicenter international surveillance study of
Details on the treatment of candidal infections in azoles found 3.2 % prevalence (increased from
general can be found in the Clinical Practice previous reports) of azole resistant A. fumigatus
Guidelines for the Management of Candidiasis: (van der Linden et al. 2015) Just as fungi form
2009 Update by the Infectious Disease Society of new resistance characteristics, new therapies are
America and the European Society of Clinical always on the horizon. A study in 2015,
100 F.K. Muakkassa and M. Ghannoum

Table 1 Abridged summary of the European Society for Clinical Microbiology and Infectious Diseases selected
implantable device related infections
Population Intention Intervention
Ventilated patients >48 h with another 72 h Prophylaxis Fluconazole 100 mg/day
expected against
Ventilated  3 days, received antibiotics, CVC, Prophylaxis Caspofungin 50 mg/day
and any of the following: parenteral nutrition, against
dialysis, major surgery, pancreatitis, systemic candidiasis/
steroids, or immunosuppression candidaemia
Candidaemia/invasive candidiasis Treatment Anidulafungin 200/100 mg, Capsofungin
70/50 mg, Micafungin 100 mg, Amphotericin
B liposomal 3 mg/kg
Candidemia with CVC Catheter Remove if possible (not over a guidewire). If
management not possible treat with Echinocandin,
liposomal amphotericin B or amphotericin B
lipid complex
Asymptomatic candiduria To clear candida None, Fluconazole 200 mg for 14 days, if
susceptible, removal of urinary catheter,
Amphotericin B deoxycholate bladder
Cystitis To cure Fluconazole, if susceptible, Amphotericin B
deoxycholate +/ flucytosine
Prosthetic valve endocarditis To cure Surgery within days, if surgery not possible,
liposomal amphotericin B 5 mg/kg or
capsofungin 70/50 mg
Pacemaker, ICD, or VAD infection To cure Removal
Prosthetic joint infection To cure Removal, if prosthesis needed, Fluconazole
400 mg for life
Cornely et al. (2012)
ICD implantable cardiac defibrillator, VAD ventricular assist device

Table 2 Abridged summary of the Infectious Disease Society of America selected implantable device related
Population therapy Alternative therapy Comments
Candidemia Fluconazole LFAmB 35 mg/kg daily or Choose an echinocandin for moderate to
(nonneutropenic 800 mg AmB-d 0.51 mg/kg severe illness or recent azole exposure
Asymptomatic None Eliminate predisposing factors
Symptomatic Fluconazole AmB-d 0.30.6 mg/kg for 17
cystitis 200 mg daily days
for 2 weeks
Endocarditis LFAmB Step-down therapy to fluconazole Valve replacement is strongly
35 mg/kg 400800 mg daily until negative recommended, if unable, chronic
blood culture results suppression with fluconazole
Infective LFAmB Step-down therapy to fluconazole Removal of pacemakers and ICDs
pacemaker, ICD, 35 mg/kg 400800 mg daily until negative strongly recommended, if unable to
or VAD blood culture results remove VAD, chronic suppression with
Pappas et al. (2009)
LFAmB lipid formulation of amphotericin B, AmB-d amphotericin B deoxycholate, ICD implantable cardiac defibril-
lator, VAD ventricular assist device
Updates on Therapeutic Strategies Against Candida (and Aspergillus). . . 101

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DOI 10.1007/5584_2016_12
# Springer International Publishing Switzerland 2016
Published online: 27 April 2016

Natural Sources as Innovative Solutions

Against Fungal Biofilms

Marion Girardot and Christine Imbert

Fungal cells are capable of adhering to biotic and abiotic surfaces and
form biofilms containing one or more microbial species that are microbial
reservoirs. These biofilms may cause chronic and acute infections. Fungal
biofilms related to medical devices are particularly responsible for serious
infections such as candidemia. Nowadays, only a few therapeutic agents
have demonstrated activities against fungal biofilms in vitro and/or
in vivo. So the discovery of new anti-biofilm molecules is definitely
needed. In this context, biodiversity is a large source of original active
compounds including some that have already proven effective in therapies
such as antimicrobial compounds (antibacterial or antifungal agents).
Bioactive metabolites from natural sources, useful for developing new
anti-biofilm drugs, are of interest. In this chapter, the role of molecules
isolated from plants, lichens, algae, microorganisms, or from animal or
human origin in inhibition and/or dispersion of fungal biofilms (especially
Candida and Aspergillus biofilms) is discussed. Some essential oils,
phenolic compounds, saponins, peptides and proteins and alkaloids
could be of particular interest in fighting fungal biofilms.

Biofilm Candida Aspergillus Natural substances Bioactive

1 Introduction

Current available antifungal agents have usually

M. Girardot (*) and C. Imbert little effect on fungal biofilms. The biodiversity
Laboratoire Ecologie et Biologie des Interactions, Equipe
represents a huge reservoir of natural
Microbiologie de lEau, Universite de Poitiers, UMR
CNRS 7267, F-86073 POITIERS, France components with variable and not yet described
e-mail: activities on human infectious diseases,

106 M. Girardot and C. Imbert

including fungal diseases. Our goal was to belonging to EO or terpenes and to saponins
review the already known components active will be described.
against fungal biofilms. Most available studies
focus on Candida yeasts and to a lesser extent 2.1.1 Essential Oils and Terpenes
Aspergillus molds. Active components will be Among substances obtained from plants, EO
described depending on their origin stemming stand out as natural, complex and variable
from plants, lichens, algae, microbes (fungi or mixtures mainly composed of terpenes and aro-
bacteria), animals as well as Humans. In each matic compounds derived from phenylpropane
case, they will be classified depending on their and characterized by strong odor and volatility.
role (for example biosurfactants or antibodies) or They are produced by aromatic plants, as second-
chemical features (for example essential oils ary metabolites. Numerous studies have already
(EO), saponins, phenolic compounds, peptides, been performed to investigate the anti-biofilm
alkaloids. . .). The development of fungal activity of EO. For example, Furletti
biofilms involves three different steps: early et al. reported that Coriandrum sativum EO
(adhesion), maturation and dispersion phases. affected the formation of Candida albicans bio-
The specific activity of natural components film causing fungal cells deformation, presenting
against these phases will be clarified when data a withering appearance, that finally resulted in a
are available. Especially, efficiency against early decreased biofilm growth (Furletti et al. 2011).
phase biofilms would suggest a prophylactic Furthermore, Cinnamomum zeylanicum EO,
benefit against biofilm-related fungal infections predominately composed of cinnamaldehyde
and efficiency against already formed mature (54 %), inhibited the formation of Candida
biofilms would suggest a both prophylactic and orthopsilosis or C. parapsilosis biofilm at
curative benefit depending on the infection loca- concentrations above 250 g/ml and was also
tion. Thus, all these data are developed below active against 24 h old biofilms of these species
and summarized in Table 1. (minimum biofilm reduction concentration: 1000
and 2000 g/ml respectively). A possible expla-
nation was that the hydrophobicity of their vola-
2 Anti-Biofilm Compounds tile compounds allowed for their insertion into
of Plant Origin the lipid bilayer of the cell membrane, causing a
disturbance and increasing its permeability to
Considering literature data, plants currently con- protons. Scanning electron microscope (SEM)
stitute the largest source of active compounds observations supported this hypothesis and
against fungal biofilms. These compounds can showed a marked decrease in cell aggregates
be classified into six groups: terpenoids; phenolic and the complete absence of an extracellular
compounds; peptides; alkaloids; biosurfactants; matrix in Candida biofilms (Pires et al. 2011).
compounds with unknown or mixed The EO extracted from leaves of Ocimum
composition. americanum, an aromatic herbaceous plant
native of Asia and Africa, also demonstrated
effectiveness against yeast biofilms. A 5-min
2.1 Terpenoids exposure of 24 h old C. albicans biofilms to
3 % v/v EO eliminated 3 log10 of biofilm cells.
This group constitutes, with steroid group, the At a lower concentration (0.3 % v/v), a 2 log10
largest known ensemble of plant secondary reduction in C. albicans was observed. Authors
metabolites. It especially includes EO, oleoresin, suggested the possibility of using this EO in oral
iridoids, pyrethrins, sesquiterpene lactones, health care products (Thaweboon and
saponins, cardiac glycosides, phytosterols, Thaweboon 2009).
carotenes, polyisoprenes (Bruneton 2008). At The EO obtained from leaves of Croton
this point only some active compounds cajucara also demonstrated anti-biofilm activity,
Table 1 Chemical or functional groups of natural origins with highlighted anti-biofilm activities against fungi
Studied phases of biofilm development
and/or Mature Tested fungal
Origins Chemical or functional groups Maturation biofilm Dispersion species References
Plant Terpenoids Essential x x C. albicans Alviano et al. (2005), Anghel et al. (2013a), (2013b), Bersan
oils/Terpenes C. dubliniensis et al. (2014), Braga et al. (2008), Carneiro et al. (2011), Chifiriuc
C. glabrata et al.(2012), Curvelo et al. (2014), Da Silva et al. (2012), Dalleau
C. orthopsilosis et al. (2008), De Campos Rasteiro et al. (2014), Doke et al. (2014),
C. parapsilosis Furletti et al. (2011), Grumezescu et al. (2012), Hsu et al. (2013),
C. tropicalis
Khan and Ahmad (2012a), (2012b), Palmeira-de-Oliveira
et al. (2012), Pemmaraju et al. (2013), Pires et al. (2011), Ramage
et al. (2012), Raut et al. (2013), Saharkhiz et al. (2012), Sroisiri
and Boonyanit (2010), Stringaro et al. (2014), Sudjana
et al. (2012), Sun et al. (2012), Taweechaisupapong et al. (2010),
Thaweboon and Thaweboon (2009), Trindade et al. (2015)
Saponins x x Aspergillus sp. Chevalier et al. (2012), Coleman et al. (2010), Sadowska
C. albicans et al. (2014), Vediyappan et al. (2013)
Phenolic Stilbenods x C. albicans Cheng et al. (2009), Li et al. (2012), Zhang et al. (2011)
compounds Tannins x x x C. albicans Bakkiyaraj et al. (2013), Evensen and Braun (2009), Feldman
C. glabrata et al. (2012), Girardot et al. (2014), Luiz et al. (2015), Rane
Natural Sources as Innovative Solutions Against Fungal Biofilms

et al. (2014)
Quinones x x C. albicans Tsang et al. (2012), Tsang et al. (2013)
C. dubliniensis
Flavonoids x x C. albicans Cao et al. (2008), Messier et al. (2011), Messier and Grenier
(2011), Onsare and Arora (2015)
Peptides x x C. albicans Delattin et al. (2014), Mandal et al. (2011), Mandal, (2012)
C. tropicalis
Alkaloids x x A. fumigatus Shao et al. (2014), Wei et al. (2011), Zhao et al. (2013)
C. albicans
C. neoformans
Biosurfactants x C. albicans Cochis et al. (2012), Rodrigues et al. (2006)
Lichen Phenolic Dibenzofuran x C. orthopsilosis Pires et al. (2012)
compounds C. parapsilosis
Flavonoids x C. albicans Singh et al. (2015)
Pyridins x C. albicans Chang et al. (2015)
Benzenediols x C. albicans Li et al. (2015)
Terpenoids Triterpens x C. albicans Chang et al. (2012)

Table 1 (continued)

Studied phases of biofilm development

and/or Mature Tested fungal
Origins Chemical or functional groups Maturation biofilm Dispersion species References
Alga Fatty acids x C. albicans Thibane et al. (2010)
C. dubliniensis
Fungus Alkaloids x C. albicans Wang et al. (2012b), You et al. (2013)
C. glabrata
C. kefyr
C. parapsilosis,
C. tropicalis
Terpenoids Terpenes x x C. albicans Cordeiro et al. (2014), Hornby et al. (2001), Ramage et al. (2002),
C. dubliniensis Weber et al. (2010)
C. metapsilosis
C. orthopsilosis
C. parapsilosis
C. tropicalis
Biosurfactants x C. albicans Monteiro et al. (2011)
Glucosides x C. albicans Wang et al. (2014a)
Bacteria Peptides/ x x C. albicans Dusane et al. (2013), Troskie et al. (2014), Wang et al. (2012a),
Proteins Yala et al. (2011)
Biosurfactants x x C. albicans Busscher et al. (1997), Ceresa et al. (2015), Dusane et al. (2011),
C. tropicalis Janek et al. (2012), Rautela et al. (2014), Rodrigues et al. (2006),
Singh et al. (2013), Zakaria Gomaa (2013)
Animal Resins x C. albicans Capoci et al. (2015), De Castro et al. (2013)
Peptides/ x x A. fumigatus De Brucker et al. (2014), Kavanaugh et al. (2014), Sengupta
Proteins C. albicans et al. (2012), Wang et al. (2014b)
C. glabrata
F. solani
Antibodies x C. albicans Bujdakova et al. (2008), Fujibayashi et al. (2009), Hodgkinson
(proteins) C. dubliniensis et al. (2007), Holmes et al. (2012), Ibrahim et al. (2008),
C. glabrata Kamikawa et al. (2014), Mishra et al. (2015)
C. tropicalis
Human Peptides/ x x C. albicans Konopka et al. (2010), Pusateri et al. (2009), Rossignol
Proteins C. glabrata et al. (2011), Tati et al. (2014)
M. Girardot and C. Imbert
Natural Sources as Innovative Solutions Against Fungal Biofilms 109

which seemed especially linked to its main com- be rich in piperitenone oxide (90 %) and to
pound: linalool. This compound induced a reduc- reduce both adherence (decrease of about 40 %)
tion in cell size and abnormal germination. The and biofilm formation (decrease of about
EO inhibited the growth of C. albicans biofilm 7080 % of the metabolic activity) of
similar to chlorhexidine (Alviano et al. 2005). C. albicans (Stringaro et al. 2014). The inhibitory
The EO obtained from rhizomes of effect on biofilm formation was confirmed by
Boesenbergia pandurata demonstrated a potent SEM observations. Besides, a synergistic effect
anti-Candida biofilm activity in vitro was observed between this EO and fluconazole.
(Taweechaisupapong et al. 2010). In addition, It was supposed that monoterpens of this EO
rhizome extract of B. pandurata inhibited passed across the cell wall, damaged lipid bilayer
C. albicans adhesion to denture acrylic surfaces of cell membrane increasing the Candida cell
in a dose-dependent manner: a pretreatment of membranes permeability and promoting flucona-
dentures with this extract at 25, 50 and 100 mg/ zole action on ergosterol, finally causing cell
ml inhibited candidal adhesion by approximately damage (Stringaro et al. 2014). Recently, a
47 %, 66 %, and 75 %, respectively (Sroisiri and novel nerolidol-rich EO derived from leaves
Boonyanit 2010). and inflorescences of Piper claussenianum was
Palmeira-de-Oliveira et al. investigated the tested on C. albicans yeast-to-hypha transition
EO obtained from aerial parts of Thymbra and on biofilm formation and stability. Leaf EO
capitata, a circum-Mediterranean plant; this EO managed to downregulate the yeast-to-hypha
was rich in carvacrol (75 %). Authors showed transition by 81 %, and reduced biofilm forma-
that T. capitata EO disrupted the biomass and tion by about 30 and 50 % after incubation for
inhibited the metabolic activity of C. albicans, 24 and 48 h, respectively. This EO also reduced
C. glabrata, C. tropicalis, C. parapsilosis and the viability of pre-formed mature biofilm (48 h)
C. guilliermondii pre-formed 24 h old biofilms. by 63.9 %. Finally, synergistic effect was
Concentrations about 0.32 l/ml (which also highlighted using association between the leaf
corresponded to the EO minimal inhibitory con- EO and fluconazole (Curvelo et al. 2014).
centration (MIC)) disrupted the biofilm matrix Melaleuca alternifolia (tea tree) EO, rich in
and inhibited the yeast metabolism up to 50 %. terpinen-4-ol (42.8 %) and -terpinene (20.4 %),
A marked metabolic inhibition (>80 %) was used at a 12.5 % was effective in vitro to eradi-
observed at 0.64 l/ml, except for C. albicans cate a 48 h old C. albicans biofilm; it also
that presented a more resistant profile. Biofilm reduced the C. albicans yeast number in an
biomass was also widely reduced at this concen- immunosuppressed mouse model (de Campos
tration, except for C. glabrata (no more than Rasteiro et al. 2014). Another study
40 % disruption) (Palmeira-de-Oliveira demonstrated that sub-inhibitory concentrations
et al. 2012). of this oil reduced C. albicans adhesion to both
Bersan et al. highlighted the interest of human cells and polystyrene, inhibited biofilm
Cyperus articulatus EO acting against the forma- formation and decreased cell surface
tion of a C. albicans biofilm and inhibiting a 72 h hydrophobicity (Sudjana et al. 2012). Comple-
old fungal biofilm (Bersan et al. 2014). mentarily, tee trea EO and two of its components:
Mentha piperita EO obtained from aerial parts terpinen-4-ol and -terpineol, displayed a potent
and rich in menthol (53.28 %), menthyl acetate activity against 69 biofilm-forming C. albicans
(15.1 %) and menthofuran (11.18 %) was tested strains. M. alternifolia EO used at 50 %
reported to completely inhibit the biofilm forma- minimal inhibitory concentration value (MIC50)
tion of C. albicans and C. dubliniensis at effectively inhibited biofilm growth when
concentrations up to 2 l/ml in a dose-dependent C. albicans cells were treated at 0, 1, and 2 h
manner (Saharkhiz et al. 2012). In the same way, post-adhesion. Interestingly, terpinen-4-ol
EO extracted from leaves of another Mentha displayed no significant toxicity at 0.5  MIC50
species, M. suaveolens, was recently shown to which gave it safety advantages over the
110 M. Girardot and C. Imbert

complete EO; authors results suggested that Casbane diterpene was isolated from the
terpinen-4-ol may be suitable for prophylaxis ethanolic extract of Croton nepetaefolius, an aro-
and treatment of established oropharyngeal matic plant used in folk medicine, native of the
candidosis (Ramage et al. 2012). Northeast of Brazil. This compound was shown
Cymbopogon nardus EO and its main com- to be effective against C. albicans and
pound (citronellal: 38 %) were recently C. tropicalis biofilm formation, causing a
investigated for their anti-adherent properties. decrease of more than 50 % of the ability to
The EO significantly inhibited C. albicans adher- form biofilm at 62.5 g/ml and 15.6 g/ml
ence to both dental implants and cover screws respectively after 24 h of treatment (Carneiro
(p <0.001) whereas citronellal only inhibited et al. 2011).
adherence to dental implants (p <0.001) Overall, these data show the anti-adherent and
(Trindade et al. 2015). anti-biofilm interest of numerous EO. However,
EOs of another species of Cymbopogon : EO are nonwettable, highly volatile and conse-
C. citratus and of Syzygium aromaticum were quently unstable. There is thus a challenge for
investigated by Khan and Ahmad and both EO applications in the biomedical field. In this
demonstrated C. albicans anti-biofilm activities. context, some galenical studies have been
These two oils were more active than performed in recent years. Literature data report
amphotericin B and fluconazole against for example the fabrication and characterization
preformed biofilms. At 0.5  MIC, C. citratus of a novel nanostructured phyto-bioactive coated
EO followed by Syzygium aromaticum EO were rayon/ polyester wound dressing surface refrac-
most inhibitory against biofilm formation. tory to C. albicans adhesion, colonization and
Deformations of the biofilms three dimensional biofilm formation, based on functionalized mag-
structure were observed by SEM after treatment netite nanoparticles and EO from Anethum
with C. citratus oil (Khan and Ahmad 2012b). graveolens and Salvia officinalis. This complex
Raut et al. recently investigated twenty-eight preparation exhibited a long term anti-biofilm
terpenoids of plant origin for their activity effect, maintained for at least 72 h. Authors
against C. albicans biofilms. Some of these concluded that the combination of stabilizing
molecules inhibited yeast-to-hypha transition at carrier properties of magnetite nanoparticles
low concentrations (0.0310.5 mg/ml), while with antimicrobial features of natural substances
adhesion to a solid surface was prevented at could be a successful approach to control and
0.52 mg/ml. Treatment with 14 terpenoids prevent fungal biofilm associated infections
resulted in significant (p < 0.05) inhibition of (Anghel et al. 2013b). The same kind of structure
C. albicans biofilm formation, and of these, lin- constituted by biohybrid nanostructured magne-
alool, nerol, isopulegol, menthol, carvone, tite nanoparticles/ Satureja hortensis EO was
-thujone, and farnesol exhibited biofilm- reported as being active against C. albicans
specific activity. Finally, thymol, carvacrol, adherence and biofilm development (Anghel
eugenol, citral, citronellol, geraniol, linalool, et al. 2013a).
and ionone inhibited 24 h old biofilms (Raut An hybrid magnetite nanoparticles/
et al. 2013). Other studies confirmed the interest Rosmarinus officinalis EO nanobiosystem or
of carvacrol, linalool, geraniol, thymol, eugenol, hybrid nanomaterial/ Eugenia carryophyllata
cinnamaldehyde, -pinene and -pinene or men- EO also displayed anti-biofilm activity against
thol against C. albicans, C. glabrata or C. albicans or C. tropicalis and authors
C. parapsilosis biofilms alone or in association suggested that they may be pelliculised on the
(one or two terpenes) with fluconazole (Braga surface of catheter pieces (Chifiriuc et al. 2012;
et al. 2008; da Silva et al. 2012; Dalleau Grumezescu et al. 2012). Finally, polyethylene
et al. 2008; Doke et al. 2014; Hsu et al. 2013; glycol (PEG)-stabilized lipid nanoparticles
Khan and Ahmad 2012a; Pemmaraju et al. 2013). sustained releasing terpinen-4-ol were also
reported. The anti-biofilm activity of these
Natural Sources as Innovative Solutions Against Fungal Biofilms 111

nanoparticles was shown to result from their Small molecules belonging to the triterpenoid
ability to disrupt cell membrane structure and saponin family of gymnemic acids, isolated from
blocking of respiration chain by the inhibition Gymnema sylvestre leaves, inhibited C. albicans
of succinate dehydrogenase which is bound to yeast-to-hypha transition and conidial germina-
the inner mitochondrial membrane of cells (Sun tion and hyphal development of Aspergillus
et al. 2012). sp. They also inhibited the formation of invasive
hyphae from C. albicans-infected
2.1.2 Saponins Caenorhabditis elegans worms and rescued
Saponins are glycosides commonly found in them from being killed by C. albicans.
plants. Their aglycone part is a triterpen or a According to their results, authors suggested
steroid group. In addition to their surface-active that these compounds may represent a valuable
properties and their common but more problem- source for the development of novel anti-biofilm
atic hemolytic properties, some saponins (for agents (Vediyappan et al. 2013).
example ivy or goldenrod saponins) previously
demonstrated antifungal activity against
phytopathogens, Human pathogens (such as
2.2 Phenolic Compounds
Candida) or some dermatophytes in vitro
(Bruneton 2008). Thus, in this context, two
Phenolic compounds form an abundant and large
saponins, one of arginoside type and another of
group containing in their structure at least one
barrigenol family were screened from the
phenolic hydroxyl ring, exclusively derived from
Analyiticon Discovery compound collection
the shikimate/phenylpropanoid and/or the poly-
housed at the Broad Institute of Harvard and
ketide pathway, and deprived of nitrogen-based
MIT; they inhibited C. albicans biofilm forma-
functions. This group is divided in numerous
tion at concentrations below the MIC (10 and
classes such as phenols, coumarins, lignans,
20 g/ml respectively); the obtained inhibition
flavonoids, anthocyanins, tannins, quinines,
level was comparable to that caused by
stilbenods (Bruneton 2008). Their activity
caspofungin and one of the tested compounds
against fungal biofilms has been widely
visibly reduced the number of C. albicans
investigated. For example, Shahzad
hyphae in the culture. Without hemolytic activity
et al. recently investigated the anti-biofilm activ-
at 100 g/ml, saponins may provide a promising
ity of 14 polyphenols. Results showed that both
source of new C. albicans anti-biofilm agents
pyrogallol, available in low amounts in cocoa
(Coleman et al. 2010).
products, coffee and beer and curcumin, avail-
Saponin-rich fractions from Medicago sativa
able in common foods such as dried turmeric and
(aerial parts and roots) and Saponaria officinalis
curry power, displayed activity against
(used as a well-known source of plant saponins)
C. albicans biofilms, without disrupting the bio-
inhibited C. albicans yeast-to-hypha transition,
film or directly affecting the cellular structure.
limited hyphal development, reduced yeast adhe-
Curcumin displayed superior anti-biofilm activ-
sion and biofilm formation, and eradicated
ity, significantly inhibiting initial cell adhesion
mature (24 h) Candida biofilms (Sadowska
following pre-coating (p <0.01), biofilm growth
et al. 2014).
(p <0.05) and gene expression (p <0.05) and
Another plant showed an interest against
could constitute an opportunity for the develop-
C. albicans: Solidago virgaurea (Goldenrod).
ment of oral care products (Shahzad et al. 2014).
Its water extract, rich in saponins (0.7 or
The freeze-dried fruit rich in polyphenols of
0.95 mg/ml depending the subsp virgaurea or
Lonicera caerulea (blue honeysuckle) and its
alpestris), decreased C. albicans yeast-to-hypha
phenolic fraction reduced biofilm formation and
transition and strongly inhibited both biofilm
cell adhesion to artificial surfaces of
growth and already formed 18 h old biofilms
C. parapsilosis (Palkova et al. 2008).
(Chevalier et al. 2012).
112 M. Girardot and C. Imbert

2.2.1 Stilbenods mature 48 h old biofilm. Green tea polyphenols,

Stilbenods are phenolic compounds with two especially tannins, have been shown to signifi-
benzenes separated by an ethane or ethene bridge cantly decrease C. albicanss ability to grow and
(stilbene, bibenzyls, bisbibenzyls) (Bruneton sustain biofilms (Evensen and Braun 2009).
2008). As they often displayed activity against These authors showed that the treatment of
planktonic cells, their anti-biofilm activity was yeasts with 1.0 mol/l of ()epigallocatechin-
studied. Some bibenzyls and bisbibenzyls 3-gallate (EGCG), the most abundant tannin,
extracted from liverwort plants (bryophytes) caused a 75 % reduction of viable cells during
have been screened for their anti-C. albicans 48 h of biofilm formation. Already formed
properties. Among them, two natural biofilms treated with EGCG were also reduced
compounds, isoriccardin C and BS-34 were by 80 %. The same concentrations of epigallo-
found to inhibit yeast-to-hypha transition and catechin and epicatechin-3-gallate also
biofilm formation in a dose-dependent manner demonstrated similar biofilm inhibition. Regard-
which was consistent with an elevated farnesol ing the mechanism of action, these authors
production and Dpp3 expression (Zhang showed that yeast treatment with catechine
et al. 2011). In the same way, riccardin D, a induced a reduction of in vivo proteasome activ-
macrocyclic bisbibenzyl isolated from Chinese ity contributing to cellular metabolic and struc-
liverwort Dumortiera hirsute was shown to dis- tural disruptions causing anti-biofilm activity
play both prophylactic and therapeutic effects (Evensen and Braun 2009).
against C. albicans biofilm formation in a dose- Some proanthocyanidins, demonstrated an
dependent manner in vitro and in vivo; SEM and efficacy against biofilms of Candida sp. For
confocal laser scanning microscope (CLSM) instance, several studies reported the anti-biofilm
showed that biofilm morphology was remarkably activity of A-type proanthocyanidins obtained
altered after riccardin D treatment, especially from Cranberry (Vaccinium macrocarpon).
hyphal development by inhibiting the Ras- These compounds prevented biofilm formation
cAMP-Efg pathway which delayed filamentation and reduced C. albicans adhesion to oral epithe-
(Cheng et al. 2009; Li et al. 2012). lial cells, saliva-coated acrylic resin discs, poly-
styrene and silicone. They also reduced
C. glabrata adhesion to polystyrene. At
2.2.2 Tannins
concentrations of at least 16 mg/l, they were
Tannins were defined as phenolic natural
shown to significantly reduce biofilm formation
products that precipitate proteins from their
of C. albicans and be additive in combination
aqueous solutions (Mole and Waterman 1987).
with conventional antifungal agents (flucona-
In plants, two groups of tannins are distin-
zole, amphotericin B and caspofungin) (Feldman
guished: hydrolyzable tannins (esters of a sugar
et al. 2012; Girardot et al. 2014; Rane
and of a variable number of phenolic acid
et al. 2014).
molecules; can be quoted ellagitannins or
The anti-biofilm activity of proantocyanidin
gallotannins) and condensed tannins
fractions obtained from Stryphnodendron
(or proanthocyanidins which are polymeric
adstringens stem bark was also recently shown
flavans). These compounds have been shown to
against C. albicans. At doses considered as safe
present antioxidant, enzymatic inhibition,
(>31.25 > 1000 g/ml), some proantocyanidin
antidiarrheal and mostly antiseptic properties
fractions inhibited biofilm formation and reduced
hence their likely interest against fungal biofilm
the metabolic activity of 24 h old biofilm cells.
(Bruneton 2008).
Interestingly, these fractions also reduced the
A study conducted by Evensen and Braun,
metabolic activity of free cells detached from
highlighted that physiologic concentrations of
the biofilm, indicating that they could contribute
tannins not only prevented biofilm formation,
to inhibit infection dissemination (Luiz
but also reduced the C. albicans population of a
et al. 2015).
Natural Sources as Innovative Solutions Against Fungal Biofilms 113

Finally, the methanolic extract of pomegran- Chinese herb Scutellaria baicalensis, inhibited
ate (Punica granatum L.) rich in ellagic acid the formation of C. albicans biofilms in a dose-
displayed anti-biofilm activity against dependent manner (inhibition of 70 % at
C. albicans: it was shown to both inhibit biofilm concentrations between 4 g/ml and 32 g/ml).
formation and disrupt pre-formed biofilms when It was active against different biofilm growth
used at 250 g/ml; it also inhibited yeast-to- stages, with 89 % and 52 % inhibition when
hypha transition. Other results showed that pure added at 0 h and 24 h of the incubation period,
ellagic acid was active against biofilm formation respectively. Baicalein may act by decreasing the
at concentrations lower than 40 g/ml which cell surface hydrophobicity as suggested by
could explain pomegranate extract activity results showing a downregulation of CSH1
(Bakkiyaraj et al. 2013). (a gene encoding the cell surface
hydrophobicity-associated protein) in baicalein
2.2.3 Quinones treated fungal cells (Cao et al. 2008).
Some quinones could also help to fight fungal A synthetic isopentenyloxychalcone of natu-
biofilms. These compounds that contain oxygen ral origin, 4-hydroxycordoin, was shown to
are essentially the oxidized homologs of aro- inhibit two important virulence factors of
matic derivatives. Some of them previously C. albicans. Indeed it strongly inhibited biofilm
demonstrated laxative or antibacterial and fungi- formation at 20 g/ml, while concentrations
cidal properties (Bruneton 2008). Purpurin which ranging from 50 to 200 g/ml significantly
is a natural red anthraquinone pigment com- reduced yeast-to-hypha transition (Messier
monly found in madder root (Rubia tinctorum et al. 2011). More recently the same research
L.), blocked C. albicans yeast-to-hypha transi- team investigated licochalcone A which is pre-
tion when used at a sub-lethal concentration dominantly present in licorice. This flavonoid
(3 g/ml) (Tsang et al. 2012; 2013). This used at 0.2 g/ml induced a 59 % inhibition of
non-toxic compound to Human cells also the C. albicans biofilm formation and a cytotox-
inhibited C. albicans and C. dubliniensis biofilm icity study showed that no toxicity was observed
formation and reduced the metabolic activity of at this concentration (Messier and Grenier 2011).
24 h old biofilms in a concentration-dependent Very interestingly, other flavonoids, extracted
manner. C. albicans or C. dubliniensis biofilms from Moringa oleifera seed coat, were recently
treated by purpurin appeared scanty and exclu- showed to inhibit early attachment, promote dis-
sively consisted of blastospore aggregates (Tsang ruption of 14 h old preformed biofilms and
et al. 2012; 2013). Regarding the mechanism of decrease metabolic activity of C. albicans
action, purpurin was shown to downregulate the biofilms, all of that being obtained at
expression of hypha-specific genes (ALS3, concentrations without toxicity or mutagenicity
ECE1, HWP1, HYR1) and the hyphal regulator (Onsare and Arora 2015).
RAS1. Purpurin triggered metacaspase-
independent apoptosis in C. dubliniensis biofilms
(Tsang et al. 2012; 2013). 2.3 Peptides

2.2.4 Flavonoids Proteins and peptides, primary metabolites, with

Some universal plant pigments, called flavonoids anti-microbial activity are produced by a wide
and possessing a basic structural element namely variety of organisms in order to protect them-
the 2-phenylchromane, previously demonstrated selves from infection (Mandal et al. 2011).
venoactive, antioxidant, enzymatic inhibitor, Some studies reported peptides with activities
antimicrobial properties (Bruneton 2008). against fungal biofilms, such as Tn-AFP1 which
Concerning an anti-biofilm activity of plant is a plant peptide, with molecular mass of
flavonoids, some examples were quoted in litera- 1230 Da. This peptide was purified from fruits
ture data. Baicalein, a flavone present in the of Trapa natans, a floating annual aquatic plant
114 M. Girardot and C. Imbert

and was able to disrupt the formation of dependent manner. Interestingly tetrandrine at
C. tropicalis biofilm in a concentration depen- 32 mg/l was able to significantly inhibit mature
dent manner; a 50 % inhibition of the metabolic biofilms (p <0.05), and here again activity
activity was observed using Tn-AFP1 at 16 g/ increased depending on this compound concen-
ml. This peptide also downregulated MDR1 and tration. These authors also investigated the anti-
ERG11 gene expression (Mandal et al. 2011). biofilm capacity against other fungal species, and
Recent results have shown that a decapeptide observed only a weak anti-biofilm activity
from the model plant Arabidopsis thaliana, against C. neoformans and no effect against
OSIP108, inhibited the biofilm formation process A. fumigatus. They hypothesized that the mecha-
of C. albicans without toxicity against various nism of action of tetrandrine could possibly be
human cell lines such as osteoblasts, mesenchy- partly linked to its ability to decrease the cell
mal stem cells and endothelial cells. Besides, surface hydrophobicity and to inhibit
OSIP108 (12.5100 M) synergistically C. albicans hyphal development through the
enhanced the activity of conventional antifungal Ras1p-cAMP-PKA pathway; this alkaloid com-
agents such as amphothericin B and caspofungin pound would thus present an interest in
against mature C. albicans biofilms (Delattin C. albicans biofilm-associated infections (Zhao
et al. 2014). et al. 2013).
Finally, a hydroxyprolin rich glycopeptide Some combinations could be interesting. For
extracted from pericarp of Datura stramonium example, berberine, a plant alkaloid from
and called datucin also displayed an anti-biofilm Hydrastis canadensis (goldenseal) combined
activity against C. albicans: the minimum bio- with miconazole were able to reduce
film eradication concentration was 2 M C. albicans biofilm formation by >91 % after
(Mandal 2012). 24 h whereas neither berberine nor miconazole
alone were active. This combination also
exhibited synergy against pre-formed
2.4 Alkaloids C. albicans biofilms (Wei et al. 2011).
Finally, matrine, one of the major alkaloid
Alkaloids are defined as an organic compound components found in sophora roots, could reduce
of natural origin, which contains a nitrogen atom, C. albicans yeast-to-hypha transition; addition-
is more or less basic, is of limited distribution, ally this compound used at 2 mg/ml would cause
and has, at low doses, marked pharmacological a 50 % decrease of the metabolic activity of
properties (Bruneton 2008). These compounds C. albicans biofilms in development after a 24 h
have been shown to perform various pharmaco- co-incubation of matrine with C. albicans cells
logical activities such as a depressant or stimu- (Shao et al. 2014).
lant of the central nervous system,
sympathomimetic or sympatholytic of the auto-
nomic nervous system, antimalarial, 2.5 Biosurfactants
antifibrillant, antitumor, local anesthetic, antimi-
crobial (Bruneton 2008). Some alkaloids are con- Biosurfactants are a surface-active heteroge-
sidered to be also active against fungal biofilms. neous group of amphiphilic molecules released
Tetrandrine is a bis-benzylisoquinoline alka- by microorganisms that exhibit surface activity
loid compound originating from several natural (Rodrigues et al. 2006). Various chemical
plant sources, including Stephania tetrandra. structures of primary metabolites, such as
Results of Zhao et al. showed that the addition glycolipids, lipopeptides, polysaccharidepro-
of tetrandrine (concentration 16 mg/l) just after tein complexes, protein-like substances,
adhesion significantly inhibited (p <0.05) the lipopolysaccharides, phospholipids, fatty acids
formation of C. albicans biofilms in a dose- and neutral lipids, have been attributed to
Natural Sources as Innovative Solutions Against Fungal Biofilms 115

biosurfactants. Due to their affinity to adhere to taken into account for an eventual future thera-
surfaces, these antimicrobial compounds are peutic use (Shuford et al. 2005).
being used as a new strategy for inhibiting the In another study, the activity of Cassia
adhesion of C. albicans in non-biological spectabilis methanol leaf extract against biofilm
surfaces (Rodrigues et al. 2006). Microbial flora formation of C. albicans was investigated using
associated to plants could also be a source of transmission electron microscopy (TEM) and
biosurfactants with anti-biofilm activity. SEM approaches. This extract used at 6.25 mg/
Endophytes help to protect their host plants by ml significantly prevented biofilm formation,
producing substances, including biosurfactants, SEM analysis revealing a reduction in adhering
which improve their natural protection against cells and biofilm development in presence of the
many environmental agents. As they are closely extract. In addition TEM analysis highlighted
linked to plants, they are included in this section some alterations in morphology and complete
instead of Sect. 4. collapse of yeast cells after 36 h of exposure to
For example, Cochis et al. demonstrated the the extract (Sangetha et al. 2009).
anti-biofilm efficacy of three biosurfactants Barbieri et al. evaluated the anti-adherent
obtained from endophyte biofilms selected from property of crude, methanol and ethyl acetate-
Robinia pseudoacacia and from Nerium olean- methanol extracts from Schinus terebinthifolius
der. Actually, C. albicans biofilm formation on and Croton urucurana on C. albicans biofilms
elastomer silicon or resin surfaces pre-coated in vitro. Results showed that S. terebinthifolius
with these biosurfactants used at low ethyl acetatemethanol and methanol extracts
concentrations (78.1 or 156 g/ml) was signifi- were the most efficient against C. albicans
cantly reduced (p <0.01) thanks to their good biofilms (p < 0.05). These extracts were
anti-adhesion activity. Interestingly these constituted by several active compounds, includ-
biosurfactants displayed a higher anti-Candida ing phenolic compounds, anthraquinones,
activity than that of chlorhexidine and a terpenoids, and alkaloids. Concerning
cytocompatibility with epithelial cells and C. urucurana extracts, higher concentrations
fibroblasts according to what was observed were needed to achieve anti-adherent activity
(Cochis et al. 2012). (Barbieri et al. 2014).

3 Anti-biofilm Compounds
2.6 Unknown or Mixed Composition
of Lichen and Algual Origin
Some literature data pointed out anti-biofilm
3.1 Lichen Origin
activities of plant extracts with various or
unspecified composition. For example, Shuford
Lichens are constituted by the association of a
et al. investigated the inhibitory activity of a
fungus (usually an ascomycete, called the
fresh garlic extract against C. albicans biofilm
mycobionte) and a green alga or a cyanobacteria
formation (treatment immediately after adher-
(called the photobionte). This symbiotic organi-
ence by concentrations ranging between 0.5 and
zation leads to the formation of lichen thalli.
1 mg/ml) and against 48 h old mature biofilms
Besides, recent molecular studies have revealed
treated for 1 h or 48 h (concentrations ranging
that lichens also present diverse microbial
between 2 and 4 mg/ml). Results showed that
communities which colonize surfaces and the
fresh garlic extract was significantly active in
inside of lichens in a biofilm-like manner. This
all cases (p <0.001). The superior activity
complex organization is at the origin of a great
observed with 1 h versus 48 h of treatment
variety of secondary metabolites, many of which
against pre-formed biofilms probably related to
are unique such as some depsides, despidones,
the half-life of the extract at 37  C and should be
dibenzofuranes or pulvinic derivatives (Grube
116 M. Girardot and C. Imbert

and Berg 2009). Besides quinones, azoles interacted synergistically to block

anthraquinones, terpenes and steroids were also C. albicans biofilm formation (Chang
found (Rundel 1978). Many secondary lichen et al. 2012).
metabolites offer protection to lichen Recently, quercetin, a dietary flavonoid
communities against other microorganisms. isolated from an edible lichen (Usnea
And some of these compounds have previously longissima), was demonstrated as strongly
demonstrated some biological activity such as suppressing the production of virulence weapons
antimicrobial (physodic, chloroatranorin, such as biofilm formation or hyphal develop-
lobastin, atranorin, olivetoric, usnic, norstictic, ment. Interestingly, treatment with quercetin
protocetraric and vulpinic acids, also increased fluconazole mediated cell death
anthraquinones), antitumoral (usnic, in fluconazole resistant C. albicans biofilms.
D-protolichesterinic and nephrosternic acids), Quercetin activity could be related to an increase
UV protectors (usnic, diffractic, barbatic acids, of farnesol production, which is known to
parietine), antioxidant (despidones, usnic, coregulate hyphal development, biofilm forma-
lecanoric acid, atranorin, orsenillic acid, tion, and virulence factor production (Singh
orcinol. . .), antiviral (usnic acid), antiprotozoal et al. 2015).
(5-propylresorcinol, isodivaricatic, divaricatic, The fungal flora inside lichens is also a source
usnic acids), smooth muscle relaxant of anti-biofilm metabolites. Pyridoxatin, a small
(fumaroprotocetraric, usnic acids, depsidones), natural product isolated from an endolichenic
inhibitors of tyrosinases (resorcinol derivatives), fungus: Tolypocladium cylindrosporum, derived
allergenic (D-usnic and evernic acids and from the lichen Lethariella zahlbruckner, could
atranorin), analgesic, antipyretic and anti- prevent the formation of biofilm. Investigations
inflammatory (diffractaic, usnic acids) (Shukla to clarify the mechanism of action revealed that
et al. 2010; White et al. 2014). Antimicrobial pyridoxatin interfered with the biosynthesis of
activity was mainly evaluated on planktonic ergosterol, which probably inhibits the cell
cells even if a few studies also focused on sessile growth of C. albicans (Chang et al. 2015).
cells. Indeed some lichen metabolites may be Similarly, diorcinol D is a natural product
useful to control biofilms formed by human obtained from an endophytic fungus: Aspergillus
pathogens as they offer protection to lichen versicolor derived from the lichen Lobaria
communities against adherent microorganisms quercizan. This compound demonstrated anti-
(Francolini et al. 2004; Lazar and Chifiriuc biofilm activity against 24 h old C. albicans
2010). Concretely, Pires et al. evaluated the biofilms at 32 g/ml, and its effect was enhanced
activity of a 48 h treatment with usnic acid by adding fluconazole (8 g/ml). In the same
against 24 h old C. orthopsilosis or way, more than 250-fold reductions of the
C. parapsilosis biofilms. They observed a 50 % MICs of fluconazole were observed when com-
reduction in the metabolic activities of the two bined with diorcinol against these mature
species biofilms using usnic acid at 3.9 g/ml; biofilms. CLSM images confirmed this activity
furthermore 31.2 and 62.5 g/ml reduced the (Li et al. 2015).
growth of the cells in 80 % respectively (Pires
et al. 2012).
Retigeric acid B, a pentacyclic triterpenoid 3.2 Algual Origin
was isolated from the lichen Lobaria kurokawae.
It demonstrated in vivo ability to inhibit Algae, photosynthetic marine or aquatic
C. albicans yeast-to-hypha transition in infected organisms, also produce active metabolites that
mice; it also inhibited in vitro adherence of are especially active against biofilms. For exam-
C. albicans to KB/VCR cells in a dose-dependent ple, Thibane et al. investigated the effectiveness
manner using concentrations ranging between against Candida biofilms of marine long chain
8 and 32 g/ml. Finally, retigeric acid B and polyunsaturated fatty acids contained in algae. It
Natural Sources as Innovative Solutions Against Fungal Biofilms 117

was found that stearidonic acid (18:4 n-3), biofilm formation, and displayed no toxicity
eicosapentaenoic acid (20:5 n-3), against humain cells up to 200 M (Wang
docosapentaenoic acid (22:5 n-3) and et al. 2012b).
docosahexaenoic acid (22:6 n-3) had an inhibi- More recently, this research team identified
tory effect on mitochondrial metabolism, two other alkaloid metabolites named
affected cellular morphology in biofilms of both shearinines D and E and produced by an
C. albicans and C. dubliniensis and significantly Alaskan-soil-derived Penicillium sp which also
inhibited biofilm biomass of C. dubliniensis exhibited anti-biofilm activity against
(Thibane et al. 2010). C. albicans. Here again the presence of these
alkaloids during both adherence step and biofilm
growth inhibited the metabolic activity of 48 h
4 Anti-Biofilm Compounds old biofilms (IC50 8.5 and 7.6 M, respec-
of Microbial Origin tively) but they blocked the outgrowth of hyphae
suggesting an effect on a relatively late stage of
Fungi and Bacteria also play an important role as biofilm development; interestingly shearinines D
producers of active compounds. They are able to and E exhibited synergistic activities with
synthesize active primary metabolites such as amphotericin B against different species of Can-
protein and peptides or glycolipids and active dida (C. albicans, C. glabrata, C. parapsilosis,
secondary metabolites such as alkaloids or C. tropicalis, C. kefyr) (You et al. 2013).
terpenes. Some literature data highlight the anti-
biofilm efficacy of some microbial compounds. 4.1.2 Terpene
Here we can again quote farnesol, a sesquiter-
pene alcohol acting as a quorum sensing mole-
4.1 Fungal Origin cule which inhibits the yeast-to-hypha transition
and compromises C. albicans biofilm formation
For a long time, fungi showed their interest as a (Hornby et al. 2001; Ramage et al. 2002).
main source of antimicrobial substances. Major Farnesol has also been shown to inhibit the for-
antifungal drugs currently employed as mation of C. dubliniensis, C. tropicalis and
echinocandins (caspofungin, micafungin and C. parapsilosis biofilms (Weber et al. 2010).
anidulafungine) were initially obtained by fer- Finally, cyclosporine A, a calcineurin inhibi-
mentation of filamentous fungi including Asper- tor with a fungal origin, was shown to exhibit
gillus sp. These fungi are also a source for new synergism with conventional antifungals
anti-biofilm compounds presented here in three (amphotericin, fluconazole, voriconazole,
non-exhaustive groups: alkaloids, terpenes and caspofungin) against C. parapsilosis species
biosurfactants/glucosides. complex. The tested combinations involving
cyclosporine A prevented biofilm formation and
4.1.1 Alkaloids showed an inhibitory effect against mature
A complex prenylated indole alkaloid named biofilms (48 h old) of C. parapsilosis sensu
waikialoid A and a polyketide metabolite, stricto, C. metapsilosis and C. orthopsilosis
waikialide A, produced by a Hawaiian-soil- (Cordeiro et al. 2014).
derived Aspergillus sp have demonstrated activ-
ity against C. albicans biofilm formation: their 4.1.3 Biosurfactants and Glucosides
presence during both adherence step and biofilm As previously mentioned, biosurfactants could
growth inhibited the metabolic activity of 48 h be used to prevent fungal adherence to non
old biofilms with IC50 values of 1.4 M and biological surfaces such as medical devices.
32.4 M respectively. Results also showed that Monteiro et al. showed that a glycolipid-type
waikialoid A inhibited yeast hyphal develop- biosurfactant produced by the yeast
ment, suggesting an effect on the early phase of Trichosporon montevideense CLOA72 used at
118 M. Girardot and C. Imbert

16 mg/ml reduced up to 87.4 % the biofilm for- formation (87 % inhibition in presence of
mation of a C. albicans strain isolated from the BL-DZ1 at 1.60 g/ml) and to disperse
apical tooth canal. Changes of cell surface C. albicans 24 h old biofilms pre-formed onto
characteristics induced by this compound could polystyrene surfaces (up to 67.2 % inhibition by
contribute to the inhibition of initial adherence of BL-DZ1 at 1.60 g/ml) (Dusane et al. 2013).
C. albicans cells. Finally this compound A cyclic lipopeptide biosurfactant constituted
presented no cytotoxic effect in mammalian by a mixture of iturin and fengycin and produced
cells (Monteiro et al. 2011). by another Bacillus: B. amyloliquefaciens
Other primary metabolites: polyketide reduced C. albicans cell surface hydrophobicity.
glycosides from Bionectria ochroleuca, a fungus It hindered yeast-to-hypha transition and reduced
cultured on Cheerios breakfast cereal exhibited the mRNA expression of hyphae-specific genes
potent biofilm inhibition against C. albicans HWP1 and ALS3. The presence of this
(Wang et al. 2014a). biosurfactant during biofilm growth (for 48 h)
inhibited C. albicans biofilm formation in the
range of 46100 % in a concentration-dependent
4.2 Bacterial Origin manner (between 1 and 6 mg/ml). Furthermore,
this compound used at similar concentrations
Similarly to fungi, bacteria also demonstrated dislodged 25100 % of 48 h old biofilm
their importance as producers of antimicrobial preformed on polystyrene surfaces (Rautela
substances. For example a current major antifun- et al. 2014).
gal drug: amphotericin B was initially produced Gramicidins were first identified as antibiotic
by filamentous bacteria of Streptomyces sp molecules produced by a soil-derived B. brevis
genus. Bacteria can also synthesize new anti- strain but could also have an anti-adherent poten-
biofilm compounds as active peptides, proteins tial. In a study of Yala et al. gramicin A was
and biosurfactants. A study conducted by Sharma covalently bound to cystamine self-assembled
and Srivastava focused on the anti-biofilm activ- monolayers on gold surface. Authors showed
ity of a spent culture filtrate of an environmental that the adsorbed gramicidin was able to reduce
isolate of lactic acid bacterium, Lactobacillus by 90 % the number of culturable C. albicans
plantarum. They observed that C. albicans bio- cells attached to the surface (Yala et al. 2011).
film formation was significantly reduced Mutanobactin A is a hybrid polyketide-
(p <0.05) when the filtrate was present (Sharma nonribosomal peptide metabolite produced by
and Srivastava 2014). Streptococcus mutans. This compound and two
Tyrocidines are cationic cyclodecapeptides new analogues, mutanobactins B and D have
produced by Bacillus aneurinolyticus. Troskie been investigated and their ability to inhibit the
et al. observed that the formation of C. albicans formation of C. albicans biofilms was
biofilm in the presence of tyrocidines for 24 h highlighted (IC50 up to 5.3 M) (Wang
caused a biofilm reduction suggesting a preven- et al. 2012a).
tive interest. They also observed that tyrocidines A study performed by Busscher et al. then
disrupted the membrane integrity of 24 h old completed by Rodrigues et al. reported that
C. albicans biofilm cells and demonstrated pro- biosurfactants produced by S. thermophilus sig-
nounced synergistic biofilm-eradicating activity nificantly reduced initial C. albicans and
in combination with amphotericin B and C. tropicalis adhesion. These biosurfactants
caspofungin (Troskie et al. 2014). were mixtures of various compounds with a
A protein designed as BL-DZ1 was obtained glycolipid-like component being the most active
from a tropical marine strain of B. licheniformis (Busscher et al. 1997; Rodrigues et al. 2006).
isolated from the surface of a green mussel, A crude biosurfactant from ten lactobacilli
Perna viridis. The characterization of BL-DZ1 isolated from Egyptian dairy products also
demonstrated its ability to decrease biofilm exhibited anti-adhesive activity against
Natural Sources as Innovative Solutions Against Fungal Biofilms 119

C. albicans (Zakaria Gomaa 2013) as well as a 5.1 Resin, Proteins and Peptides
biosurfactant produced by Lactobacillus brevis,
which was shown to reduce both fungal adhesion Numerous properties including antimicrobial,
and biofilm formation of C. albicans on silicone anti-inflammatory, immunostimulatory or anti-
elastomer surfaces (Ceresa et al. 2015). septic effects have been previously described in
A di-rhamnolipid biosurfactant produced literature for a complex resin produced by honey
from Pseudomonas aeruginosa was able to dis- bees and called propolis (Capoci et al. 2015).
rupt C. albicans biofilm formed on polystyrene Besides a study performed using 30 clinical
surfaces by modifying the extracellular matrix C. albicans strains demonstrated that an ethanol
(Singh et al. 2013). extract of propolis used at 273 g/ml inhibited
Pseudofactin II is a biosurfactant cyclic ( p  0.05) the biofilm formation of 93.34 % of
lipopeptide secreted by P. fluorescens and strains (Capoci et al. 2015). Another study con-
obtained from freshwater from the Arctic Archi- firmed the ability of propolis to reduce
pelago of Svalbard. This biosurfactant at C. albicans biofilm formation in vitro and also
0.25 mg/ml has been shown by CLSM identified some genes involved in cell adhesion,
observations to reduce C. albicans adhesion to biofilm formation or filamentous growth that
glass, polystyrene and silicone surfaces. On poly- contributed to propolis tolerance (de Castro
styrene surfaces, this surfactant used at et al. 2013).
0.0350.500 mg/ml caused adhesion inhibition Other compounds of interest were mucins
in the range of 4599 % (Janek et al. 2012). obtained from pig stomachs. These biopolymers,
Finally, another biosurfactant, a glycolipid heavily glycosylated proteins, were the main
composed of glucose and palmitic acid and pro- gel-forming constituents of mucus. Mucins may
duced by a tropical marine strain of Serratia suppress C. albicans surface adhesion by
marcescens isolated from the hard coral, downregulating ALS1 and ALS3 genes and
Symphyllia sp. was also investigated; its ability decreasing its biofilm formation. Besides, a new
to both prevent adhesion (up to 76 % and 82 % at oval-shaped form of cells was observed which
50 and 100 g/ml) and disrupt 24 h old was less able of stably integrating into an
preformed biofilms of C. albicans (polystyrene emerging biofilm. The yeast-to-hypha transition
surfaces; up to 55 % at 50 g/ml) was appeared to be also perturbed (Kavanaugh
demonstrated. CLSM and SEM confirmed the et al. 2014).
effective removal of biofilms from glass surfaces A 26-amino-acid truncated form of the
(Dusane et al. 2011). 34-amino-acid cathelicidin-related peptide was
produced by murine pancreas and named P318;
its use at 0.15 M was shown to inhibit
C. albicans biofilm formation (De Brucker
5 Anti-Biofilm Compounds
et al. 2014).
of Animal Origin
A study performed by Sengupta et al. revealed
that lactoferricin B, an antimicrobial peptide
Literature data highlighted that insects such as
derived from a milk protein, could improve bio-
bees or wasps and mammals such as pigs, mice,
film susceptibility to antifungal agents and be a
cows or chickens also produced anti-biofilm
promising candidate as an antibiofilm-antifungal
compounds. These compounds are of natural
additive in lens care solution against three com-
origins meaning without human intervention
mon keratitis-associated fungal pathogens:
(resin, proteins and peptides) or of artificial
C. albicans, Fusarium solani and Aspergillus
origins requiring human action (production of
fumigatus (Sengupta et al. 2012).
antibodies). Some of them will be developed
Finally, polybia-MPI, an amphipathic cationic
peptide (14 amino-acids) originally isolated from
120 M. Girardot and C. Imbert

the venom of a social wasp Polybia paulista was biofilm formation of C. albicans in nutrient-poor
recently investigated for its anti-C. glabrata condition (medium without serum), but inhibi-
activity (Wang et al. 2014b). These authors tion was slightly restored in medium containing
underscored the anti-biofilm activity of polybia- 10 % serum (Fujibayashi et al. 2009). According
MPI : the first 24 h of C. glabrata biofilm forma- to authors, anti-CA IgY may be considered as a
tion were inhibited in a dose-dependent manner prophylactic immunotherapy under limited
in presence of polybia-MPI (864 M) (Wang conditions (Fujibayashi et al. 2009; Ibrahim
et al. 2014b). et al. 2008). A study has recently completed
these results, demonstrating an anti-adhesion
activity of anti-CA and anti-C. glabrata IgY
5.2 Antibodies against C. albicans and C. glabrata cells to den-
ture base material (Kamikawa et al. 2014).
Another strategy to fight Candida biofilm may be Furthermore, bovine milk produced by
the use of antibodies. Nowadays, cell-surfaces, immunized cow was studied, and contained
cell walls, and secreted proteins are targeted by anti-C. albicans immunoglobulin A antibodies.
many antifungal therapies (Mishra et al. 2015). This milk decreased adherence of C. albicans to
Antibodies are believed to stimulate anti-Can- silicone in vitro. However, this effect was not
dida activity by different mechanisms, such as replicated in a small clinical pilot study
inhibition of adhesion and neutralization of (Hodgkinson et al. 2007; Holmes et al. 2012).
virulence-related antigens (Mishra et al. 2015).
Recently, these authors found that a monoclonal
antibody (MAb 7D7) generated in mice against
C. albicans biofilm cell surface antigen 6 Anti-Biofilm Compounds
(47.2 kDa) could prevent C. albicans adhesion of Human Origin
and biofilm formation, although MAb 7D7 activ-
ity appeared to be strain dependent (Mishra Finally, even Humans appear to be a promising
et al. 2015). source of compounds with anti-biofilm activity
Another study presented the activity of a poly- especially Human-derived peptides and proteins.
clonal serum obtained after immunization of First, intuitively, Human-derived antimicro-
rabbits with a peptide corresponding to a frag- bial peptides should have reduced toxicity.
ment of the Candida antigen CR3-RP. Authors Rossignol et al. investigated a 18-amino-acid
observed that ability of C. albicans to adhere to cationic, tryptophan-rich ApoEdpL-W peptide
buccal epithelial cells and to form biofilms were derived from human ApoE apolipoprotein
reduced by up to 35 % (p <0.001), and 28 % (Rossignol et al. 2011). This peptide
(p <0.001) respectively, in presence of the stud- (concentrations ranging between 2.5 and
ied serum. Changes in biofilm thickness and 20 M) added once C. albicans was adhered to
integrity were also observed. The Candida anti- polystyrene (early-stage biofilm), inhibited the
gen CR3-RP may have the potential for vaccine biofilm development. In addition, ApoEdpL-W
development (Bujdakova et al. 2008). adsorbed on polyurethane and polydimethyl-
Additionally, polyclonal anti-C. albicans siloxane substrates partially prevented the forma-
antibodies (anti-CA IgY) in chicken egg yolks tion of biofilms by promoting biofilms that had a
were proved to significantly reduce the adher- strong tendency to detach from polymer,
ence of C. albicans, C. dubliniensis and suggesting that this peptide could be promising
C. tropicalis to human buccal epithelial cells coating for medical devices. Unfortunately, a
and human pharynx carcinoma cells in a dose- significantly lower activity was observed against
dependent manner (Fujibayashi et al. 2009; mature biofilms. This study also suggested that
Ibrahim et al. 2008). Fujibayashi et al. also ApoEdpL-W mode of action was dependent
observed that anti-CA IgY (2 mg/ml) inhibited upon vacuolar targeting (Rossignol et al. 2011).
Natural Sources as Innovative Solutions Against Fungal Biofilms 121

Histatins, a family of basic peptides secreted involving dermatophyte fungi as Trichophyton

by the major salivary glands in humans, espe- or yeasts such as Malassezia sp also involved in
cially histatin 5, were investigated and some infection. Up to now, investigations have
demonstrated an ability to inhibit both mainly focused on the anti-adhesion or anti-
C. albicans biofilm formation and C. albicans maturation activity of natural compounds. It
and C. glabrata mature 48 h old biofilms devel- would be of course essential to characterize
oped on denture acrylic (Konopka et al. 2010; components able to inhibit dispersion or to erad-
Pusateri et al. 2009). icate already formed biofilms. Anyway, all these
However a recent study also reported that a natural sources are a huge reserve for new drugs
large glycofragment of the Msb2 surface protein and are worth exploring in order to develop new
was released by C. albicans into the growth envi- anti-biofilm strategies.
ronment, and protected against histatin-5 activity
(Swidergall et al. 2013). Besides, it has been Acknowledgments The authors would like to acknowl-
shown that histatins were rapidly degraded edge Mrs Deborah Bell Grascoeur for revising the
English text.
in vivo, limiting their interest as therapeutic
agents (Tati et al. 2014). A conjugate peptide
was constructed using spermidine linked to the
active fragment of histatin-5 (Tati et al. 2014). References
This component appeared significantly more
effective in killing C. albicans and C. glabrata Alviano WS, Mendonca-Filho RR, Alviano DS
through the biofilm development than histatin-5 et al (2005) Antimicrobial activity of Croton cajucara
Benth linalool-rich essential oil on artificial biofilms
used alone. Finally, topical application to tongue and planktonic microorganisms. Oral Microbiol
surfaces of immunocompromised mices caused Immunol 20(2):101105
reduction of C. albicans colonies recovered from Anghel I, Grumezescu AM, Holban AM et al (2013a)
tongue tissues, highlighting the potential of this Biohybrid nanostructured iron oxide nanoparticles
and Satureja hortensis to prevent fungal biofilm devel-
new class of peptides (Tati et al. 2014). opment. Int J Mol Sci 14(9):1811018123
Anghel I, Holban AM, Andronescu E et al (2013b) Effi-
cient surface functionalization of wound dressings by
7 Conclusion a phytoactive nanocoating refractory to Candida
albicans biofilm development. Biointerphases 8(1):12
Bakkiyaraj D, Nandhini JR, Malathy B et al (2013) The
Nature shows a crucial interest in the fight anti-biofilm potential of pomegranate (Punica
against fungal biofilm. Literature data are numer- granatum L) extract against human bacterial and fun-
ous concerning original compounds of various gal pathogens. Biofouling 29(8):929937
Barbieri DS, Tonial F, Lopez PV et al (2014)
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A lower airways, 34
ABC. See ATP-binding cassette (ABC) medical devices, 5
AFM. See Atomic force microscopy (AFM) microorganisms, 1
AFST. See Antifungal susceptibility testing (AFST) pharmacological inhibition, Hsp90, 7
A. fumigatus MDR (AfuMDR), 6 polymicrobial, 7
Agglutinin-like sequence (ALS) family, 95 prokaryote and eukaryote, 8
Algual origin, 116117 transcriptional and metabolomics, 8
ALS family. See Agglutinin-like sequence (ALS) family transcription factors, 2
AMN. See Anti-mannan antibody (AMN) triazoles, 7
Amphogel, 98 upper airways, 23
Animal origin wounds, 45
antibodies, 120 Aspergillus sp., 106, 111, 117, 121
resin, proteins and peptides, 119120 A. fumigatus, 3, 2526, 40
Anti-biofilm compounds A. nidulans, 3
algual origin, 116117 Atomic force microscopy (AFM), 23
alkaloids, 114 ATP-binding cassette (ABC), 6, 41
animal origin, 119120
bacterial origin, 118119 B
biosurfactants, 114115 Bacillus subtilis, 22
chemical structure, 121 Bacterial origin, 118119
classification, 106 Bacterium-fungus interactions
fungal origin chemical interaction, 1617
alkaloids, 117 ecosystem, 15
biosurfactants and glucosides, 117118 metabolic interactions, 1718
terpene, 117 physical interactions, 1516
human origin, 120121 BAL. See Bronchoalveolar lavage (BAL)
lichen origin, 115116 BCA. See Bicinchoninic acid (BCA)
peptides, 113114 Bicinchoninic acid (BCA), 23
phenolic compounds (see Phenolic compounds) Biofilm related infections
terpenoids (see Terpenoids) alkylphospholipid, 9899
unknown/mixed composition, 115 ALS, 95
Antifungal susceptibility testing (AFST), 65 anti-fungal medications, 98
Anti-mannan antibody (AMN), 6768 Apergillus and Fusarium, 99101
Aspergillus biofilms bench-work research, 98
A. fumigatus, 7 biology and resistance, 97
AmB and azole agents, 8 EPS, 96
antifungal treatments, 56 eukaryotic fungi, 99
broad-spectrum antibiotics, 1 flufenamic acid, 98
and Candida albicans, 2 gene MET3, 96
ECM, 67 human commensal organism, 95
efflux pumps, 6 invasive candidiasis, 96
glucan rich polymeric matrix, 2 miltefosine, 98
influence filamentation, 2 patients with candidemia, 98
limb, 7 physicians, 97

128 Index

Biofilm related infections (cont.) E

prokaryotic bacteria, 99 EAPCRI. See European Aspergillus PCR Initiative
therapy, American and European guidelines, 99 (EAPCRI)
thigmotropism, 96 ECM. See Extracellular matrix (ECM)
urinary and intravascular catheters, 96 ECVs. See Epidemiological cutoff values (ECVs)
Biosurfactants, 114115 eDNA. See Extracellular DNA (eDNA)
Broad-spectrum antibiotics, 1 EORTC. See European Organization for Research and
Bronchoalveolar lavage (BAL), 69 Treatment of Cancer (EORTC)
Epidemiological cutoff values (ECVs), 74
C Epigallocatechin-3-gallate (EGCG), 112
C. albicans germ tube antibody (CAGTA) assay, 68 ESCMID. See European Society of Clinical Microbiology
Candida sp. and Infectious Diseases (ESCMID)
anti-Candida biofilm activity, 109 European Aspergillus PCR Initiative (EAPCRI), 72
C. albicans, 2, 2627, 40 European Organization for Research and Treatment of
biofilm, 106 Cancer (EORTC), 67
keystone organisms theory, 18 European Society of Clinical Microbiology and Infectious
oral cavity, 1314 Diseases (ESCMID), 67
oral ecology, 14 Exopolymeric substances (EPS), 96
oral fungi, 1415 Exopolysaccharide (EPS), 22
CR3-RP, 120 Extracellular DNA (eDNA), 7, 40
human pathogens, 111, 115 Extracellular matrix (ECM), 67
Candidiasis, Clinical Practice Guidelines for the Aspergillus fumigatus, 39, 40
Management, 97, 99 biofilm-embedded cells, 39
Central line-associated BSI (CLABSI), 72 Candida albicans, 39, 40
Central venous catheters (CVC), 98 chemical composition, 39
Cerebrospinal fluid (CSF), 69 Cryptococcus neoformans, 39
Chlorhexidine, 86 Candida parapsilosis, 39
CLABSI. See Central line-associated BSI (CLABSI) Candida tropicalis, 39
Clinical Microbiology and Infectious Diseases, 100 drug diffusion, 39
Confocal laser scanning microscope (CLSM), 112 efflux-pumps activity, 41
Cryptococcus neoformans, 26 extracellular polymeric substance (EPS), 38, 39
CSF. See Cerebrospinal fluid (CSF) in vitro and in vivo models, 39
CVC. See Central venous catheters (CVC) microbial biofilms, 38
Cystic fibrosis (CF), 25 spatial heterogeneity, 39
Czapek medium, 52
D Fungal biofilms
Deep-seated candidiasis (DSC), 6465 alginate lyase, 42
Diagnosis alginate oligomer and OligoG, 42
Aspergillus infections anti-biofilm compounds (see Anti-biofilm
culture approaches, 6970 compounds)
nonculture approaches, 7072 Aspergillus fumigatus, 2526, 38
biofilm-associated bacterial infections, 76 Bacillus subtilis, 22
biofilm formation, 64 biofilm-related invasive fungal diseases, 38
bloodstream and deep tissues, 64 Candida albicans, 2627, 38
Candida infections Candida yeasts, 106
AFST, 65 Cryptococcus neoformans, 26
DSC, 65 definition, 21
entities, 6465 development of, 106
FilmArray BCID panel, 65 DNase treatment, 42
Luminex xTAG fungal assay, 65 drug resistance, 2829
MALDI-TOF MS system, 65, 66 drug tolerance, 4243
nonculture approaches, 6668 extracellular matrix components, 2930
PNA FISH and multiplex approaches, 6566 extracellular polymeric matrix, 37
candidemia/invasive candidiasis, 64 fungal matrix, 2324
clinical mycology laboratory, 64 fungal resistance mechanisms, 38
invasive fungal disease, 64 host immune cells and antifungal drugs, 37
DSC. See Deep-seated candidiasis (DSC) mechanisms, 4142
Index 129

mucolytic agents, 42 Mass spectrometry (MS), 23

natural components, 105106 Matrix-assisted laser desorption ionization time-of-flight
PGA, 22 mass spectrometry (MALDI-TOF MS), 65, 66, 70
physical barrier, 2122 MDR1. See Multidrug resistant transporter genes (MDR1)
planktonic cells, 38 Metabolic interactions, 1718
prophylactic and curative, 106 MFS. See Major facilitator superfamily (MFS)
Saccharomyces cerevisiae, 2425 MGCx. See Mannan-glucan complex (MGCx)
Staphylococcus aureus, 22 MS. See Mass spectrometry (MS)
Vibrio cholerae, 22 MSD. See Mass selective detector (MSD)
Fungal origin Multidrug resistant transporter genes (MDR1), 97
alkaloids, 117
biosurfactants and glucosides, 117118 N
terpene, 117 Natural substances, 110
Fungi isolation NCAC species. See Non-Candida albicans Candida
biofilm production, 7274 (NCAC) species
water NMR. See Nuclear magnetic resonance (NMR)
asexual/anamorph, 53 Non-Candida albicans Candida (NCAC) species, 84
Aspergillus genus, 53 Nosocomial blood stream infection, 96
black yeasts, 54 Nuclear magnetic resonance (NMR), 23, 97
DNA sequences, 53
filamenteous fungi, 53 O
fungal colonization, 55 Oral Candida biofilms
fungal contamination, 55 biomaterials, 83
in hemodialysis units, 5455 candidal biofilms, 84
hospital and community environments, 54 Candida species, 83
microbiological quality, 55 denture surfaces, 84
non-standardized methods, 55 fungaemia, 84
pathogenic species, 5354 human oral infection, 84
quantitative evaluation, 53 oral cavity, 83
traditional phenotype-based methods, 53 oral disinfectants (see Oral disinfectants)
scanning electron microscopy images, 85
G sequence, 84
Galactosaminogalactan (GAG), 25, 40, 101 Oral cavity, 1314
Oral disinfectants
H antifungal agents, 85
Haemophilus influenzae, 3 benzalkonium chloride, 8687
Human origin, 120121 biotic and abiotic surfaces, 85
chlorhexidine, 8586
I LLLT, 90
Infectious Disease Society of America, 100 microwave irradiation, 90
Invasive fungal disease (IFD), 64 nystatin, 87
Invasive fungal infections (IFIs), 64 povidone iodine, 89
silver nanoparticles, 8789
K Oral ecology, 14
Keystone organisms theory, 18
L PDT. See Photodynamic therapy (PDT)
Lichen origin, 115116 Peptide nucleic acid fluorescence in situ hybridization
Low-level laser therapy (LLLT), 90 (PNA FISH), 65
Luminex xTAG fungal assay, 65 PGA. See Poly-DL-glutamic acid (PGA)
Phenolic compounds
M Candida albicans biofilms, 111
Macromolecules, 23 flavonoids, 113
Major facilitator superfamily (MFS), 6 Lonicera caerulea, 111112
MALDI-TOF MS. See Matrix-assisted laser desorption quinones, 113
ionization time-of-flight mass spectrometry shikimate/phenylpropanoid, 111
(MALDI-TOF MS) stilbenoids, 112
Mannan-glucan complex (MGCx), 27 tannins, 112113
Mass selective detector (MSD), 23 Phenzines, 99101
130 Index

Photodynamic therapy (PDT), 90 Cymbopogon nardus, 110

Physical interactions, 1516 Cyperus articulatus, 109
Planktonically-growing isolate tests, 7475 Melaleuca alternifolia, 109
Platelia GM assay, 71 Mentha piperita, 109
PNA FISH. See Peptide nucleic acid fluorescence in situ minimal inhibitory concentration, 109
hybridization (PNA FISH) Ocimum americanum, 106
PNAG. See Poly-N-acetyl glucosamine (PNAG) Piper claussenianum, 109
Poly-DL-glutamic acid (PGA), 22 plants, 106
Poly-N-acetyl glucosamine (PNAG), 22 Rosmarinus officinalis, 110
Porphyromonas sp. Satureja hortensis, 110
P. aeruginosa, 3, 4 SEM, 106
P. gingivalis, 18 terpinen-4-ol, 110111
Thymbra capitata, 109
Q saponins, 111
Quinidine drug resistance (QDR), 97 T2 magnetic resonance (T2MR), 68
Transmission electron microscopy (TEM), 114
R Triazoles, 7
Ras1p-cAMP-PKA pathway, 114 Tricarboxylic acid cycle (TCA), 43
Reactive oxygen species (ROS), 43
Rhinosinusitis, 2 U
Universal ribosomal DNA region ITS, 53
Saccharomyces cerevisiae, 2425, 30 V
Saponins, 111 Vibrio polysaccharide (VPS), 22
SAXS. See Small-angle X-ray scattering (SAXS)
Scanning electron microscope (SEM), 104, 112 W
Scedosporium inflatum infection, 7 Water
Serum BDG assay, 67 aerocontamination, 50
Sessile/biofilm-growing isolate testing, 7576 aerosol inhalation, 55
Sinusitis, 2 A. fumigatus, 56
Small-angle X-ray scattering (SAXS), 23 air quality control in hospitals, 58
Staphylococcus aureus, 3, 16 anti-biofilm strategies, 59
Stilbenoids, 112 Aspergillus conidia, 50
biofilms, 5051
T dental unit waterlines, 5657
Tannins, 112113 environmental origin, 50
TCA. See Tricarboxylic acid cycle (TCA) epidemiological information, 58
Terpenoids fungi isolation (see Fungi isolation, water)
essential oils and terpenes hydric fungal contamination, 50
anti-adherent and anti-biofilm, 110 microorganisms, 50
Boesenbergia pandurata, 109 potential vector, 58
Candida albicans, 106 prophylactic and curative actions, 5758
Cinnamomum zeylanicum, 106 Pseudallesheria sp., 50
Coriandrum sativum, 106 rigorous control, 50
Croton cajucara, 106, 109