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INTRODUCTION TO ANAEROBIC BACTERIA Facultative Anaerobe: grow in neutral aerobe

Dr. Ayochok/ February 23, 2017 or anaerobe conditions
PRELIM: LAB PRACTICALS Microaerophilic: require oxygen as terminal
electron acceptor to grow aerobically (21% O2)
Isolation of Anaerobes however it cannot be cultivated aerobically
Aerobes: require molecular O2 as terminal electron Minimal growth anaerobically
acceptor Gram (+) bacilli
Anaerobic: use fermentative pathways (organic Spore formers (Clostridium)
acids, alcohols, and other obligate anaerobes) Others: non-spore formers
Strict obligate : O2 beyond 0.5% = unable to
grow EPIDEMIOLOGY
Moderate obligate: tolerates O2 @ 2-8% Endogenous infections
Aerotolerant anaerobes
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Came from normal flora (i.e. C. difficile
gastroenteritis)
Indigenous microflora
Skin: Propionibacterium, Peptostreptococcus
There is an existing normal flora, of which the
anaerobes are part, if you disturb the milieu or
the normal environment, you are giving a chance
for the anaerobes to proliferate, when they
proliferate, that is the time they become
pathogenic.
Although not all of them are evil. B. fragilis
contributes to the normal host physiology by
production of Vit. K. and it also helps
deconjugate bile acids
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Exogenous infections
From outside the body
Tetanus, Botulism, C. perfringens
ROLE OF ANAEROBES
Prevent colonization and infection by pathogens
Bacterial interference through elaboration of toxic
metabolites, low pH, depletion of nutrients
Interference with adhesion
Whenever there is an abcess, lahat ng klase ng
abcess, kahit saan sa katawan brain abcess, liver
abcess, empyema, it is normal to suspect na
anerobic in nature
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Elaboration of toxins (worsens the already

VIRULENCE FACTORS
Attachment and adhesion: polysaccharide capsule
and pili
Invasion: alteration in host tissue (trauma or a pre
existing co-morbid condition)
Aerotolerance
Establishment of infection:
Polysaccharide capsule (B. fragilis)
Spore formation (Clostridium)
Maintenance of reduced environment (Low
Redox Potential)
Tissue damage
existing tissue damage)
CLINICAL FEATURES OF ANAEROBIC INFECTIONS
The source of infecting microorganisms is the
endogenous flora of host
Alteration of host tissue provide suitable conditions
for development of opportunistic anaerobic infections
Anaerobic infections are generally polymicrobial in
nature
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Abscess formation
Exotoxins formation
Usually with gas
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Immunosuppressed like for example, chronic renal

failure patients, those on hemodialysis, HIV patients ;
Cytotoxic drugs like chemotherapeutic drugs ;
people with collagen vascular diseases tend to suffer
from tissue hypoxia.
Low redox potential will hasten the proliferation of
your anerobes ; Calcium salts like in pancreatits
Post-op patients especially those procedures
involving GI tract, thats why metronidazole is given
as prophylactic drug for the probable anaerobic
infection brought by the spillage of your intestinal
contents ; Trauma such as stab wounds and gun shot
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wounds ; Bites (not so common) at risk for
Veillonella infection
Human Infection
Important factors that help potentiate anerobic
infection includes : Stasis, necrosis, low O 2
tension, low redox potential
Polymicrobial : obligate anaerobe, facultative
anaerobe, microaerophilic (usual story : Myonecrosis
facultative anaerobe paves way / produces the
correct environment for obligate anaerobes to
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grow; that is why in a single specimen, it is
normal to isolate more than one organism)
B. fragilis is the most common infection
intraperitoneal infection
EXOGENOUS SOURCES
Clostridium botulinum (Botulism)
Clostridium perfringens (gastroenteritis)
Clostridium tetani (Tetanus)
Crepitans cellulitis
Septic abortion
Infections of animal or human bites
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ENDOGENOUS SOURCES No normal flora: body fluids other than urine,

Clostridium difficile (pseudomembranous colitis and exudates from deep abscesses, tissue biopsy,
antibiotic gastroenteritis) aspirates (examples: pleural fluid, CSF, synovial,
Abscess formation, Actinomycosis, Antibiotic pericardial fluid, ascitic fluid)
associated diarrhea, Aspiration or Necrotizing Should not be cultured
Pneumonia Throat / nasopharyngeal/ gingival swab
Dental infections Sputum (bronchoscopic)
Endocarditis, meningitis, Ggastric / small intestine / feces / rectal swabs
Otitis media, peritonitis, empyema Surfaces of decubitus ulcers/ mucosal lesions/
Tetanus septic arthritis eschars
Urine / vaginal / cervical swabs
SPECIMENS
DIRECT EXAMINATION
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Foul odor (due to exudates = mixture of necrotic Inhibits swarming of a potential pathogen
tissue, pus cells, inflammatory cells, sometimes the (Proteus)
organism), purulent appearance, necrotic tissue, gas, No. 1 enemy in isolation = contaminants
sulfur granules (Actinomyces only)
3. AnKanamycin-Vancomycin Blood Agar
Gram stain (initial step): shape, arrangement, spores
Kanamycin: inhibits facultative anaerobic Gram
(-) rods
CULTURE MEDIA
Vancomycin: inhibits Gram (+) Porphyromonas
1. AnBAP, non-selective for primary isolation
Add Yeast extract or Vitamin K or L-cysteine Selective isolation of Bacteroides, Prevotella,
Prevotella and Fusobacterium Fusobacterium, Velionella
4. AnParomomycin-Vancomycin Blood Agar Plate
2. AnPEA (Anaerobic Phenylethyl Alcohol Agar)
Laked blood (produced by freezing and thawing),
Selective isolation from mixture of bacteria
early recognition of pigmented Prevotella
Growth of obligate facultative anaerobe
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Selective isolation of B. fragilis, pigmented or

non-pigmeted Prevotella, Fusobacterium,
Velionella
5. Cycloserin Cefoxitin Fructose Agar
Antibiotics inhibit GI flora
Selection or isolation of C. difficile from stool,
intestine
6. Enriched Thioglycolate Medium ANAEROBIC SYSTEMS
Enriched liquid medium (No. 1 5 = solid culture 1. Jar Technique
media) 2. Evacuation Replacement
Primary isolation of actinomycetes (A. israelii) 3. Gas Pak (currently used)
Example : Robertsons Cooked Meat Medium 4. Anaerobic Glove Box technique
Takes longer for anaerobes to grow
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5. Pre reduced Anaerobic sensitized medium (PRAS)

6. Aerobic disposable plastic bags INCUBATION
35-37C at least 48 hours (for other bacteria, you
can appreciate growth as early as 18 hours)
avoid prolonged exposure of freshly inoculated plates
to ambient air
Broth cultures: 5-7 days

ANAEROBIC COCCI
EPIDEMIOLOGY
Normal flora of skin, mouth, intestines, and genito-
urinary tract
Peptostreptococcus
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P. magnus Brain abscess

- Notorious in causing Brain abcess Pneumonia, skin and soft tissue infection
- Chronic bone and joint infection Intra-abdominal infections
especially in prosthetics
Staphylococcus PEPTOSTREPTOCOCCUS
Saccharolyticus Non-pigmenting colonies
Streptococcus intermedius Indole (+): P. asaccharolyticus, P. hydrogenalis, P.
** Pepto, staph, sapro, strepto: Gram (+) indoliticus
Indole (-): P. anaerobius, P. prevotrii, P. tetradius
PATHOGENESIS
Opportunistic pathogen VELIONELLIA
Often involved in polymicrobial infection Gram (-) cocci
Virulence factors are not well characterized Indole (-)
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Nitrate (+)
Urease (-)
Non fermenter

*** Definitive Test to identify anaerobes: GAS

LIQUID CHROMATOGRAPHY. In this test, you can
identify the SPECIFIC SHORT CHAIN FATTY ACID
PRODUCTS that are elaborated by the anaerobe during
its metabolism. Culture is not definitive.
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ANAEROBIC GRAM POSITIVE BACILLI organism - Gardnerella vaginalis to grow and cause
NO SPORE FORMATION SPORE the bacterial vaginosis.
FORMATION
Propionibacterium (P. acnes) C. perfringes CHEMICAL CHARCTERISTICS
Actinomyces (A. isrealli) C. difficile ACTINOMYCES
Lactobacillus C. tetani Sulfur granules
Mobiluncus (Bacterial C. botulinum Spider colonies
vaginosis) Molar tooth colonies
Products of metabolism: Lactic Acid,
***Clue cells are diagnostic for bacterial vaginosis, Succinic Acid, Acetic Acid
not pathognomonic for G. vaginalis. When EUBACTERIUM
Lactobacillus, which is part of the normal flora of the Acetic and butyric acid
vagina, dies or is wiped out, it will allow another
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PROPIONIBACTERIUM
Propionic acetic acid, catalase production
BIFIDOBACTERIUM
Acetic, lactic acid
PROPIONIBACTERIUM ACNES
Causative agent of inflammatory acne : chronic
disease
Resides in sebaceous follicles, release LMW peptide,
stimulates an inflammatory response
Opportunistic infections: prosthetic devices
Anaerobic or aerotolerant produces propionic acid as
major by-product of fermentation
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Colonize skin, conjunctiva, external ear, oropharynx,
female genito-urinary tract
ACTINOMYCES
Facultative or strict anaerobe
Colonize the URT, GI and female genito-urinary tract
VIRULENCE:
Low virulence (when isolated from a specimen =
already pathogenic), development of disease
when normal mucosal barriers are disrupted
DIAGNOSIS:
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Macroscopic colonies Dental abcess

of organism Slowly evolving painless process
resembling grains of Chronic granulomatous lesion that becomes
sand (sulfur suppurative and forms sinus tracts
granules). These TREATMENT:
granules have Surgical debridement and prolonged antibiotics
different color for (Penicillin)
different species.
Some produce black, others yellow or orange. MOBILUNCUS
Culture Obligate anaerobe
Gram variable : Classified as Gram (+) but Gram (-)
ACTINOMYCOSIS: in young colonies
Cervico-facial actinomycosis
Colonizes GI tract in low numbers
Orofacial trauma
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Associated with Bacterial vaginosis
LACTOBACILLUS
Facultative, strict anaerobe
Part of normal vaginal flora and maintains acidic pH
Clinically significant when wiped out : Prone to
bacterial vaginosis
Produces H2O2 which is bacteriacidal to Gardnerella
Colonizes GI and GU tract
CLOSTRIDIUM
Ubiquitous, rapid growth in nutritionally enriched
environments with low redox potetial
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Spore formation: resistant to heat dessication
Causes disease via toxin elaboration
Clostridium perfringens
Gas Gangrene (a.k.a Clostridial myonecrosis)
Shows myonecrosis on X-ray (normally,
mucle is radiopaque / radiodense / white but
necrotic muscles appear as radioluscent /
black)
TX : Adequate debridement
Cellulitis, fasciitis, gastroenteritis
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Non motile Esculin/ gelatin hydrolyzed

Lecithinase (+) Indole (-), Urease ()
Double zone of hemolysis Ferments glucose, mannitol, mannose
(inner layer = beta Products of Metabolism: acetic, propionic,
hemolysis = theta toxin ; butyric, isobutyric valeric, isovaleric, isocaproic
outer layer = alpha acid
hemolysis = alpha toxin)
Saccharolytic, proteolytic Clostridium tetani
Tetanospasmin (heat labile neurotoxin): produce
Clostridium difficile spastic paralysis by blocking GABA
Pseudomembranous colitis (TX: Discontinue Lipase (-), lecithinase ()
antibiotics, give Metronidazole / Vancomycin) Asaccharolytic
Colonies: speckled opalescence Products of Metabolism: Acetic, butyric acids
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Gelatin Hydrolysis test (+)

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Clostridium botulinum Products of Metabolism: Acetic, butyric, isovaleric

Flaccid paralysis acid
Food borne
Infant botulism follows the ingestion of spores, NON SPORE FORMING ANAEROBIC GRAM NEGATIVE
germination of the spores, and toxin production BACILLI
in vivo; honey is a common vehicle for spread of Colonize human body in great numbers, part of
the spores in infants. normal flora
Demonstrates toxin in serum, feces, vomitus, Intra-abdominal infection (MC causative agent: B.
gastric contents fragilis)
MOA: Prevents release of acetylcholine Brain abscess
Motile, Lipase (+)
Saccharolytic, proteolytic BACTEROIDES
Indole (-): B. fragilis, B. distasonis, B. vulgatus
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Indole (+): B. ovatus, B. uniformis, B.

thetaiotamicron PREVOTELLA
Bacteroides fragilis Inhibited by bile
Growth is enhanced by bile Ferments glucose
Resistant to Penicillin, Kanamycin Brick red fluorescence
Susceptible to Rifampicin Pigment production: Black colonies (P.
Nonmotile, small to medium, pleiomorphic melaninogenica)
Non hemolytic, gray, concentric whorls Lipase production: (P. intermedia)
Polysaccharide capsule = important virulence Gelatin hydrolyzed (P. bivia, P. disiens, P. intermedia)
factor (allows protection from phagocytosis and
adherence to peritoneal surfaces) PORPHYROMONAS
Intraabdominal infections, especially those Pigmenting (red colonies)
associated with the colon Brick red Fluoresce
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Inhibited by Vancomycin, bile, Penicillin and

Rifampicin
Resistant to Kanamycin just like Bacteriodes
Indole (+)
Does not ferment glucose, assacharolytic just like C.
tetani

A and B: Dark Pigmented Colonies of Porphyromonas

gingivalis
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FUSOBACTERIUM
Product of metabolism: Butyric acid
Colonies: speckled opalescence, fried-egg
appearance (larger than mycoplasma)
Esculin not hydrolyzed, resistant to Rifamipin (F.
varium)
Esculin hydrolyzed, resistant to Rifampin (F.
mortiferum)
Propionic Acid from threonine (F. nucleatum)
Lipase production: (F. necrophorum)
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