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Biocidal agents: modes of action and correlation with antibiotic resistance

Use of biocides can be traced back to ancient times, but their application in the modern era continues to prove invaluable. Nowhere is this more important than in the healthcare setting, but there are concerns about the potential impact on resistance to antimicrobial drugs, as Fiona Myers explains.
A biocide can be a pesticide or an antimicrobial agent. Biocides have various uses in areas such as medicine, agriculture, forestry and mosquito control. A pesticide is a substance, preparation or organism used to control or destroy a pest. Pesticides are used to protect the supply of food, to safeguard public health and to protect buildings and other structures from harmful pests. Biocides are toxic to both prokaryotic and eukaryotic cells. Antimicrobial biocides (antiseptics, detergents and preservatives) are used in a wide range of household items including soaps, cosmetics and cleaning products. They are also used extensively in clinical settings as disinfectants and surgical scrubs to improve hygiene. Antiseptics are generally less toxic than detergents and are used on the skin, whereas detergents tend to be used on inanimate surfaces. Examples of antimicrobial biocides include phenols, quaternary ammonium compounds (QACs) and glutaraldehydes. There is growing concern that the widespread use of antimicrobial biocides may select for antibiotic resistance. Mechanisms for resistance to antibiotics are well-recognised and understood processes. In contrast to antibiotic resistance, biocide resistance is less well understood. In this article, the mode of action of antimicrobial biocides and the mechanisms by which bacteria become less susceptible are considered, and the potential relationship between biocidal and antibiotic resistance in bacteria is discussed. drinking water, while in more recent times silver products have been used as antimicrobials on burns and trauma wounds and on diabetic ulcers.1 Syphilis was originally treated with mercury and the arseniccontaining drug Salvarsan before effective treatment became available. Iodine, chlorine and phenol were introduced much later in clinical applications. Quaternary ammonium compounds have been used as biocides since the 1930s,2 and subsequently various other biocides have been introduced. Many were discovered and described long before their introduction or application (Table 1).

REGULATION OF BIOCIDES HISTORICAL PERPECTIVES

The use of antimicrobial biocides is centuries old, with early applications including their use in food preservation (salting and spices). Aromatic oils were used by the Romans, Greeks and Egyptians, and balsams were used as natural preservatives to aid mummification. Silver products have been used for thousands of years for their beneficial effects. The Romans used silver vessels to preserve Pesticides include active and inert ingredients that may be carcinogens or toxic substances. In the UK, the Health and Safety Executive (HSE) acts as the regulatory authority for non-agricultural pesticides and biocides. Nonagricultural pesticides include products such as insecticides for public hygiene use, rodenticides, wood preservatives, surface biocides and antifouling agents. Agricultural pesticides and plant protection products are regulated by the Pesticides Safety Directorate

Table 1. Biocides and their introduction into clinical practice (adapted from reference 2).
Biocide (as antiseptic or disinfectant) Alcohols Chlorine Iodine Formaldehyde Phenol Triclosan QACs Mercuric compounds Chlorohexidine Discovery/description BC 1774 1812 1867 1834 1906 1856/1916 Some BC 1946 Introduction/application 1847 1847 1816 1894 1867 Early 1970s 1933 Unknown 1954

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(PSD), and such agents include herbicides, insecticides, fungicides and soil sterilants used in agriculture and forestry to protect plant materials.

The Romans used silver vessels to preserve drinking water, while in more recent times silver products have been used on burns and wounds
components. Penetration of the outer cell wall allows the biocide to reach other target sites within the cytoplasm.

nature, state and composition of the outer envelope of the microorganism (bacterial endospores are most resistant to disinfectants).

MODES OF ACTION
Many biocides attack several target areas with differing susceptibilities depending on concentration. The lethal action for various pathogens (viruses, bacteria, fungi, protozoa) depends on the chemical composition of the biocide and the make-up of the organism. The initial action of biocides involves interaction with components of the outer cell wall. The damaging effects of high concentrations of triclosan on the cell membrane have been demonstrated.3 Bactericidal concentrations of biocides interact with outer cell components to produce changes in hydrophobicity and disruption of the cytoplasmic membrane, causing dissipation of the proton motive force involved in the active transport, oxidative phosphorylation and synthesis of adenine triphosphate (ATP) in bacteria. The activity of membrane-associated enzymes is also inhibited.4 The action of biocides (Table 2) may be due to interactions with outer cellular components, the cytoplasmic membrane or a combination of interactions with intracellular

DETERMINING BIOCIDE ACTIVITY

Determination of the decimal reduction time (D-value), which is the time taken to reduce a population of organisms by 90%, is the key parameter used to measure disinfectant efficacy.6 The D-value provides information on the contact time required at a specific concentration to achieve the reduction in microbial load required. A 5-log reduction in bacterial numbers is required by standard European suspension tests to demonstrate efficacy.

FACTORS AFFECTING EFFICACY

Various factors affect the efficacy of biocides5 and these include the following: the physical environment (ie porous or cracked surfaces) contact time (no disinfectant works instantly) biocide concentration presence of moisture organic material (prevents contact with the disinfectant) ability of the microorganism to inactivate the agent pH and temperature

BIOCIDE RESISTANCE
It has been suggested that the widespread use of antimicrobial biocides may impact on the prevalence of antibiotic-resistant bacteria.7 High concentrations of biocide are usually bactericidal, but the use of biocides as preservatives or in products at low concentrations is driving studies to understand microbial target sites.4 Similarities in the way that bacteria can circumvent the action of these antimicrobial agents have arisen whereby a selective target site for biocides is shared by a therapeutic agent or agents. Where simultaneous changes in susceptibility to antibiotics and to antimicrobial biocides have been investigated, the resistance determinants involved mostly are genes encoding for multidrug pumps either plasmid-borne in Gram-positive species or chromosomally encoded in Gram-negative species.8

Table 2. Bacterial target sites for biocides (adapted from reference 4).
Target Cell wall Outer membrane (Gram negatives) Mechanism(s) Cross-linking Increased permeability Biocides Glutaraldehyde Chlorohexidine, QACs, chlorine-releasing agents, phenolics Organic acids and parabens, alcohols, chlorohexidine, QACs, phenolics Organic acids and parabens, QACs, phenolics Chlorohexidine, ethylene oxide Chlorohexidine, QACs, phenolics Chlorohexidine,QACs, gluteraldehyde Organic acids and parabens, acridines, formaldehyde, glutaraldehyde, chlorine-releasing agents Hydrogen peroxide, organomercurial compounds Ethylene oxide, glutaraldehyde, hydrogen peroxide, chlorine-releasing agents, iodine and iodophors Ethylene oxide, glutaraldehyde Ethylene oxide, glutaraldehyde, hydrogen peroxide, chlorine-releasing agents, metallic salts Hydrogen peroxide, agents causing damage to the cytoplasmic membrane

Cytoplasmic membrane

Increased permeability

EFFLUX MECHANISMS
Efflux systems function via an energydependent mechanism (active transport) to pump out unwanted toxic substances through specific efflux pumps. Xenobiotic metabolism is the set of metabolic pathways that chemically modify xenobiotics (compounds foreign to an organisms normal biochemistry) such as drugs and poisons. The Escherichia coli AcrAB efflux system has the physiological role of pumping out bile acids and fatty acids to lower their toxicity. Antibiotic-susceptible and antibioticresistant bacteria carry and express efflux genes, and there is some debate about their normal physiological function. Bacterial efflux mechanisms have a big impact on antimicrobial resistance, and this is usually attributed to several factors outlined below. The genetic elements encoding efflux pumps may be encoded on chromosomes and/or plasmids, thus contributing to intrinsic (natural) and acquired resistance, respectively. As an intrinsic mechanism of resistance, efflux pump genes can survive a hostile environment (eg the presence of antibiotics), and this permits the selection of mutants that over-express these genes.

Membrane potential and electron transport chain Adenosine triphosphate synthesis Inhibition of enzyme activity Cytoplasmic constituents General coagulation Nucleic acids

Ribosomes Interaction with specific groups Thiol groups

Amino groups Sulphydryl groups

Biocide-induced autocidal activity

Accumulation of free radicals

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Their location on transposable genetic elements such as plasmids or transposons is also advantageous for the microorganisms as it enables the easy spread of efflux genes between distant species. Antibiotics can act as inducers and regulators of the expression of some efflux pumps. Expression of several efflux pumps in a given bacterial species may lead to a broad spectrum of resistance when considering the shared substrates of some multidrug efflux pumps, where one efflux pump may confer resistance to a wide range of antimicrobials.
Table 3. Classification of bacterial efflux transporters into five major superfamilies.
Major facilitator superfamily (MFS) Multidrug and toxic compound extrusion family (MATE) ATP-binding cassette superfamily (ABC) Small multidrug resistance family (SMR) Resistance-nodulation-cell division superfamily (RND)

BACTERIAL EFFLUX PUMPS

Bacterial efflux pumps are ubiquitous in nature, and advances in DNA technology have led to the identification of several new members of the families listed in Table 3. Efflux pumps are proteinaceous transporters localised in the cytoplasmic membrane of all kinds of cell. As active transporters they require a source of chemical energy to perform their function. Bacterial efflux transporters are classified into five major superfamilies (Table 3), based on the amino acid sequence and the energy source used to export their substrates.8 Efflux systems that contribute to antibiotic resistance have been described in a number of clinically important bacteria. Pseudomonas aeruginosa is ubiquitous in the environment and is intrinsically resistant to a broad range of substances due to multidrug efflux pumps.9 The level of expression of an outer membrane protein, OprR, correlates with the level of resistance of P. aeruginosa to QACs.10 The multiple antibiotic resistance (mar) locus and mar regulon in E. coli and other Enterobacteriacae control the cells response to multiple toxic substances and can regulate expression of efflux pumps.11 One such pump, AcrAB, in E. coli can extrude various biocides (eg triclosan, QACs, chlorohexidine) as well as antibiotics.11 Triclosan can also select for mutant strains of Stenotrophomonas maltophilia that overproduce the SmeDEF multidrug efflux pump.12 Resistance to QACs was studied initially in clinical isolates of staphylococci from humans. A range of genes mediating resistance to QACs have been identified in methicillin-resistant Staphylococcus aureus (MRSA) and there is concern that the spread of resistance genes under selective pressure could occur at a rapid rate.13 Various QAC genes have been

characterised. Cloning and nucleotide sequencing of the qac(A) and qac(B) genes show that they differ by only seven nucleotide substitutions.14 The smr gene, formally known as qac(C), encodes for multidrug efflux proteins involved in the efflux of lipophilic drugs.15 Furthermore, qac(A/B), smr and additional resistance genes (qac[G] and qac[H]) are also distributed in staphylococci from the food industry.16 Resistance patterns of the QAC-resistant strains to various antibiotics have been examined. The results indicate that there may be a dominance of certain strains in environments where QACs are used on a regular basis. Antiseptics and disinfectants based on QACs are active components in various preparations used in veterinary medicine, and they play an important role in the control of infectious diseases in animals.17 A new QAC gene, designated qac(J), has been identified in equine staphylococcal isolates. In three of the four horses involved, a QAC-containing skin preparation had been used extensively for several years.18 Elsewhere, an increase in resistance to QACs was detected in S. aureus isolates from dairy cows in a herd in which QAC-containing teat cream had been used to treat mastitis for several years.17 A further study suggests that the widespread distribution of QAC resistance genes among staphylococci of bovine and caprine origin in Norway is the result of both intraspecies and interspecies spread of QAC resistance plasmids and also the clonal spread of QAC-resistant strains.18

Studies on the target site of triclosan in S. aureus provide evidence that the mode of action of triclosan in S. aureus is also via inhibition of FabI.22 Further studies of S. aureus, using higher triclosan minimum inhibitory concentrations (MICs) of 12 g/mL were shown to over-express FabI, and this correlated with the observed decrease in sensitivity to triclosan.23 InhA is a mycobacterial enoyl reductase shown to be a target for triclosan.24 InhA is the mycobacterial homologue of E. coli FabI, so it appears that the action of triclosan is the same in both organisms.22 Reductions in the isoniazid sensitivity of Mycobacterium smegmatis can be conferred by mutation in the inha gene.25 Similar phenomena have not been demonstrated for M. tuberculosis, which remains sensitive to triclosan.25 This suggests that although the two agents share the same target, their interactions with it are not the same. Methicillin-resistant S. aureus isolates with reduced susceptibility to triclosan have been described, but genetic loci other than fabI may be involved.26 Evidence has been presented to suggest that low concentrations of triclosan affect the growth of several bacteria, while higher concentrations are bactericidal; however, its effect cannot be explained solely on the inhibition of metabolic pathways (eg enoyl reductase).27 It has been suggested that the mechanisms involved in such pathways may be significant to the effect of triclosan at low concentration.

LINKAGE
Some laboratory studies support the hypothesis that antibiotic and biocide resistance may be linked;28 however, others refute this idea.29 The linkage between biocide resistance and antibiotic resistance was first suggested in 1997 when it was found that E. coli mutants selected for resistance to pine oil over-expressed the marA gene (multiple antibiotic resistance) and showed low-level resistance to tetracycline, ampicillin, nalidixic acid and chloramphenicol.30 Later, it was shown that mutants of S. aureus selected for resistance to pine oil cleaner demonstrated reduced susceptibility to the cell wall-active antibiotics vancomycin and oxacillin.31 A more recent study investigated the development of bacterial resistance to a range of biocides using standard bacterial strains and laboratory mutants.32 Using E. coli gradient plate mutants, they observed a low-level cross-resistance between didecyldimethylammonium chloride, eugenol and thymol, as well as reduced susceptibility to antibiotics, most notably chloramphenicol. A further study investigated the potential of Salmonella enterica and E. coli 0157 strains adapted for resistance to commonly used biocides and demonstrated reduced susceptibilities to various antibiotics.33 Strains of MRSA resistant to

TARGET SITES
As stated previously, many biocides are multitargeted with differing susceptibilities depending on concentration. Triclosan, which is found in a wide range of hygiene products, has a more specific mechanism of action than previously realised.2 It targets fatty acid synthesis by inhibition of enoyl(acyl carrier protein) reductase (FabI) in E. coli. Mutations in the enoyl reductase gene (fabI) gave rise to resistance to triclosan.20 Exposure of E. coli to this agent markedly decreases susceptibility, but this phenomenon is not widespread and appears to be confined to certain enteric flora, and especially E. coli.21

Antimicrobial biocides are used in a wide range of household items including soaps, cosmetics and cleaning products
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benzalkonium chloride, a disinfectant commonly used in the clinical setting, have been reported, and mutations conferring resistance to benzalkonium chloride caused an increase in resistance of MRSA to -lactam antibiotics.28 Some of the benzalkoniumresistant mutants were found to have MICs to oxacillin eight-fold higher than the MICs for the parent strains. The mutants showed no remarkable increase to other antibiotics tested, the lethal mechanisms of which differ from those of -lactams. It was suggested that resistance to oxacillin and benzalkonium are related and may be caused by the mutation of genes encoding elements regulating the expression of methicillin resistance, or in a non-specific manner that affects efficiency of uptake or the activation of an efflux pump. The widespread use of triclosan has raised concerns that it may exert a selective pressure for antibiotic-resistant strains of staphylococci.2 Strains of MRSA with elevated MICs to triclosan have been isolated from patients treated with daily triclosan baths.34 However, another study investigated the susceptibility of MRSA to triclosan and found no evidence to support the contention that the widespread use of triclosan in various products will select for resistance in MRSA.35 An assessment of triclosan susceptibility in clinical isolates of MRSA and methicillinsensitive S. aureus (MSSA) was carried out using an agar dilution method to determine the MIC to triclosan. No differences between triclosan resistance were reported in strains of MRSA and MSSA.36 Reduced triclosan susceptibility has been shown to be more prevalent among methicillin-resistant S. epidermidis than among methicillinsensitive S. epidermidis isolates.37 A recent study analysed antibiotic and antimicrobial biocide susceptibility data in clinical isolates of MSSA, MRSA and P. aeruginosa.29 It was suggested that alterations in susceptibility data to antimicrobial biocides had little to do with methicillin resistance in S. aureus, and that it is unlikely to be linked to increased antibiotic resistance in S. aureus or P. aeruginosa from clinical isolates.

Efflux systems that contribute to antibiotic resistance have been described in a number of clinically important bacteria
Currently, antibiotic resistance is assumed to be caused mainly by antibiotic use in clinical practice. Antibiotic-resistant strains of bacteria are not generally more resistant to bactericidal concentrations of biocides than are sensitive strains; however, low concentrations may select for bacteria containing efflux genes and the development of antimicrobial resistance. Over-expression of these genes among highly resistant clinical isolates is of concern, and the intrinsic antibiotic resistance of certain species may be largely due to efflux pumps.8 The efflux of drugs needs to be considered when agents are developed, in order to maximise their overall efficacy and to minimise the likelihood of the development of resistance. Biocides form an integral part of infection prevention because, in healthcare settings, adequate disinfection is essential in order to prevent the spread of hospital-acquired infection. Thus, biocides must be used responsibly in the domestic and healthcare setting to avoid the possibility of any impact on future antimicrobial resistance. A small change in susceptibility may confer an advantage in growth for a bacterial strain, and therefore it is important that biocides be used at concentrations and under conditions that result in a rapid and effective kill. Reactive biocides (eg peroxide and hypochlorite bleach) and those that evaporate (alcohols) rapidly should be preferred, as they leave bacteria with no residue in which to develop tolerance. However, in view of ongoing concerns about antibiotic resistance, it is important to continue with research and to monitor these issues.

CONCLUSIONS
When assessing the significance of antimicrobial resistance, one must bear in mind that the term resistance is relative. Owing to the multitarget nature of many biocides, the differences in MIC values between biocide-sensitive and -resistant strains are often much smaller than those associated with antibiotics.38 Two- to four-fold changes in the MIC of biocides are often referred to as resistant, even though they are used in hygiene applications at concentrations that are much higher than the minimum biocidal concentration. Thus, it is unclear how significant these changes are from a clinical standpoint, as toxicity problems associated with high-level antibiotic therapy are not generally applicable to biocides.

REFERENCES
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Roitt I, Wakelin D, Zucherman M. Medical microbiology (3rd edn). Europe: Elsevier Mosby, 2004: 5645. 6 Mazzola PG, Penna TC, Martins AM. Determination of decimal reduction time (D-value) of chemical agents used in hospitals for disinfection purposes. BMC Infect Dis 2003; 3: 24. 7 Levy SB. The 2000 Garrod Lecture. Factors impacting on the problem of antibiotic resistance. J Antimicrobial Chemother 2002; 49: 2530. 8 Webber MA, Piddock LJV. The importance of efflux pumps in bacterial antibiotic resistance. J Antimicrobial Chemother 2003; 51: 911. 9 Chauchen R, Beinlich K, Hoang TT, Becher A, Karkhoff-Schweizer RR, Schweizer HP . Cross resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxb mutants overexpressing MexCD-OprJ. Antimicrob Agents Chemother 2001; 45: 42832. 10 Tabata A, Nagamune H, Maeda T, Murakami K, Miyake Y, Kourai H. Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR. Antimicrob Agents Chemother 2003; 47 (7): 20939. 11 Levy SB. Active efflux, a common mechanism for biocide and antibiotic resistance. J Appl Microbiol 2002; 92 (Suppl): 65S71S. 12 Sanchez P , Moreno E, Martinez JL. The biocide triclosan selects Stenotrophomonas maltophilia mutants that overproduce the Sme DEF multidrug efflux pump. Antimicrob Agents Chemother 2005; 49 (2): 7812. 13 Fraise AP . Susceptibility of antibioticresistant cocci to biocides. J Appl Microbiol 2002; 92 (Suppl): 158S62S. 14 Paulsen IT, Brown MH, Littlejohn TG, Mitchell BA, Skurray RA. Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity. Proc Nat Acad Sci USA. 1996; 93: 36305. 15 Paulsen IT, Skurray RA, Tam R et al. The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs. Mol Microbiol 1996; 19: 116775.

Cloning and nucleotide sequencing of the qac(A) and qac(B) genes show that they differ by only seven nucleotide substitutions
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The lethal action for various pathogens depends on the chemical composition of the biocide and the make-up of the organism
16 Heir E, Sundheim G, Holck AL. Identification and characteristation of quaternary ammonium compound resistant staphylococci from the food industry. Int J Food Microbiol 1999; 48: 2119. 17 Bjorland J, Sunde M, Waage S. Plasmidborne smr gene causes resistance to quaternary ammonium compounds in bovine Staphylococcus aureus. J Clin Microbiol 2001; 39 (11): 39994004. 18 Bjorland J, Steinum T, Sunde M, Waage S, Heir E. Novel plasmid-borne gene qacJ mediates resistance to quaternary ammonium compounds in equine Staphylococcus aureus, Staphylococcus simulans and Staphylococcus intermedius. Antimicrob Agents Chemother 2003; 47: 304652. 19 Bjorland J, Steinum T, Kvitle B, Waage S, Sunde M, Heir E. Widespread distribution of disinfectant resistance genes among staphylococci of bovine and caprine origin in Norway. J Clin Microbiol 2005; 43 (9): 43638. 20 McMurray LM, McDermott PF, Levy SB. Genetic evidence that InhA of Mycobacterium smegmatis is a target for triclosan. Antimicrob Agents Chemother 1999; 43: 7113. 21 Ledder RG, Gilbert P , Willis C, McBain AJ. Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex situ environmental and human isolates. J Appl Microbiol 2006; 100 (5): 113240. 22 Slater-Radosti C, Van Aller G, Greenwood R et al. Biochemical and genetic characterization of the action of triclosan on Staphylococcus aureus. J Antimicrob Chemother 2001; 48: 16. 23 Fan F, Yan K, Wallis GS et al. Defining and combating the mechanisms of triclosan resistance in clinical isolates of Staphylococcus aureus. Antimicrobiol Agents Chemother 2002; 46: 33437. 24 Parikh SL, Xiao G, Tonge PJ. Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid. Biochemistry. 2000; 39 (26): 764550. 25 Parikh S, Moynihan DP , Xiao G, Tonge PJ. Roles of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoylACP reductase from Mycobacterium tuberculosis. Biochemistry 1999; 38 (41): 1362334. 26 Brenwald NP , Fraise AP . Triclosan resistance in methicillin-resistant Staphylococcus aureus (MRSA). J Hosp Infect. 2003; 55 (2): 1414. 27 Escalada MG, Russell AD, Maillard JY, Ochs D. Triclosan-bacteria interactions: single or multiple target sites. Letts Appl Microbiol 2005; 41: 47681. 28 Akimitsu N, Hamamoto H, Inoue R et al. Increase in resistance of methicillinresistant Staphylococcus aureus to -lactams caused by mutations conferring resistance to benzalkonium chloride, a disinfectant widely used in hospitals. Antimicrob Agents Chemother 1999; 43 (12): 30423. 29 Lambert RJW. Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000. J Appl Microbiol 2004; 97: 699711. 30 Moken MC, McMurray LM, Levy SB. Selection of multiple antibiotic-resistant (mar) mutants of Escherichia coli by using the disinfectant pine oil: roles of the mar and acrAB loci. Antimicrob Agents Chemother 1997; 41 (12): 27702. 31 Price CTD, Singh VK, Jayaswal RK, Wilkinson BJ, Gustafason JE. Pine oil cleaner-resistant Staphylococcus aureus: reduced susceptibility to vancomycin and oxacillin and involvement of SigB.

Biocides form an integral part of infection prevention in healthcare settings because adequate disinfection is essential
Appl Environ Microbiol 2002; 68 (11): 541721. 32 Walsh SE, Maillard JY, Russell AD et al. Development of bacterial resistance to several biocides and effects on antibiotic sensitivity. J Hosp Infect 2003; 55: 98107. 33 Braoudaki M, Hilton AC. Adaptive resistance to biocides in Salmonella enterica and Escherichia coli 0157 and cross resistance to antimicrobial agents. J Clin Microbiol 2004; 42 (1): 738. 34 Cookson BD, Farrelly H, Stapleton P , Garvey RPJ, Price MR. Transferable resistance to triclosan in MRSA. Lancet 1991; 337: 15489. 35 Al-Doori Z, Morrison D, Edwards G, Gemmell C. Susceptibility of MRSA to triclosan. J Antimicrob Chemother 2003; 51: 1856. 36 Bamber A, Neal TJ. An assessment of triclosan susceptibility in methicillinresistant and methicillin-sensitive Staphylococcus aureus. J Hosp Infect1999; 41: 1079. 37 Schmid MB, Kaplan N. Reduced triclosan susceptibility in methicillin-resistant Staphylococcus epidermidis. Antimicrob Agents Chemother 2004; 48: 13979. 38 Gilbert P , McBain AJ, Bloomfield SF. Biocide abuse and antimicrobial resistance: being clear about the issues. J Antimicrob Chemother 2002; 50: 1379.
Fiona Myers (fiona.myers@hdft.nhs.uk) is a biomedical scientist in the Department of Microbiology, Harrogate District Hospital, Harrogate, North Yorkshire.

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