Journal of Applied Microbiology Symposium Supplement 2002, 92, 98S110S

Biolms in vitro and in vivo: do singular mechanisms imply cross-resistance?

P. Gilbert, D.G. Allison and A.J. McBain
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, UK

1. Summary, 98S 2. Introduction, 99S 3. Biolm resistance to antimicrobial agents, 99S 3.1 Resistance and the exopolymeric matrix, 100S 3.1.1 Extracellular polymers as diffusion and reaction barriers, 101S 3.1.2 Enzymes as moderators of diffusion barrier property, 101S 3.1.3 Biolm matrix and resistance: conclusions, 101S 3.2 Resistance associated with growth rate and nutrient availability, 102S 3.3 Resistance associated with the adoption of resistance phenotypes, 103S 3.3.1 Efux pumps and antimicrobial resistance, 103S

Attachment-specic resistance phenotypes, 103S 3.3.3 Programmed cell death and biolm persistence, 103S 3.4 Biolm resistance: conclusions, 104S 4. Cellular resistance to biocides and antibiotics, 104S 4.1 Current perspectives, 104S 4.2 Biocide and antibiotic resistance through target modication, 104S 4.2.1 Triclosan: antibiotic or biocide?, 105S 4.2.2 Could other non-specic biocides select for drug resistance?, 105S 4.3 Biocide and antibiotic resistance through permeability change and efux, 106S 5. Cross-resistance: a public health time bomb?, 107S 6. References, 107S

3.3.2

1. SUMMARY Microbial biolm has become inexorably linked with mans failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria. This failure relates to a localized concentration of biolm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells. Biolms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive. Such clones might include efux mutants in addition to genotypes with modications in single gene products. Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population. This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specic cellular targets. Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones,
Correspondence to: P. Gilbert, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK (e-mail: pgilbert@man.ac.uk).

modied either in efux or in their most susceptible target. The latter might also confer resistance to antibiotics where the target is shared. Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specically targets the enzyme enoyl reductase that is involved in lipid biosynthesis. Triclosan may, therefore, select for mutants in a target that is shared with the anti-E. coli diazaborine compounds and the antituberculosis drug isoniazid. Although triclosan may be a uniquely specic biocide, sublethal concentrations of less specic antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents. Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efux pumps and efux mutants. Whilst efux does not confer protection against use concentrations of biocidal products it is sufcient to confer protection against therapeutic doses of many antibiotics. It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities. Whilst such notions are supported by laboratory studies that utilize pure cultures, recent
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evidence has strongly refuted such linkage within the general environment where complex, multispecies biolms predominate and where biocidal products are routinely deployed. In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benets bestowed by the changes in an individuals antimicrobial susceptibility. 2. INTRODUCTION Biocides and antibiotics have traditionally been regarded as distinct and separate groups of antibacterial agents distinguished by the extent of their pharmacological specicity and the degree of mammalian toxicity. Thus, the ideal antibiotic possesses a single biochemical target that is absent from the host organism, whilst biocides generally possess a number of distinct targets, with varied susceptibilities, that may also occur in eukaryotic systems. It is widely recognized that the indiscriminate and often inappropriate use of antibiotics has led to the emergence of multidrug-resistant strains and a general reduction in their therapeutic effectiveness. Such resistance relates to changes in the biochemical target, enzymic neutralization of the agent or changes in accessibility of the target through reduction in cellular permeability or by the induction of efux pumps. Similar reductions in effectiveness have not been so apparent for chemical biocides (i.e. oxidizers, quaternary ammonium compounds (QACs), bisbiguanides and isothiazolones). Indeed, some of these have been widely deployed for over 100 years without any apparent loss of effectiveness (Bloomeld, this volume). For such biocidal molecules, small changes in susceptibility, indicated by an altered minimum inhibitory concentration (MIC), can be generated by sublethal exposure to the agents but these changes are generally insufcient to alter the outcome of treatments. This reects not only the multiplicity of biochemical targets possessed by such agents but also use concentrations that, unlike the therapeutic use of antibiotics, are often several thousand times higher than the MIC (Russell et al. 1999). Recently, there has been a massive expansion in the marketing and sales of various household products incorporating antibacterial biocides. Whilst the concentrations of the active agents at the point of application can generally be controlled there will be an inevitable dilution effect as they dissipate to the environment. It can be postulated that subeffective concentrations would prevail some distance from the site of use. The selection pressure caused by the incorporation of antibacterial agents in such a broad range of products is now undeniable. Such effects will be more marked for the less chemically reactive molecules (i.e. QACs and triclosan) than for oxidizing agents, such as hypochlorite, that will be rapidly dissipated. Whilst the activity of most

biocidal molecules would be unaffected by such selection pressure, some concerns have been expressed that, where this involves targets that are shared with third-party therapeutic agents, resistance might emerge towards the latter (Levy 2000). Sublethal stress with inimical agents might also induce general defence mechanisms in microbial cells, such as efux pumps (McMurry et al. 1998a, 1998b). If this led to a constitutive expression of multidrug efux then, whilst the effectiveness of biocides would not be affected, it may be sufcient to confer multidrug resistance in a clinical setting. In the majority of natural habitats micro-organisms grow and survive as adherent biolms. Of particular concern is that our ability to eradicate biolms from infected hosts, and the fouled surfaces of industrial plant, is substantially lower than that for dispersed, planktonic bacteria (Allison et al. 2000). As such, biolms have been reported as possessing susceptibilities towards biocides and antibiotics that are 100 1000 times less than equivalent populations of planktonic bacteria. This means that not only are biolms frequently encountered but they are difcult to kill and ineffective treatments are more likely to be deployed against them. Biolms are often comprised of many different species and genera, associated with surfaces and generally enveloped within a matrix of polymers of microbial origin (Costerton et al. 1987, 1994). The physiology, ecology and commercial and medical impact of microbial biolms are distinct from those of the same organisms growing singly or in planktonic culture. Not only do such differences contribute much to their problematic nature, i.e. infection, biofouling, biocorrosion, biodeterioration, etc., but they also make biolms the most likely environmental nidus of resistance development and selection. In the present article the mechanisms associated with the insusceptibility of biolm communities towards chemical and antibiotic treatments will be considered in the context of their potential to generate and maintain less susceptible phenotypes. Shared mechanisms of action and susceptibility modulation for biocides and antibiotics will be reviewed against the potential for cross-resistance development. The extent to which chronic, sublethal exposure of biolm communities to environmental biocides might lead to the selection of antibiotic resistance will then be discussed. 3 . B I O FI L M R E S I S T A N C E T O ANTIMICROBIAL AGENTS Almost without exception, surfaces within aqueous and humid environments become colonized with micro-organisms growing as biolm. In medicine, biolms have been widely implicated in many chronic infections (Costerton et al. 1987, 1994). In industry and the environment they have been associated with the biofouling and corrosion of

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pipe-work (Characklis 1990; Little et al. 1990), where increased frictional resistance to uid ow greatly increases energy consumption. To these problems can be added those of contamination and spoilage in food processing (Holah et al. 1994; Eginton et al. 1998). In all of these situations chemical agents and antibiotics form a major component of any control/eradication regime. Paradoxically, biolms are broadly resistant to such chemical agents and may be 100 1000 times less susceptible than their free-living counterparts (Allison et al. 2000). Such resistance is demonstrated not only towards antibiotics and antiseptics, but also towards highly reactive chemicals, including isothiazolones (Costerton and Lashen 1984), QACs (Costerton and Lashen 1984; Evans et al. 1990a), halogens and halogen-release agents (Favero et al. 1983; Huang et al. 1995). A biolm comprises a functional consortium of cells enveloped within a matrix of extracellular polymers (glycocalyx) and the concentrated products of their own metabolism. The latter might include ions and nutrients sequestered from the environment and extracellular enzymes such as polysaccharases, proteases, b-lactamases, etc. There appears to be no unique physiology associated with the individual cells within the community, rather the localized high cell density and the conditioning of their immediate extracellular environment causes the expression of a plethora of phenotypes (growth rate, nutrient deprivation, stringent response, etc.) spatially organized across the matrix. This is in contrast to planktonic cultures where phenotypic variation occurs mainly on a temporal rather than a spatial basis. With the exception of biolm-related infections, biolms are generally made up of a wide diversity of species. This further complicates the phenotypic heterogeneity through the establishment of mutualistic and antagonistic partnerships. The breadth of phenotypic heterogeneity represented within the biolm, therefore, reects the extent of chemical and biological heterogeneity within the lm (McBain et al. 2000). Paramount, in the context of resistance development, is the notion that the concentration of many antimicrobial agents is moderated by the presence of the extracellular polymeric matrix. The outcome of any antimicrobial treatment of the biolm community will, therefore, reect the susceptibility of the most resistant phenotype represented within the consortium. As the biolm matures, and exopolymer deposition increases, the magnitude of the nutrient and gaseous gradients within them will increase and the net growth rate of the community will become further reduced. This has been shown to cause onset of dormancy in some cells and trigger the expression of stringent response genes (Zambrano and Kolter 1995). Proposed explanations for the observed resistance of biolm communities include diffusional resistance of the extracellular matrix, augmented by chemical/enzymatic modication of the agent (reaction-diffusion limitation),

physiological changes due to slow growth rate and starvation responses and the induction of attachment-specic, drugresistant physiologies (Gilbert and Allison 1999; Allison et al. 2000). Whilst there is evidence to support each of these explanations no single mechanism can account for the general observation of resistance. Rather, these mechanisms act in concert within the biolm and amplify the effect of small variations in susceptibility phenotype. 3.1 Resistance and the exopolymeric matrix A diagnostic characteristic of biolm communities is a close proximity of cells maintained through the presence of extracellular matrices. Since these constitute the bulk of the biomass, their role in the moderation of antimicrobial treatments must be considered. Direct light and electron microscopy show the exopolymeric matrix of biolms to provide a relatively thick coating of the community with an ordered array of ne bres comprised of gelled and highly hydrated exopolysaccharides (EPS; Sutherland 1997). Sequestered within the EPS arrays are other macromolecules, such as nucleic acids, proteins, globular glycoproteins and lipids (Sutherland 1995). Whether the matrix polymers differ from those associated with planktonically-grown cells and also whether they differ from foot-print polymers that cement the primary colonizers to the substratum is undetermined (Sutherland 1997; Allison 1998). Nevertheless, matrix polymers determine the physical properties of the biolm. Up-regulation of EPS biosynthesis generally occurs within minutes of the irreversible attachment of a cell to a surface (Allison and Sutherland 1987; Davies et al. 1993) and proceeds with the development of a microcolony over a period of several hours. It is notable that, with the exception of the alginates produced almost exclusively by members of the Pseudomonadaceae, most extracellular polysaccharides are, to a certain degree, soluble in water. Biolm structure is, therefore, dynamic with solubilization of polymers occurring at the periphery being compensated by increased polymer production within the depths (Evans et al. 1994). Recently, it has been suggested that regulation of EPS, under the control of signal substances such as N-acyl homoserine lactones (HSL; Davies et al. 1998) is responsible for the early transcriptional events associated with biolm formation. Such global regulators are responsive to increases in cell density, beyond critical threshold values, and may be general regulators of biolm-specic physiology (Williams et al. 1992; Gambello et al. 1993; Cooper et al. 1995; Heys et al. 1997). In biolms, signal substances, such as HSL, would become concentrated within the geometric centre of the microcolonies/biolm, thereby increasing exopolymer production. This would alter the distribution and density of cells throughout the matrix and confer some level of structural organization upon the community to provide

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customized microniches at various points within the biolm (Costerton et al. 1994). The biolm matrix has the potential to modify the response of the enveloped cells to antimicrobial treatments through its action as a diffusion barrier and reaction sink (neutralizer). The latter function is augmented by the retention of extracellular products and enzymes. 3.1.1 Extracellular polymers as diffusion and reaction barriers. The presence of a charged, hydrated exopolymer matrix around individual cells and microcolonies profoundly affects the access of solutes. Restricted diffusion from the surrounding medium, by a combination of ionic interaction and molecular sieving events, will occur for appropriate classes of molecule. The polymers of the extracellular matrix thereby act as would an ion exchange resin and actively remove strongly charged molecules (i.e. glycopeptides; Hoyle et al. 1992) from solution (Slack and Nichols 1981, 1982; Costerton et al. 1987). Curiously, macrolide antibiotics, which are also positively charged and very hydrophobic, are relatively unaffected by the presence of exopolymers (Ichimiya et al. 1994). Poor penetration through anionic matrices might, therefore, be a phenomenon restricted to the more hydrophilic, positively charged agents. Alternatively, the matrix polymers might react chemically with and directly neutralize reactive molecules. The latter effects would be most pronounced with biocides, such as iodine and iodine polyvinylpyrollidone complexes (Favero et al. 1983), and chlorine and peroxygens (Huang et al. 1995), that react directly in a consumptive manner with the exopolymer and cellular materials. Total penetration failure will only occur, however, when the reaction sites are sufcient to deplete the bulk concentration of treatment agent or where replenishment of the matrix proceeds at a faster rate than does adsorption/reaction and diffusion (Suci et al. 1994; Stewart 1996; Stewart et al. 1998). Such explanations of resistance cannot be universally applied since reduction in the diffusion of antibiotics such as tobramycin and cefsulodin, within biolms or microcolonies, is insufcient to account for the observed change in susceptibility (Gordon et al. 1988; Nichols et al. 1988, 1989). Thus, Gristina et al. (1987) and Evans et al. (1991) concluded that differences in susceptibility towards antibiotics of slime-producing and non-slime-producing strains of Staphylococcus epidermidis and Pseudomonas aeruginosa, respectively, could not be attributed to reductions in diffusion coefcient. At equilibrium, the concentrations at the cell surface and in the surrounding bulk aqueous phase would be equal. Diffusion limitation studies have generally focused on antibiotics rather than biocides and upon medically relevant biolm populations rather than biofouling situations. The thickness of biolms

in vivo is in the order of tens of micrometres whilst, for industrial biolms, it may be in the order of tens of centimetres (Nichols et al. 1989). Whilst the thickness will not affect diffusion properties per se it will affect the mass ux of charged antimicrobial agents within the depths of industrial biolms (Nichols 1993). Stewart (1996) has also reached similar conclusions, albeit using a theoretical model, that the extent of retardation of antibiotic diffusion due to sorption is not sufcient to account for observed changes in susceptibility. 3.1.2 Enzymes as moderators of diffusion barrier property. In addition to the potential of the exopolymer matrix to react directly and chemically quench reactive moieties, retention of drug-inactivating enzymes within the glycoclayx will amplify its barrier properties with respect to the diffusion of suitable substrates. Thus, the sequestration of b-lactamase enzymes (Giwercman et al. 1991) or formaldehyde lyase and dehydrogenase (Sondossi et al. 1985), which would cause the degradation of b-lactam antibiotics and formaldehyde, respectively, greatly enhances the protection afforded. In such a fashion, the b-lactam resistance of Ps. aeruginosa biolms has been attributed to b-lactamase retention in the biolm matrix (Lambert et al. 1993) but with the levels of enzyme induction that are paradoxically lower than in corresponding planktonic cells (Giwercman et al. 1991; Lambert et al. 1993). 3.1.3 Biolm matrix and resistance: conclusions. Clearly, whether or not the exopolymeric matrix constitutes a physical barrier to antimicrobial penetration depends greatly upon a number of features. These relate to the nature of the agent, the binding capacity of the polymeric matrix towards it, the levels of agent used therapeutically (Nichols 1993), the distribution of biomass and local hydrodynamics (DeBeer et al. 1994), the rate of turnover of the microcolony relative to antibiotic diffusion rate (Kumon et al. 1994) and the production and retention of extracellular products. Resistance of this fashion was exemplied in a study of the resistance of Klebsiella pneumoniae and Ps. aeruginosa biolms towards monochloramine (Huang et al. 1995). These workers grew biolms of the two organisms, together on stainless steel surfaces using a continuous ow annular reactor. Biolms were treated with 2 mg l1 monochloramine for 2 h and stained using a uorogenic redox indicator that could differentiate respiring from non-respiring cells. Epiuorescent micrographs of frozen cross-sections taken at regular time intervals revealed gradients of respiratory activity after the monochloramine treatment of biolms. Cells near the biolmbulk uid interface lost respiratory activity early in the treatment whereas residual respiratory activity persisted near the substratum or in the centre of

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small, viable cell clusters even after 2 h treatment. In a further study, Stewart et al. (1998) exposed biolm constructs of alginate-entrapped Enterococcus faecalis to both oxidizing and non-oxidizing biocides (chlorine, glutaraldehyde, isothiazolone and QAC biocides). They demonstrated a similar lack of penetration and action against the entrapped cells. At sites where biocide formulations were actively deployed, however, the volume and reactive capacity of a biolm would be insufcient to deplete the bulk availability of biocide and interactive sites within the matrix polymers would become saturated with adsorbed/reacted biocide. The net effect would, therefore, be to delay, rather than prevent, the inhibitory process (Huang et al. 1995). At sites remote from the point of application, particularly for recalcitrant biocides, the exposures within biolm communities would be brief and subeffective. Under such circumstances reaction diffusion limitation would allow the survival of less susceptible clones at the base of the biolm, which would ourish and be subject to clonal expansion once the biocide was removed, a situation that is analogous to laboratory resistance-training experiments (Brown et al. 1969; Gilleland et al. 1989). 3.2 Resistance associated with growth rate and nutrient availability Since mature biolms are composed of multiple layers of bacteria embedded in EPS matrices, diffusion and transport of nutrients through the biolm becomes an important consideration. One consequence of the localized high cell density within a biolm is that deep-lying cells are exposed to concentrations of substrates, and redox potentials, that are substantially altered from those experienced by cells at the periphery or grown planktonically. Growth rates will, therefore, be reduced within biolms through the imposition of nutrient deciencies and gradients. These may or may not reect the composition of the bulk aqueous phase. Nutrient and gaseous gradients ensure that, at any particular time, a plethora of phenotypes is represented within the community that reect the chemical heterogeneity of the biolm. Whilst this has long been assumed to be the case within biolms (Brown et al. 1990; Gilbert et al. 1990), it is only recently that it has been visualized. Wentland et al. (1996) used acridine orange stain to show that the regions of fastest growth occurred in the outer 30 lm of a Kl. pneumoniae biolm. Dispersed regions of slow growth, tending towards the substratum, were also noted. Such heterogeneity is important. A multitude of publications, over the last 25 years, has rmly established links between nutrient status/growth rate and the susceptibility of bacteria towards antibiotics, biocides and preservatives (Brown and Williams 1985; Williams 1988; Brown et al. 1990). The general

observation is that susceptibility towards most inimical agents varies as a direct function of growth rate and that much of the resistance associated with biolms might be associated with slow-growing, starved community members. Such phenomena have been widely demonstrated using the perfused biolm fermenter (Gilbert et al. 1989). This device enables the growth rate to be controlled in thin, relatively homogeneous biolm populations. The decreased susceptibility of Staph. epidermidis (Duguid et al. 1992) and E. coli biolms to tobramycin (Evans et al. 1990b) and cetrimide (Evans et al. 1990a) could be explained largely in terms of growth rate. Cells resuspended from growth rate-controlled biolms and planktonic cells at the same growth rate possessed virtually identical susceptibilities towards these agents. Stewart (1994) developed mathematical models, which incorporated the concepts of metabolism-driven oxygen gradients and growth rate-dependent killing, to examine the susceptibility of Staph. epidermidis biolms to various antibiotics. The models accurately predicted that susceptibility would be reduced in thicker biolms due to oxygen limitation. Oxygen gradients within the biolm may also directly inuence the activity of some antibacterial agents (Shepherd et al. 1988; Zabinski et al. 1995). Since nutrient and gaseous gradients will increase in extent as biolms thicken and mature, growth rate effects on susceptibility, such as these, will become particularly marked in aged biolms (Anwar et al. 1989; Anwar and Costerton 1990). Whilst the contribution of reduced growth rate within biolm communities cannot be denied, it is not the sole explanation of the reported resistance, as with diffusion limitation. This is because the existence of physiological gradients across biolms depends on growth and metabolism of cells at the periphery to consume nutrients before they permeate to the more deeply placed cells. The peripheral cells will have growth rates and nutrient proles that are similar to those of planktonic cells. They will, therefore, be relatively sensitive to the treatments imposed and will quickly die. The lysed products of the killed cells will feed the cells within the depths of the biolm which, in turn, will step up their metabolism and growth rate, adopt a more susceptible phenotype and die. Should the supply of antimicrobial agent cease, then the biolm could re-establish almost as rapidly as it was destroyed because of the relative abundance of nutrients. A major contributor towards the failure of biolms to rapidly succumb to antimicrobial treatments must, therefore, be associated with physiological heterogeneity (Allison et al. 2000). Slow-growing, deeply recessed cells in a biolm will not only be subject to a delayed and progressive contact with biocide or antibiotic (see above) but will also display a less sensitive phenotype. Neither mechanism, however, can provide a complete explanation of the long-term tolerance of

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antimicrobial agents displayed by biolm communities. Collectively, they provide processes by which viability might be temporarily maintained during the early phases of treatment. For long-term tolerance towards antimicrobial agents to occur, the biolm population must adapt to a resistant phenotype during the window of opportunity provided. For those populations inadvertently (i.e. peripheral to the main treatment site) exposed to sublethal, or transient, concentrations of biocides and antibiotics, such protective mechanisms would be sufcient for the survival and clonal expansion of the least susceptible phenotypes. 3.3 Resistance associated with the adoption of resistance phenotypes Long-term survival of biolm communities must relate to the adoption, or clonal expansion, of a more resistant phenotype during the delayed action of the treatment agent. Such phenotypes might relate to growth of a biolm per se, the so-called biolm phenotype, non-specic responses to a localized high cell density (quorum sensing) or the proximity of a surface and/or subinhibitory concentrations of the treatment agent. Resistance might relate to active efux of the treatment agent or to other less well-understood mechanisms. 3.3.1 Efux pumps and antimicrobial resistance. Sublethal concentrations of antibiotics and biocides might act as inducers/transcriptional activators of more tolerant phenotypes, such as those expressing the multidrug resistance operon mar and efux pumps such as acrAB (George and Levy 1983; Ma et al. 1993). Activators of mar include subeffective concentrations of antibiotics (tetracycline and chloramphenicol), biocides (QACs, triclosan and pine oil) and xenobiotics such as salicylate. Mar is chromosomal, variously induced and represented within a wide range of Gram-negative bacteria. The importance of mar would be greatly increased if it were induced by growth as a biolm per se and conferred a more resistant phenotype upon the cells prior to exposure (Maira-Litran et al. 2000a). This has been investigated using biolms of a group of isogenic E. coli mar and acrAB mutants with ciprooxacin as test agent. In this organism, exposure to ciprooxacin does not induce mar or acrAB in E. coli but its expression does confer a limited protection. Results showed that mar and acrAB mutants (constitutive) had reduced susceptibility to ciprooxacin but that there was little or no difference between the wild-type and mar- or acrAB-deleted strains (Maira-Litran et al. 2000a). Clearly, neither mar nor acrAB is specically induced within biolm populations but their expression in response to appropriate inducer substances might be enhanced. In this respect, mar expression has been shown to be inversely related to specic growth rate (Maira-Litran

et al. 2000b). Following exposure of biolms to sublethal levels of b-lactams, tetracyclines and salicylates, mar expression will, therefore, be greatest within the depths of the biolm where growth rates are suppressed. Treatment with antimicrobial agents that were not inducers might, however, lead to the clonal expansion of mutant cells constitutive in efux pump expression. Similar systems, under the regulation of different inducer agents, might extend this explanation of biolm tolerance to include other treatment agents. It is noteworthy that resistance operons such as mar have also been reported to be up-regulated in response to exposures to many commonly occurring, natural inhibitory substances, such as garlic, chilli peppers, herbs and spices (Whyte et al. 2001). Possible selection pressures towards efux mutants would not, therefore, be restricted to chemical interventions by man. 3.3.2 Attachment-specic resistance phenotypes. Bacteria can sense the proximity of a surface, up-regulate production of EPS and rapidly alter their susceptibility towards antibiotics (Ashby et al. 1994) and biocides (Das et al. 1998) after binding. Das et al. (1998) showed that the susceptibility of Ps. aeruginosa and Staph. aureus to a range of different biocides changed rapidly after cellular attachment and biolm formation. In some instances, three- to vefold decreases in susceptibility occurred immediately on attachment in the presence of biocide that exceeded the MIC for planktonic cells. Gilbert et al. (2001) later showed that the bactericidal mechanisms of the same biocides were unchanged in mature biolms. This indicated that active efux of the agent or decreases in penetrability of the community had caused the reduced susceptibility rather than a change in target. In a similar study, Fujiwara et al. (1998) demonstrated that, after a 1-h incubation, the minimum bactericidal concentrations towards adherent Ps. aeruginosa, Serratia marcescens and Proteus mirabilis were markedly elevated. The magnitude of the decreases in susceptibility observed immediately after bacterial attachment, but before biolm formation, is generally far less than that observed in mature biolms and is insufcient to account for the reported levels of resistance in biolm communities. Whatever the extent or implication of such change, the possibility exists that they are mediated through the accumulation of HSL-like signal substances at the occluded surface (Davies et al. 1998) and might be circumvented by HSL antagonists such as the furanones. 3.3.3 Programmed cell death and biolm persistence. A recent and novel hypothesis for the considerable recalcitrance of biolm relates to the potential of damaged bacterial cells to undergo apoptosis or programmed cell death (PCD). In this respect, Lewis (2000, 2001) suggested that death of cells following treatment with bactericidal agents results not

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from direct action of the agent but from a programmed suicide mechanism and cellular lysis (Moyed and Bertrand 1983; Black et al. 1991). The biocide literature of the last 50 years has been punctuated with reports of low-level, persistent survival of antimicrobial treatments (tailing) where the agent has not been quenched and where the survivors do not demonstrate resistance when recultured or cloned (Bigger 1944). The recent evidence suggests that such cells, rather than being resistant to the agent (Koch 1987), are actually defective in PCD (Brooun et al. 2000). Following removal of an inimical stress, these damaged persistent cells (persistors) would grow rapidly in the presence of nutrients released from their lysed community partners and the community would become restored. It is also postulated that biolm populations are enriched in persistor cells, either as a biolm-specic phenotype or due to their protection within the biolm from immune responses or inimical agents (Lewis 2000, 2001). These cells would survive treatment phases and proliferate in the post-treatment phase, thereby engendering considerable recalcitrance upon the biolm community. 3.4 Biolm resistance: conclusions Resistance of microbial biolms to a wide variety of antimicrobial agents is clearly associated with the organization of cells within an extensive exopolymer matrix. Such organization is able to moderate the concentrations of antimicrobial agents and antibiotics to which the more deeply lying members of the biolm community are exposed. Such cells are coincidentally slow-growing, starved and express stressed phenotypes that may include the up-regulation of efux pumps. The expressed phenotype of the deeply seated biolm community reduces their susceptibility to the treatment agents and exacerbates the likelihood of their being exposed sublethally. The deeper-lying cells will out-survive those at the surface and multiply if the bulk of the treatment agent is depleted or the exposure transient. At the fringes of action, selection pressures will enrich the populations with the least susceptible genotype. It is possible under such circumstances for repeated chronic exposure to sublethal treatments to select for a more resistant population. Interestingly, such enrichments are not dissimilar to in-vitro training experiments whereby microorganisms that survive a low concentration of an antimicrobial agent are gradually exposed to increasing concentrations in a step-wise manner. In this fashion cells can be trained to acquire resistance (Brown et al. 1969; Gilleland et al. 1989). This process has often been dismissed as being articial on the grounds that it could not possibly occur in nature. However, biolms exposed to antimicrobial agents in a pulsed fashion will provide at least one mechanism by which such training could occur in the real world.

4 . C E L L U LA R R E S I S T A N C E T O B I O C I D E S A N D A N TI B I O T I C S 4.1 Current perspectives The same general mechanisms of resistance apply to planktonic cells as for biolm communities. These include deletion or modication of the target site, chemical or enzymic inactivation of the agent or reduction of the concentrations at a critical intracellular target (Bryan 1988; Okusu et al. 1996). The difference between the resistance of clinical isolates and communities is that the former is dependent on genotypic change, reected in the properties of the individual cell, whereas the latter reects phenotypic heterogeneity and spatial organization within the community. Spatial organization in biolm communities provides for the exposure and sublethal stress of a large proportion of the community with the associated selection/enrichment of genotypes with reduced susceptibility. Where biochemical targets are shared between chemical biocides and therapeutic agents, cross-resistant forms might be selected by subeffective biocide treatment (Gilbert and McBain 2001). A second concern is that, where the selected genotype involves a change in cell envelope permeability mediated through porin proteins or energetic efux, subeffective deployment of biocides might select for antibiotic-resistant phenotypes (McBain and Gilbert 2001). 4.2 Biocide and antibiotic resistance through target modication Antibiotic resistance genes existed long before the rst therapeutic applications of antibiotics (Dancer et al. 1997). Their rapid evolution and spread in clinical isolates together with the marked decline in the rate of discovery of new agents has, however, raised considerable concern that the antibiotic era might be coming to an end (Hart 1998). Much of these developments are unambiguously a consequence of antibiotic overuse (Levy 1991; Swartz 1997; Hart 1998). Whilst therapeutic failure in the clinic may result from misdiagnosis or inappropriate treatment, changes in the bacterial phenotype, rendering the target micro-organism clinically resistant, are also implicated. Antibiotic resistance is an arbitrary endpoint representing a change in sensitivity, for a previously susceptible organism, that is sufcient to cause therapeutic failure. Unlike biocides, the desired selective toxicity of therapeutic agents dictates that they affect unique, often single, pharmacological targets that are not represented in the host (Russel and Day 1996). Such pharmacological specicity means that there is potential for minimal mutational events within the target organism to render a bacterium clinically resistant to an antibiotic (Cohen and Tartasky 1997). This concept of drug resistance, as

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applied to antibiotics in clinical settings, cannot be applied to biocides. The multitarget nature of many biocides means that mutation within a single target is unlikely to result in treatment failure. Indeed, reductions in sensitivity of up to 50-fold have been noted (Chapman 1998; McDonnell and Russell 1999), but even these changes are much smaller than those associated with antibiotics. Furthermore, toxicity problems associated with high level antibiotic therapy do not generally apply to biocide applications, such that marginal increases in MIC do not translate to application failure. Biocides tend to have a broader spectrum of activity than do antibiotics. This does not necessarily reect a lack of target specicity, rather it reects a multiplicity of specic targets (Russell and Russell 1995). Our current mechanistic understanding of biocide action is poor, the common view being that cellular effects occur by gross membrane damage (phenolics and QACs), oxidation (peroxygens and halogens), protein coagulation (biguanides and aldehydes) and/or by a general cytoplasmic poisoning (mercurials, aldehydes and isothiazolones). Given the exquisite detail in which we understand therapeutic drugtarget interactions, it is unsurprising that, until recently, little thought has been given to the possibility of biocidal agents and antibiotics having shared targets. Shared targets imply that genetic modications selected by the deployment of one agent might lead to changes in susceptibility toward both. 4.2.1 Triclosan: antibiotic or biocide? Triclosan, a broad-spectrum bisphenol biocide, is widely used in a variety of household and personal products as an antibacterial agent, as well as having therapeutic applications in the treatment of topical methicillin-resistant Staph. aureus infection (Bloomeld, this volume). Recent studies have demonstrated that triclosan selects for mutants in the fabI gene of E. coli at sublethal concentrations. FabI encodes an enoyl-acyl carrier protein reductase, an essential enzyme involved in the synthesis of fatty acids (Heath and Rock 1995). Triclosan, therefore, appears to be a very potent inhibitor of enoyl reductase (McMurry et al. 1998a, 1998b; Levy et al. 1999). Somewhat worryingly, triclosan shares this target with some current therapeutic agents, suggesting that triclosan could select for resistance to third-party antimicrobial agents. In this respect, the enoyl reductase of Mycobacterium tuberculosis is the target for isoniazid, currently the most widely used antituberculosis drug (Miesel et al. 1998). Accordingly, McMurry et al. 1999) demonstrated that partial isoniazid resistance in M. smegmatis can be conferred by mutations in InhA, the homologous gene to fabI. The experimental diazaborine family of antibacterial agents also acts on enoyl reductase in E. coli (Roujeinikova et al. 1999). Furthermore, the enoyl reductase

of E. coli is also a target of the biocide hexachlorophene (Heath et al. 2000). The clinical importance of triclosan lies primarily in its efcacy against Gram-positive skin infections (Cookson et al. 1991) whilst the majority of reported triclosan resistance has been in Gram-negative bacteria. Heath et al. (2000) have shown, however, that there is considerable homology between the enoyl reductase of E. coli and Staph. aureus, that these enzymes are functionally interchangeable and that mutations in the Staph. aureus fabI confers triclosan resistance. It has now been demonstrated that, like other biocidal agents, triclosan possesses a number of interactive targets and that changes in fabI are generally insufcient to confer resistance, in the targeted Gram-positive bacteria, at typical use concentrations. The possibility exists, however, that the widespread use of triclosan has contributed to antibiotic resistance development and that other widely used biocides might have similar sublethal specicity (Bloomeld, this volume). 4.2.2 Could other non-specic biocides select for drug resistance? Studies of triclosan action have suggested that some biocides may not be multitargeted, but act in a similar fashion as antibiotics and specically affect one or two cellular targets. Even for those biocides that are known to affect a multiplicity of targets, susceptibility for each is variable and dependant upon the concentration of biocide. Sublethal concentrations might, therefore, have only a single biochemical target and mutations in that target could confer a selective advantage upon the sublethally exposed bacteria (i.e. remote from the site of use or within a biolm). Survivors of a sublethally treated biolm might, therefore, possess gene modications that could confer resistance to third-party agents that have, as their sole target, the most susceptible biocide target. Specic examples of biocides with previously described specic cellular effects include phenoxyethanol (and its analogues, including the water treatment agent 2,4 dichlorphenoxyethanol (dichlorophen); Gilbert et al. 1978). Although generally considered to act by non-specic membrane disruption, these compounds actually affect a multitude of intracellular targets, dependent on concentration (Gilbert et al. 1977a, 1977b). At sublethal levels, a number of concentration-dependant inhibition processes occur. These vary from action as a potassium : proton antiporter and respiration uncoupler through to competitive inhibition of NADH binding by malate dehydrogenase. DNA biosynthesis is slowed relative to general anabolism in the cell at sub-MIC (Gilbert et al. 1977b). Similarly, isothiazolone biocides also have a more complex effect on cellular machinery than generally accepted. Whilst it is recognized that the primary action is mediated through thiol interactions (Collier et al. 1990a), specicity is seen towards

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a number of respiratory enzymes (Collier et al. 1991). At sub-MIC they have been shown to distribute variously around and in the cell (Collier et al. 1990b) and to induce lamentation (Fig. 1) in both bacteria and the ssion yeast Shizosaccharomyces pombe (Collier et al. 1990b). Whilst lethal doses affect thiol groups on proteins, sublethal doses more specically affect DNA (Gilbert et al. 1980) and bacterial cell walls (Collier et al. 1990b). It is interesting to note that other biocides, i.e. phenylethanol (Silver and Wendt 1967), are known to inhibit the initiation of DNA replication and to cause a similar lamentation to that of the isothiazolones. If these sublethal effects were directed through a DNA gyrase or a specic penicillin-binding protein then it is not difcult to imagine how the action of quinolone or b-lactam antibiotics might also be affected. At present, such links between sublethal exposure to multitargeted biocides and the development of resistance in third-party agents can be speculated but have not been substantiated either by laboratory studies or surveillance studies (Suller and Russell 2000; Bloomeld, this volume). 4.3 Biocide and antibiotic resistance through permeability change and efux The majority of antimicrobial agents must gain access to the cytoplasm in order to exert their effect. Polycationic agents, such as the aminoglycosides, gain access to the cell through a self-promoted mechanism (Hancock 1981; Taber et al.

1987). In self promotion, the agent destabilizes cations associated with the cell envelope causing reorganization of the LPS to facilitate antibiotic entry. It is notable that many biocides, particularly polymeric biguanides (Wilkinson and Gilbert 1987), share this mechanism of cellular uptake. Adaptations against such agents might, therefore, demonstrate cross-reaction between biocide and antibiotic. An increasingly observed resistance mechanism is the expression and over-production of multidrug efux pumps (Nikaido 1998). Expression of such pumps is induced, in Gram-negative bacteria, through sublethal exposure to a plethora of agents. These include not only small hydrophilic antibiotics but also other xenobiotics, such as pine oil, salicylate and triclosan (Miller and Sulavick 1996; McMurry et al. 1998a; Levy, this volume). Mutations that increase the expression of such efux pumps result in elevated levels of resistance. Whilst efux pumps are operational in a wide variety of Gram-negative organisms, and may be plasmid or chromosomally encoded (Nikaido 1996), multidrug efux pumps qacAG also contribute to biocide tolerance in Staph. aureus (Rouch et al. 1990). Maira-Litran et al. (2000a, 2000b) showed that, whilst the efux system acrAB was not required for biolm resistance, its constitutive expression signicantly enhanced the levels of survival within E. coli biolm communities exposed to ciprooxacin. Moken et al. (1997) and McMurry et al. (1998a, 1998b) have shown that mutations causing over-expression of marA or acrAB are associated with exposure and reduced susceptibility

(a)

(b)

Fig. 1 Morphological changes induced in (a) Escherichia coli by sublethal exposure to ciprooxacin (005 lg ml)1) and (b) Pseudomonas by exposure to a sublethal concentration of 4-chloro, N-methyl isothiazolone (5 lg ml)1)
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towards triclosan. Similarly, mutations in the mexAB operon of Ps. aeruginosa leads to over-expression of this efux system (Rella and Haas 1982) and triclosan can select for mutants that hyper-express mexCD efux in mexAB-deleted mutants (Chuanchuen et al. 2000). Many xenobiotics will induce the expression of efux systems. Indeed, under conditions of chronic sublethal exposure, such as can be generated in the laboratory, they may also select hyper-expressing mutants. It must be borne in mind that the primary function of energetic efux is to defend the cell against naturally occurring environmental toxicants (Miller and Sulavick 1996). Concentration of research upon induction and selection by antibiotics and biocides neglects the fact that many food substances, such as mustard, chilli and garlic, can also induce such systems (Whyte et al. (2001). Also often neglected is that hyperexpressing mutants, whilst resistant to antibiotics in laboratory cultures, will also pump out key metabolites and be relatively non-competitive in mixed microbial communities. This is especially the case when the antimicrobial selection pressure is removed or transient. 5. CROSS-RESISTANCE: A PUBLIC HEALTH TIME BOMB? Many biocides have retained their therapeutic efcacy during 100 years of use. However, in the case of triclosan, current studies strongly suggest that misuse could select for a more generalized resistance. Since enoyl reductase is a major target of this compound, triclosan abuse may also jeopardize the development of any future agent for which enoyl reductase is a primary target. With respect to other biocides, there exists worrying possibilities that changes in biocide susceptibility may be reected in antibiotic resistance, especially where a sole antibiotic target is shared with the biocide. Currently, there is insufcient understanding as to whether the indiscriminate use of biocides might select resistance to current antibiotics or hinder development of new antibiotics. Further urgent investigation is clearly required. 6. REFERENCES
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2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 98S110S