Thien-Fah Mah Department of Biochemistry, Microbiology ...

106110.2217/FMB.12.76 © 2012 Future Medicine Ltd ISSN 1746-0913Future Microbiol. (2012) 7(9), 1061–1072

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Introduction to biofilms & biofilm-specific antibiotic resistance

Biofilms are communities of bacteria that form on solid surfaces. In medical settings, biofilms can have a profound negative effect on human health. Examples of bacterial biofilms are chronic Pseudomonas aeruginosa infections in the lungs of cystic fibrosis (CF) patients, Staphyloccocus aureus and Staphylococcus epidermidis medical implant-related infections and plaque formation on teeth [1].

Bacterial biofilms are formed in response to environmental signals, resulting in the transi-tion from unicellular, planktonic (free-swim-ming) cells to a multicellular population that is attached to a solid surface and encased in a poly-saccharide matrix [1]. Biofilms exhibit a complex structure made up of pillar-like, mature macro-colonies surrounded by fluid-filled channels. This developmental cycle is completed when cells from the biofilm break away to resume a planktonic existence.

Biofilm cells differ from their planktonic counterparts in several ways. First, they are high-density, heterogeneous communities on a surface rather than uniform planktonic popu-lations. This heterogeneity is a key attribute of biofilms, resulting in subsections of the biofilm where different conditions exist (Figure 1) [2]. Second, the pattern of gene expression is altered in biofilm populations [3–7]. Examples of altered gene expression in surface-attached populations include the upregulation of a number of osmoti-cally regulated genes and the genes required for exopolysaccharide production. Third, cells growing in surface-attached communities are more resistant to the effects of antimicrobial agents, including antibiotics such as tobramycin (Tb) and biocides such as chlorine [8–11]. In some

cases, a 1000-fold increase in resistance has been reported [12]. Once biofilms have formed on a surface, they become almost impossible to eliminate [13,14]. These biofilms can then serve as a reservoir of bacteria that can be shed into the body to cause repeated episodes of acute infection.

Historically, study of biofilms was based on observation of these communities in their natu-ral environments [1]. Resident species were iden-tified and their functions within the communi-ties were analyzed. In the late 1990s, researchers focused on the molecular mechanisms of biofilm formation, resulting in a systematic characteriza-tion of genes important for biofilm formation [15]. A similar history of the study of antibiotic resistance of biofilms has occurred. Early studies were focused on documentation, specifically in the clinic, bringing a stronger awareness of this medically pertinent aspect of biofilm physiol-ogy. More recent studies have focused on the molecular mechanisms of biofilm-specific anti-biotic resistance. In this review, I will discuss the different molecular mechanisms of biofilm resis-tance that contribute to the overall high level of resistance found within a biofilm.

Definition of biofilm-specific antibiotic resistance

There are some issues regarding the use of the terms ‘resistance’ and ‘tolerance’ in referring to the survival of biofilm cells after exposure to anti-biotics. Antibiotic resistance is classically defined as an increase in the MIC of an antibiotic due to a permanent change in the cells (e.g., a muta-tion or the acquisition of resistance functions by horizontal gene transfer). These mutations have been well-studied and include the alteration of antibiotic targets, increases in the expression

Biofilm-specific antibiotic resistanceThien-Fah MahDepartment of Biochemistry, Microbiology & Immunology, University of Ottawa, Ottawa, ON, K1H 8M5, Canada n [email protected]

Bacterial biofilms are the basis of many persistent diseases. The persistence of these infections is primarily attributed to the increased antibiotic resistance exhibited by the cells within the biofilms. This resistance is multifactorial; there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. These mechanisms are based on the function of wild-type genes and are not the result of mutations. This article reviews the known mechanisms of resistance, including the ability of the biofilm matrix to prevent antibiotics from reaching the cells and the function of individual genes that are preferentially expressed in biofilms. Evidence suggests that these mechanisms have been developed as a general stress response of biofilms that enables the cells in the biofilm to respond to all of the changes in the environment that they may encounter.

Keywords

n biofilm n biofilm-specific antibiotic resistance mechanism n heterogeneity

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of drug efflux pumps or decreased permeability of the cell due to alterations in the outer mem-brane and the action of antibiotic-modifying enzymes [16]. By contrast, antibiotic tolerance is defined as the ability of cells to survive antibiotic exposure due to a reversible phenotypic state. These definitions work well when applied to a classical example using planktonic cultures – if a cell acquires a mutation that allows it to resist a higher concentration of antibiotic, then it has become more resistant.

These definitions are less precise when deal-ing with biofilms. As discussed in this review, the mechanisms of biofilm-specific resistance or tolerance that have been identified are the result of the expression of wild-type genes within bio-films. In other words, biofilm-specific antibiotic resistance is not the result of mutations; thus, the

classical definition of resistance does not apply to biofilms. However, another set of definitions has been presented: resistance mechanisms prevent the antibiotic from accessing its target, while tolerance mechanisms shut down the targets of antibiotics [17]. The biofilm-specific antibiotic resistance mechanisms discussed in this article encompass both of these definitions.

Study of biofilm-specific resistance has been confusing, likely because there is no single mechanism that encompasses all resistance. Given the heterogeneous nature of biofilms, it is likely that multiple mechanisms of resis-tance and/or tolerance act together to provide an overall high level of resistance.

For the purposes of this review, biofilm- specific resistance includes tolerance to anti-microbial agents and means that the resistance

Figure 1. Physiological heterogeneity in a Pseudomonas aeruginosa biofilm imaged by inducing a green fluorescent protein in a mature flow cell biofilm. Cells exhibiting de novo protein synthesis appear green; areas of red (rhodamine B counterstain) contain inactive cells. Zones of activity are seen at the periphery of cell clusters where biomass adjoins the nutrient medium and also in the hollowing centers of some clusters. Image courtesy of E Werner, B Pitts and PS Stewart, Center for Biofilm Engineering, MT, USA.

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(or tolerance) mechanism occurs predominantly in biofilm cells, not in planktonic cells. However, it is expected that the aforementioned classical resistance mechanisms provide additional resis-tance to biofilm cells. These mechanisms and those responsible for biofilm-specific resistance would synergistically contribute to the overall level of resistance observed in biofilms.

Biofilm infectionsWhile there is some debate over designation of an infection as being a biofilm-based infection, Parsek and Singh have laid out a set of criteria that is useful in defining biofilm-based infections. These criteria are:

“(a) The infecting bacteria are adherent to some substratum or are surface-associated. (b) Direct examination of infected tissue shows bacteria

living in cell clusters, or microcolonies, encased in an extracellular matrix. The matrix may often be composed of bacterial and host components.

(c) The infection is generally confined to a partic-ular location. Although dissemination may occur, it is a secondary phenomenon. (d) The infection is difficult or impossible to eradicate with anti biotics despite the fact that the responsible organisms are

susceptible to killing in the planktonic state.”

– Parsek & Singh [18]

Infections such as chronic lung infections of CF patients, chronic wounds, bladder infections and otitis media adhere to the Parsek and Singh crite-ria. Bacteria growing in biofilms can contribute to the chronic phase of an infection, whereas the same bacterium, when released from the biofilm, can then also contribute to the acute phase of the infection.

P. aeruginosa is one of the best-studied in vitro models of biofilm formation [19]. In vivo, P. aeru-ginosa is thought to form biofilms in the lungs of CF patients, resulting in a chronic infection that lasts several years to decades [20,21]. This infection leads to an inflammatory response by the host immune system that ultimately results in lung failure [22,23]. The CF model provides a framework for studying genetic adaptation of P. aeruginosa over the course of chronic infec-tions. Typical changes are the transition to mucoidy (overproduction of the matrix glucan, alginate) and accumulation of mutations that result in increases in antibiotic resistance [22,23]. The failure to effectively treat P.aeruginosa infec-tions is a major cause of morbidity and mortality in CF patients.

While it is tempting to imagine that it is a monospecies biofilm coating the lungs of a CF patient, it is unlikely that P. aeruginosa is the only bacterium residing in the gooey mucous layer that coats a CF patient’s lungs. More sen-sitive assays are being used to document the variety of organisms that comprise the CF microbiome [24,25]. These studies have gener-ated an astounding list of bacteria associated with the CF microbiome. For instance, using culture-dependent, as well as culture-indepen-dent methods, one study found 48 different bacterial families associated with sputum from six different CF patients [24].

Urinary tract infections (UTIs) are considered to be one of the most common bacterial infec-tions worldwide. The majority of these infec-tions are caused by uropathogenic Escherichia coli, which is capable of forming biofilms [26]. While UTIs can be acute and self-limiting, they often recur within 6 months. This recurrent nature strongly suggests that biofilms contribute to their persistence. Accordingly, studies of blad-der infections in mouse models have implicated a novel form of E. coli biofilms called intra cellular bacterial communities (IBCs) [27,28]. IBCs exhibit the standard criteria used to designate a biofilm: they are high-density communities of E. coli surrounded by a bacterially derived sugar-rich matrix. E. coli IBCs are attached to the wall of the bladder and serve as a resevoir of bacteria for repeated rounds of acute infection despite antibiotic therapy [29–31].

S. aureus and S. epidermidis are commonly isolated species from the skin and mucous mem-branes of humans. The majority of medically related research has been focused on S. aureus because of its obvious virulence capacity. S. aureus biofilms have been implicated in dis-eases such as osteomylitis, medical implant-related infections, chronic wound infections and endocarditis [32]. Until recently, S. epidermidis was considered an inocuous skin organism, but it is now recognized as one of the most common causes of nosocomial infections [33]. This change in status is directly related to its ability to form biofilms on medical implants such as catheters, heart valves and artificial joints [33]. Thus, genes important for biofilm formation and persistence are considered major virulence factors of this organism.

These examples of biofilm-based infections serve as a reminder of the importance of under-standing biofilm-specific antibiotic resistance. However, they also illustrate the complicated nature of these infections where the bacteria are

Biofilm-specific antibiotic resistance Review


part of a multifaceted community that somehow survives in a complex environment such as our bodies.

Gene expression differencesA key concept that can confound biofilm gene expression studies is the existence of multiple microenvironments within biofilms. More specifically, concentration gradients of oxy-gen, metabolites and signaling molecules, have been measured (reviewed in [2]). Thus, differ-ent cells within the biofilm experience varying levels of nutrients, waste products and signaling molecules. These differences may be small, but they can lead to alterations in gene expression profiles, which in turn, can further change the micro environment of any given cell within the biofilm. If we consider that biofilm-specific anti-biotic resistance is not due to mutations, it could emerge from differential expression of wild-type genes in biofilms relative to planktonic cells.

Several studies have investigated differential gene expression within a biofilm using fluores-cent microscopy. For instance, the agr locus encodes the S. aureus quorum-sensing system [34]. Using time-lapse confocal microscopy, Yarwood et al. monitored agr-gfp expression in a developing biofilm over 40 h [35]. They found that agr was expressed in a subset of cells within microcolonies and that the expression occurred in three waves, where expression peaked and then waned [35].

In P. aeruginosa, rhamnolipids are surfac-tants that are important for biofilm develop-ment. Expression of the rhamnolipid synthesis genes, rhlAB, is under the control of quorum sensing. Using an rhlA-gfp fusion, Lequette and Greenberg documented heterogeneous expres-sion of GFP within a biofilm, where expression was limited to the stalks of mature macro colonies [36]. Furthermore, using an unstable form of GFP driven by the pqsA promoter, Yang et al. demonstrated that pqsA expression was initially spread throughout the biofilms in 1-day-old bio-films but then localized to the exterior layer of biofilm macrocolonies by day 2. By day 4, pqsA expression was almost nonexistent [37]. Thus, gene expression in biofilms varies over both time and space. Even cell death in biofilms occurs in a temporal and spatial manner. Biofilms in a flow-ing system were stained with propidium iodide, a viability probe, and cell death was observed within localized regions of macrocolonies [38].

In an effort to study interactions between sub-populations of cells within a developing biofilm, Yang et al. analyzed mixed biofilms that were

marked by different fluorescent markers [39]. The authors demonstrated that while the expression of pvdA (required for the production of the sid-erophore pyoverdine) was limited to the stalks of macrocolonies, expression of pvdA in this loca-tion was required for the proper formation of the cap-like structures of mature macrocolonies [39].

Given the heterogeneous nature of biofilm environments, overall conclusions about gene expression based on transcriptomic and pro-teomic approaches might be misleading, since they present averages over a whole biofilm. Researchers at the Center for Biofilm Engineering (MT, USA) have utilized a technique called laser capture microdissection microscopy (LCMM) that begins to address biofilm heterogeneity by enabling the isolation of specific subsections of a biofilm [40]. LCMM followed by quanti-tative PCR allows for the characterization of gene expression within each subsection. Using this approach, Williamson and colleagues iso-lated cells located at the top and the bottom of a P. aeruginosa biofilm and reported that tran-scripts indicative of active metabolism were abundant at the top of biofilms, whereas they were not abundant at the bottom of biofilms [41]. Furthermore, the metabolically inactive cells at the bottom of the biofilm were less susceptible to Tb and ciprofloxacin, compared with the cells within the active section [41].

Mechanisms of biofilm-specific antibiotic resistance

MatrixThe biofilm matrix is composed of extracellular polysaccharides, DNA and proteins that serve to provide structure and protection to the cells in the biofilm [42,43]. The matrix can serve as a bar-rier to antimicrobial agents such as bleach and antibiotics, where these compounds are bound or consumed by the components of the matrix. However, this attribute varies with the type of matrix, the type of antimicrobial agent and the age of the biofilm. For instance, using a colony biofilm model system, Walters et al. demon-strated that the fluoroquinolone antibiotic cip-rofloxacin was able to penetrate P. aeruginosa biofilms, whereas the aminoglycoside antibiotic Tb was unable to penetrate the biofilm [44]. Conversely, other studies suggest that cipro-floxacin is consumed by matrix components and therefore does not diffuse through the matrix very rapidly [45]. These studies utilized different experimental conditions and therefore it is likely that these conflicting conclusions are due to the heterogeneity of biofilms.

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PolysaccharidesP. aeruginosa biofilm matrix is composed of at least three exopolysaccharides – alginate, Psl and Pel [43]. Mutants producing alginate but deficient in both Psl and Pel are unable to form biofilms [46]. Studies have shown that Pel contributes to antibiotic resistance in biofilms. A DpelA mutant is more sensitive to Tb and gentamicin in the 96-well microtitre plate-based MBC-B assay [47]. Furthermore, in a colony biofilm assay, PA14 DpelB deletion mutant is more sensitive to Tb and gentamycin, compared with the wild-type strain. This sensitivity was not observed when ciprofloxacin was used in the assay. This result is strain-specific; deletion of pelB in the stan-dard laboratory P. aeruginosa strain, PA01, has no effect on resistance [48]. Overexpression of pel in planktonic cells resulted in an increase in planktonic resistance, suggesting that Pel expres-sion is a resistance mechanism [48]. However, the molecular basis of Pel-specific resistance has not been worked out.

Despite not being essential for biofilm for-mation [49], overproduction of alginate results in mucoid strains that are more resistant to Tb compared with an isogenic non-mucoid strain [50]. Furthermore, alginate has been implicated in protection against macrophage killing [51].

The S. epidermidis biofilm matrix is mainly composed of an exopolysaccharide called poly-saccharide intercellular adhesion (PIA; reviewed in [52]). S. epidermidis mutants that do not pro-duce PIA are unable to establish an infection in the intestine of Caenorhabditis elegans, a model system for host–pathogen interactions [53,54]. Interestingly, PIA mutants had no defect in the C. elegans model when C. elegans with impaired immune responses were used, suggesting that PIA protects against the C. elegans immune system [54].

These studies all support a role for biofilm sug-ars protecting biofilms against antibiotics. While the exact mechanism is unclear, the authors speculate that they bind or sequester antibiotics, similar to ndvB-derived glucans (see below) [48].

DNAExtracellular DNA (eDNA) is an abundant component of the biofilm matrix. The DNA originates from dead cells, outer membrane vesicles and quorum-sensing regulated release of DNA [42,55]. eDNA was initially identified as a structural component of the biofilm matrix [42]. However, Mulcahy et al. recently described a novel cation chelating property [56]. They found that adding eDNA to a planktonic cul-ture of P. aeruginosa had a negative impact on

growth. Investigation into this phenomenon led to the understanding that the eDNA was chelat-ing cations, which disrupted the bacterial cell membranes, resulting in cell lysis. Interestingly, eDNA induces the expression of an operon (PA3552–3559) required for resistance to cat-ionic antimicrobial peptides in biofilms, and this induction is the basis for eDNA induction of resistance to cationic antimicrobial peptides and aminoglycosides [56].

Colony variantsIn P. aeruginosa, different colony morphology variants have been described. Small colony vari-ants (SCVs) are small, slow-growing bacteria that can be isolated from CF patients and also from in vitro-grown biofilms. These variants are often extremely resistant to antibiotic treat-ment. Recent phenotypic and genome ana lysis of one SCV has revealed that this resistance is multifactorial with multiple mutations resulting in increases in the expression of two multidrug efflux pumps and alterations in genes affecting the outer membrane composition [57].

Rugose SCVs are typically isolated from CF patients with a chronic infection and also from laboratory-grown biofilms. They produce small, wrinkled colonies on solid media and have an increased capacity for biofilm formation [58,59]. Furthermore, these variants display an increased resistance to Tb when grown as biofilms [59]. Detailed ana lysis of rugose SCVs isolated from both in vitro biofilms and CF patients revealed an increased expression of psl and pel synthesis genes, increased levels of the intracellular sig-naling molecule, cyclic di-GMP, and decreased motility [60].

While it is not completely clear why these col-ony variants are more resistant to antibiotics, it is likely that the heterogeneity that exists within the biofilm provides selective pressure that then leads to diversification within the biofilm. This diversification could lead to the selection of colony variants.

PersistersPersisters are dormant cells that exist in both planktonic and biofilm cultures. They contrib-ute to the persistence of biofilms. Essentially, cell metabolism is slowed or shut down in persist-ers, creating a situation where antibiotics can-not act because their targets are not active [17]. Persisters were identified in 1944, but recently work from Lewis’ group has sparked molecular ana lysis of this tolerant subpopulation [61]. In order to isolate persisters and study their gene



expression profile, Shah et al. took advantage of the fact that persisters could be identified within a population of cells by sorting bright versus dim (the persisters) GFP-expressing cells [62]. This study, as well as other recent studies, has linked toxin–antitoxin genes in persister formation.

Plasmid-encoded toxin–antitoxin genes sta-bilize plasmids due to a system where the stable toxin targets an essential cellular function and the labile antitoxin prevents toxin function [63]. Chromosomally encoded toxin–antitoxin sys-tems exist but they are less-well characterized (reviewed in [64]). Various stress conditions including biofilm and antibiotic exposures can induce toxin–antitoxin systems [64].

RelE and MazF are two chromosomally encoded toxins that have been identified as being important for persister formation [17,64]. Both proteins inhibit protein synthesis through their function as mRNA endonucleases. Expression of their antitoxins, RelB and MazE, respectively, inhibit the destructive effects of the toxins [65].

yafQ is another toxin gene in E. coli. Deletion of yafQ did not affect biofilm formation and resulted in a decrease in cell survival in bio-films after exposure to cefazolin and Tb, but not doxycycline or rifampin [66], suggesting that the mechanism of resistance is specific.

An additional suggestion for why persisters are less sensitive to antibiotics is that they do not produce hydroxyl radicals in response to exposure to bactericidal antibiotics [67]. This observation is linked to the recent discovery that bactericidal antibiotics, despite having different cellular targets, kill cells through a common mechanism based on the production of reactive oxygen species [68,69].

Biofilm-specific antibiotic resistance genesThe mechanisms of resistance discussed so far contribute to more passive mechanisms. For instance, persisters ‘shut down’ antibiotic targets and the biofilm matrix retards/prevents the dif-fusion of antibiotics. The mechanisms discussed below represent more active mechanisms of resis-tance. These genes are expressed in response to growth in a biofilm and their wild-type function is required for resistance.

In order to determine if there were specific genes that are important for biofilm-specific antibiotic resistance, we took a genetic approach and screened a random transposon insertion mutant library for mutants that were more sensi-tive to Tb, compared with the wild type P. aeru-ginosa PA14 strain [70]. The screen yielded six

novel genes that we have begun to characterize. Deletion of each locus, individually, resulted in a two- to eight-fold reduction in biofilm-specific antibiotic resistance to Tb while having no effect on growth or biofilm formation [70–72] [Zhang L,

Mah TF, Unpublished Data]. Interestingly, all six genes are more highly expressed in a biofilm compared with rapidly growing planktonic cells, supporting the assertion that they are important for biofilm-specific antibiotic resistance. These genes are discussed below.

ndvBThe ndvB gene product of P. aeruginosa is 37% identical and 58% similar to Bradyrhizobium japonicum NdvB. Based on this sequence similarity to B. japonicum, the P. aeruginosa ndvB gene was predicted to encode a glucosyl-transferase that is required for the synthesis of cyclic-b-(1,3)-glucans [73,74]. This predic-tion was confirmed by the determination of the structure of P. aeruginosa glucans by NMR [75]. P. aeruginosa cyclic glucans are composed of 12–16 glucose molecules that are linked by b-(1,3) linkages and modified by phospho-glycerol [75]. It was demonstrated that peri-plasmic glucans from a partially pure glucan fraction from the wild-type strain interact with Tb [70]. There was no interaction detected between an equivalent fraction from the ndvB mutant strain and Tb [70]. Furthermore, an independent group confirmed our observa-tion and reported that P. aeruginosa glucans interact with aminoglycosides [75]. Based on these data, it was proposed that glucans confer resistance to antibiotics by sequestering these anti biotics in the periplasm and thus away from their cellular targets [70]. A similar mechanism of interaction has been proposed for Brucella abortus cyclic glucans and cholesterol [76], as well as Candida albicans extracellular glucans and antifungals [77,78].

We have recently identified another role that ndvB has in antibiotic resistance in biofilms. A DNA microarray experiment revealed that the presence of ndvB is important for the expres-sion of ethanol oxidation genes in biofilms; these genes are not expressed in a DndvB biofilm [79]. However, addition of wild-type periplasmic extract, but not DndvB periplasmic extract, was able to restore wild-type expression of represen-tative ethanol oxidation genes. Interestingly, purified wild-type glucans lacked this activity, suggesting that a signal that is important for the expression of ethanol oxidation genes is associ-ated with wild-type glucans. Furthermore, we

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confirmed the importance of the ethanol oxida-tion genes biofilm-specific antibiotic resistance, although their role in this resistance is unclear. Thus, ndvB has at least two roles in biofilm-specific antibiotic resistance: by sequestering antibiotic molecules before they reach their cel-lular targets and by affecting the expression of several P. aeruginosa genes [79].

PA1875–1877A second locus, PA1875–1877, identified in the screen has been characterized [71]. This three-gene operon encodes, respectively, an outer mem-brane protein (OpmL), an ATP-binding cas-sette (ABC) transporter and a membrane fusion protein (HlyD homolog) [71,80]. In E. coli, an ABC transporter complex comprised of HlyB–HlyD–TolC is involved in the export of hemo-lysin [81]. It is possible that PA1875–1877 form a multi component efflux complex that is similar to that of the resistance–nodulation–cell division (RND) transporters, which contribute to multi-drug resistance of clinical relevance [82]. Indeed, deletion of these genes resulted in a mutant P. aeruginosa strain that was sensitive to Tb and ciprofloxacin, when the cells were grown in bio-films. Furthermore, the deletion mutant biofilm accumulated more Tb than the wild-type biofilm, suggesting that PA1875–1877 does function as an efflux pump. Consistent with these results, cloned PA1875–1877 also increased the MIC of wild-type planktonic cells to Tb [71]. While efflux pumps are known to increase the antibiotic resis-tance of planktonic cells, this is the first time that an efflux pump has been shown to be important for biofilm-specific resistance to antibiotics.

tssC1The first type VI secretion (T6S) system was characterized in Vibrio cholera in 2006 [83]. Since that time, T6S systems have been identi-fied in several additional pathogenic organisms, including Francisella tularensis, Burkholderia species, E. coli and P. aeruginosa [84,85]. T6S systems have been implicated in virulence, fit-ness in chronic infection and toxin delivery to bacteria [85–89]. In P. aeruginosa, there are three T6S loci: HSI-I, HSI-II and HSI-III [90]. tssC1 was identified in our screen for biofilm-specific antibiotic resistance genes. It is a component of HSI-I. Its homolog in V. cholera, VipA, along with the homolog of TssB1 (VipB) form a com-plex similar to a bacteriophage tail sheath [91]. Deletion mutants of either tssC1 or hcp1, another component of the HSI-I T6S system, had defects in biofilm-specific sensitivity phenotype,

suggesting that this secretion system has a role in biofilm-specific antibiotic resistance [72].

Other genes identified in the screenOther genes identified in our screen for poten-tial biofilm-specific resistance include PA0757, PA2070 and PA5033. The function of these genes in antibiotic resistance is still under investigation. PA0757 is predicted to encode a two-component sensor histidine kinase that is located within an operon with PA0756, a pre-dicted two-component response regulator [80]. In bacteria, two-component regulatory systems act to convey information from the environ-ment to the cell, thus it is possible that this two- component regulatory system responds to signals that indicate a potential threat to the biofilm cells. PA2070 is predicted to encode a hypothetical protein with transmembrane transporter activity [80]. PA5033 is predicted to encode a protein of unknown function with a predicted type 1 secretion signal [80].

Efflux pump genesThe P. aeruginosa genome contains genes encod-ing a total of 12 RND efflux pumps, several of which have been implicated in clinical resistance [82,92] and stress responses [93] in planktonic cells. A microarray ana lysis comparing gene expres-sion of planktonic cultures versus biofilms did not reveal differential expression of any RND pumps [94]. These results are consistent with another recent transcriptional ana lysis demon-strating that RND pumps were not activated in biofilms [95]. Interestingly, under standard con-ditions, expression of two major P. aeruginosa efflux pumps, MexAB–OprM and MexCD–OprJ, decreases as biofilms develop [96], further suggesting that RND pumps are not involved in biofilm-specific antibiotic resistance.

Despite the above observations, certain drug-specific resistance of biofilm cells in P. aeru-ginosa appears to be associated with RND pumps. In response to the macrolide antibiotic azithromycin, Gillis and colleagues determined that P. aeruginosa biofilms exhibited increased expression of several efflux pumps, including MexCD–OprJ and MexXY [97]. Analysis of the ability of different efflux pump deletion mutants to form biofilms in the presence of azithromycin, as well as documentation of the expression pat-terns of the efflux pumps led to the conclusion that the mexCD–oprJ efflux pump represents a biofilm-specific antibiotic resistance mechanism [97]. Taken together, these data suggest that care-ful ana lysis should be performed before making



conclusions about the role of drug efflux pumps in biofilms.

RapARapA was identified in a screen for E. coli genes that are important for biofilm-specific resistance to penicillin G [98]. The function of RapA in bacteria has not been clearly defined; however, loss of the rapA gene from the E.coli genome results in the differential expression of 22 genes. One of these genes, yhcQ, is predicted to encode an efflux pump, suggesting that RapA controls the expression of genes that might have a direct effect on antibiotic resistance [98].

HeterogeneityUsing differentially tagged wild-type and motility mutants of P. aeruginosa, Klausen et al. discovered that there are different types of cells within the biofilm that act to produce the specific structures that are characteristic of a mature biofilm [99]. Motile cells in biofilm become the cap of the macrocolonies, while the cells in the stalk are non-motile [99]. Using confocal laser scanning microscopy, GFP (to mark live cells) and propidium iodide (to mark dead cells), Haagensen et al. showed that the motile cells in the cap are tolerant to colistin and SDS while the non-motile cells in the stalk are sensitive to antibiotics [100]. They determined that the motility is based on type IV pili and that the pmr operon is important for tolerance. Furthermore, they demonstrated that the pmr operon was expressed in the cap in response to colistin [100]. Using their expertise in con- Using their expertise in con-Using their expertise in con-focal laser scanning microscopy, Pamp et al. extended these observations by establishing that the metabolically active cells localize to the periphery of biofilms using an unstable GFP. They then showed that different antimicrobial agents target cells that exhibit different levels of metabolic activity. They also build on the observation that this tolerance is dependent on the pmr operon and also demonstrated that the tolerance also involves the mexAB–oprM efflux pump [101]. mexAB–oprM are not expressed in biofilms initially, but after exposure to colis-tin, mexAB–oprM expression and sensitivity to colistin are co-expressed. Together, these stud-ies use microscopy to beautifully illustrate how metabolically active cells become resistant and that this is dependent on specific genes. In other words, these experiments suggest that active cells are needed for resistance, as compared with other mechanisms of resistance or tolerance that are based on dormancy.

Other important considerationsThis review has focused on the intrinsic mecha-nisms of resistance found in biofilm communi-ties. However, mutations do influence antibiotic resistance in biofilms: in vivo environments tend to be stressful (lack of nutrients, presence of anti-microbial agents) and stressful conditions result in an increased rate of mutation. For instance, a recent in vitro ana lysis of P. aeruginosa popula-tions in the CF lung found that upon exposure to sublethal levels of ciprofloxacin, the wild-type strain was able to develop a single mutation (e.g., in an efflux pump) that would increase resis-tance by a certain level [102]. However, mutator strains (mutations in the DNA mismatch repair system) were more likely to develop two-step mutations that increased resistance by a greater amount [102].

Many medically important bacteria can exhibit a social type of motility called swarm-ing. Lai et al. analyzed the antibiotic resistance phenotype of P. aeruginosa, E. coli, Serratia marcescens, Burkholderia thailandensis and Bacillus subtilis swarm cells and found that, like biofilm cells, they were all more resistant than their planktonic counterparts [103]. It is not clear why swarm cells are more resistant. However, the authors suggest that multicellularity leads to resistance [103].

Most in vitro studies focus on monospecies biofilms since this leads to a more tractable system. However, monospecies biofilms are unlikely to exist in nature. Therefore, cells in biofilms are surrounded by a ‘soup’ of signals produced by all of the other inhabitants of the particular ecosystem. Ryan et al. addressed the likelihood that a signal from one bacterium can influence the antibiotic resistance level of another. This group cultured dual-species bio-films comprised of Stenotrophomonas maltophilia and P. aeruginosa [104]. They discovered that a diffusible signal factor produced by S. malto-philia induces a stress response in P. aeruginosa that results in increased resistance to polymyxins [104]. Polymyxins are produced by soil microbes, so induction of resistance to this particular class of antimicrobial agent is important in the soil environment.

ConclusionAntibiotic resistance in biofilms is a complex phenomenon that is the result of multiple mechanisms of resistance working together. The evidence suggests that the resistant bio-film cells are simply responding to stress. For instance, eDNA is linked to stress: the DNA

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creates a cation-limited environment [56], ndvB involved in regulating the expression of ethanol oxidations genes [79], and subinhibitory concen-trations of antibiotics induce biofilm formation in a variety of bacteria [105,106]. Furthermore, Nguyen et al. demonstrated that inactivation of the stringent response in biofilms decreased resistance to four different classes of antibiotics [107]. It has also been shown that universal stress genes are more highly expressed at the top of a thick biofilm [41]. These mechanisms of resis-tance were not necessarily developed in response to the presence of antibiotics. They were devel-oped in response to the myriad of different environments that biofilm bacteria find them-selves in. For example, in the soil environment, there are countless molecules produced by the countless soil organisms sharing the ecosystems. These molecules include antimicrobial agents produced by the other organisms that can also act as signals at low concentrations [108]. Plus, access to nutrients and water is limited. Bacteria have to adapt to changes in the environment and respond to these various signals in order to per-sist. Thus, it makes sense that there would be so many different mechanisms that act to protect biofilm cells.

Future perspectiveGiven the innate heterogeneity in biofilms, it is likely that there is a temporal and spatial aspect

to expression of these resistance mechanisms. Microscopy will be important in driving this research forward. The majority of results dis-cussed in this review have been derived from experiments performed with single-species bio-films. Future work should focus on multispecies biofilms and resistance. Finally, it is clear that bacteria respond to their environment. Biofilms are rich with signaling interactions between their various component species. These signals (in some cases antibiotics) serve to inform the residents of the community who else is out there and demand a response.

Executive summary

Bacterial biofilms n Bacteria transition from free-swimming, planktonic cells to surface-attached biofilms in response to environmental signals. n Biofilms are complex structures that are composed of bacterial cells surrounded by a polymeric matrix. n Heterogeneity is a key attribute of biofilms, resulting in subsections where different conditions exist. n Bacteria in a biofilm can be up to 1000-times more resistant to antibiotics than their planktonic counterparts. n Medically important biofilms include chronic Pseudomonas aeruginosa infections on the lungs of cystic fibrosis patients, Staphylococcus

aureus and Staphylococcus epidermidis medical implant-related infections and Escherichia coli-based urinary tract infections.

Biofilm-specific antibiotic resistance n Definition: biofilm-specific resistance includes tolerance to antimicrobial agents and means that the resistance (or tolerance) mechanism

occurs predominantly in biofilm cells, not in planktonic cells.

Mechanisms of biofilm-specific antibiotic resistance n The components of the biofilm matrix can prevent antibiotics from reaching the cells within the biofilm. n Colony variants arise from biofilms and display increased resistance due to altered expression of specific genes. n Persisters are dormant cells found in planktonic and biofilm cultures. Cell metabolism is slowed or shut down in persisters, creating a

situation where antibiotics do not work because their targets are not active. n Several biofilm-specific antibiotic resistance genes have been identified. These genes are more highly expressed in biofilms compared

with planktonic cells and represent novel mechanisms of antibiotic resistance. Examples include ndvB, PA1875–1877 and tssC1. n Much of the evidence suggests that these mechanisms represent a general stress response in biofilms.

Future perspective n Heterogeneity needs to be addressed. n Antibiotic resistance needs to be studied in the context of multispecies biofilms.

AcknowledgementsThe author would like to thank G Anderson, A Hinz and X-Z Li for critical review of the manuscript, and B Pitts, PS Stewart and E Werner for contributing Figure 1.

Financial & competing interests disclosureResearch in T-F Mah’s laboratory has been supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and Cystic Fibrosis Canada. The author has no other relevant affiliations or financial involvement with any organi-zation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.



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