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The Role of Efflux and Physiological Adaptation in Biofilm Tolerance and Resistance*

  • Heleen Van Acker
    Affiliations
    Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium
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  • Tom Coenye
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium
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  • Author Footnotes
    * This work was supported by FWO-Vlaanderen and the Interuniversity Attraction Poles Programme initiated by the Belgian Science Policy Office. This is the fifth article in the Thematic Minireview series “Biofilms.” The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Figs. S1 and S2.
Open AccessPublished:April 21, 2016DOI:https://doi.org/10.1074/jbc.R115.707257
      Microbial biofilms demonstrate a decreased susceptibility to antimicrobial agents. Various mechanisms have been proposed to be involved in this recalcitrance. We focus on two of these factors. Firstly, the ability of sessile cells to actively mediate efflux of antimicrobial compounds has a profound impact on resistance and tolerance, and several studies point to the existence of biofilm-specific efflux systems. Secondly, biofilm-specific stress responses have a marked influence on cellular physiology, and contribute to the occurrence of persister cells. We provide an overview of the data that demonstrate that both processes are important for survival following exposure to antimicrobial agents.

      Introduction

      Microbial biofilms are surface-attached communities, consisting of cells embedded in an extracellular polymeric matrix that is at least partially composed of polymers produced by the microorganism themselves (
      • Costerton J.W.
      The Biofilm Primer.
      ). Biofilms are omnipresent in natural and man-made environments (
      • Costerton J.W.
      The Biofilm Primer.
      ,
      • Hall-Stoodley L.
      • Costerton J.W.
      • Stoodley P.
      Bacterial biofilms: from the natural environment to infectious diseases.
      ), and biofilm-associated bacteria are involved in a wide range of infections, including respiratory tract infections in cystic fibrosis (CF)
      The abbreviations used are: CF
      cystic fibrosis
      RND
      resistance-nodulation-division
      PAβN
      phenyl-arginine-β-naphthylamide
      c-di-GMP
      cyclic-di-guanosine monophosphate
      (p)ppGpp
      guanosine tetra- or pentaphosphate
      ROS
      reactive oxygen species
      TA
      toxin antitoxin
      TCA
      tricarboxylic acid.
      patients, chronically infected wounds, and device-related infections (
      • Wolcott R.D.
      • Rhoads D.D.
      • Bennett M.E.
      • Wolcott B.M.
      • Gogokhia L.
      • Costerton J.W.
      • Dowd S.E.
      Chronic wounds and the medical biofilm paradigm.
      ,
      • Bjarnsholt T.
      The role of bacterial biofilms in chronic infections.
      ). One of the hallmarks of these biofilm-associated infections is the frequent failure of antimicrobial chemotherapy. Although it is often postulated that sessile cells are more resistant to antimicrobial agents, these cells typically do not grow better than planktonic cells in the presence of antibiotics; for example, biofilm-associated and stationary-phase planktonic Burkholderia cepacia complex bacteria showed similar susceptibilities to antibiotics (
      • Peeters E.
      • Nelis H.J.
      • Coenye T.
      In vitro activity of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole against planktonic and sessile Burkholderia cepacia complex bacteria.
      ). However, it is much more difficult to kill biofilm-associated cells than planktonic cells (
      • Lewis K.
      Multidrug tolerance of biofilms and persister cells.
      ), and various mechanisms that potentially could be involved in this have been described in the literature (see Refs.
      • Lebeaux D.
      • Ghigo J.M.
      • Beloin C.
      Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics.
      and
      • Van Acker H.
      • Van Dijck P.
      • Coenye T.
      Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms.
      for recent reviews as well as Refs.
      • Sheppard D.C.
      • Howell P.L.
      Biofilm exopolysaccharides of pathogenic fungi: lessons from bacteria.
      and
      • Gunn J.S.
      • Bakaletz L.O.
      • Wozniak D.J.
      What's on the outside matters: The role of the extracellular polymeric substance of Gram-negative biofilms in evading host immunity and as a target for therapeutic intervention.
      in this minireview series). Avoiding exposure to (sufficiently high concentrations of) antibiotics, and the presence of a small population of specialized survivor cells that are tolerant toward particular antimicrobial agents (i.e. they are not killed upon exposure to the product) are two important mechanisms that will be discussed in this review.

      The Role of Efflux in Biofilm Resistance

      Bacteria use specialized membrane-associated proteins to expel a wide range of compounds from the cytoplasm (
      • Routh M.D.
      • Zalucki Y.
      • Su C.C.
      • Zhang Q.
      • Shafer W.M.
      • Yu E.W.
      Efflux pumps of the resistance-nodulation-division family: a perspective of their structure, function, and regulation in Gram-negative bacteria.
      ). Combined with reduced influx of these compounds and/or enzymatic degradation, efflux pumps are responsible for keeping the cytoplasmic concentrations of certain antimicrobial compounds below a critical threshold (
      • Routh M.D.
      • Zalucki Y.
      • Su C.C.
      • Zhang Q.
      • Shafer W.M.
      • Yu E.W.
      Efflux pumps of the resistance-nodulation-division family: a perspective of their structure, function, and regulation in Gram-negative bacteria.
      ,
      • Soto S.M.
      Role of efflux pumps in the antibiotic resistance of bacteria embedded in a biofilm.
      ). Bacterial efflux pumps can be divided into several superfamilies, with members of the RND family being the most studied when it comes to their involvement in bacterial biofilm resistance and/or biofilm formation. They are composed of an inner membrane protein, a periplasmic membrane fusion protein, and an outer membrane protein (
      • Tseng T.T.
      • Gratwick K.S.
      • Kollman J.
      • Park D.
      • Nies D.H.
      • Goffeau A.
      • Saier Jr., M.H.
      The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins.
      ,
      • Nikaido H.
      Structure and mechanism of RND-type multidrug efflux pumps.
      ). This complex spans the Gram-negative cell envelope and allows the efficient translocation of a wide range of molecules (supplemental Fig. S1).

      Enterobacteriaceae: Escherichia coli, Klebsiella pneumoniae, and Salmonella

      In biofilms formed by two E. coli strains, a considerable fraction of genes up-regulated in biofilms as compared with planktonic cells was shown to be involved in efflux and transport (128 out of 600 up-regulated genes) (
      • Kvist M.
      • Hancock V.
      • Klemm P.
      Inactivation of efflux pumps abolishes bacterial biofilm formation.
      ). It was claimed that this up-regulation was a direct consequence of the “waste management problem” occurring in the “cramped conditions” encountered in the biofilm. When cells of various E. coli strains and a strain of K. pneumoniae were exposed to the efflux inhibitors thioridazine, phenyl-arginine-β-naphthylamide (PAβN), or 1-(1-naphthylmethyl)-piperazine (NMP), biofilm formation was significantly repressed, suggesting that functional efflux systems are required for full biofilm formation. In addition, these efflux pump inhibitors increased the activity of tetracycline against biofilms (
      • Kvist M.
      • Hancock V.
      • Klemm P.
      Inactivation of efflux pumps abolishes bacterial biofilm formation.
      ). In enteroaggregative E. coli, the TolC efflux pump is required for adherence to HEp-2 cells and biofilm formation, probably because it plays an important role in secreting a yet unidentified factor (
      • Imuta N.
      • Nishi J.
      • Tokuda K.
      • Fujiyama R.
      • Manago K.
      • Iwashita M.
      • Sarantuya J.
      • Kawano Y.
      The Escherichia coli efflux pump TolC promotes aggregation of enteroaggregative E. coli 042.
      ).
      Salmonella enterica serovar Typhimurium mutants lacking any of the known efflux systems also showed a marked reduction in biofilm formation (
      • Baugh S.
      • Ekanayaka A.S.
      • Piddock L.J.
      • Webber M.A.
      Loss of or inhibition of all multidrug resistance efflux pumps of Salmonella enterica serovar Typhimurium results in impaired ability to form a biofilm.
      ). It was observed that in these mutants, the expression of several curli genes was down-regulated, and these mutants effectively failed to produce curli. As curli fimbriae are an important component of the Salmonella biofilm matrix, and have previously been shown to be involved in adhesion, cell aggregation, and biofilm formation (
      • Barnhart M.M.
      • Chapman M.R.
      Curli biogenesis and function.
      ), this presents a functional connection between efflux and biofilm formation. Also, in this study, the anti-biofilm effects of inactivating particular efflux systems could be mimicked by adding efflux pump inhibitors, including PAβN, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and chlorpromazine. In a subsequent study, the same authors focused on the S. enterica serovar Typhimurium AcrAB-TolC system and showed that AcrA and TolC are involved in biofilm formation, whereas AcrB is not, and in both the acrA and the tolC mutant, the expression of structural curli genes and regulatory genes involved in curli biosynthesis was significantly down-regulated as compared with the wild type strain (
      • Baugh S.
      • Phillips C.R.
      • Ekanayaka A.S.
      • Piddock L.J.
      • Webber M.A.
      Inhibition of multidrug efflux as a strategy to prevent biofilm formation.
      ).

      Pseudomonas aeruginosa

      The genome of the gamma-proteobacterium P. aeruginosa contains at least 12 RND pump-encoding operons (
      • Kumar A.
      • Schweizer H.P.
      Bacterial resistance to antibiotics: active efflux and reduced uptake.
      ,
      • Fernández L.
      • Hancock R.E.
      Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance.
      ). Although initial studies on the mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY efflux systems in P. aeruginosa did not point to a major role in biofilm resistance, expression of these efflux systems in biofilms was found to be heterogeneous, with cells closest to the substrate showing the highest expression levels (
      • De Kievit T.R.
      • Parkins M.D.
      • Gillis R.J.
      • Srikumar R.
      • Ceri H.
      • Poole K.
      • Iglewski B.H.
      • Storey D.G.
      Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms.
      ). This should not come as a surprise, as it is currently well established that different populations can be found in microbial biofilms, and these subpopulations of sessile cells are exposed to different chemical and physical environments, and hence show differences in physiology (for reviews on this topic, see Refs.
      • Stewart P.S.
      • Franklin M.J.
      Physiological heterogeneity in biofilms.
      and
      • Coenye T.
      Response of sessile cells to stress: from changes in gene expression to phenotypic adaptation.
      ). Subsequent investigations revealed that mexAB-oprM and mexCD-oprJ are essential for P. aeruginosa biofilm formation in the presence of azithromycin, as a mutant in which both systems were knocked out was not capable of forming biofilms in the presence of this macrolide (
      • Gillis R.J.
      • White K.G.
      • Choi K.H.
      • Wagner V.E.
      • Schweizer H.P.
      • Iglewski B.H.
      Molecular basis of azithromycin-resistant Pseudomonas aeruginosa biofilms.
      ). Remarkable in this regard is that mexC was only expressed in azithromycin-exposed sessile cells, whereas mexA expression was observed in both exposed and unexposed planktonic and sessile cells, suggesting that the P. aeruginosa MexCD-OprJ pump is a biofilm-specific defense mechanism against azithromycin (
      • Gillis R.J.
      • White K.G.
      • Choi K.H.
      • Wagner V.E.
      • Schweizer H.P.
      • Iglewski B.H.
      Molecular basis of azithromycin-resistant Pseudomonas aeruginosa biofilms.
      ). Similarly, it was suggested that the P. aeruginosa MexAB-OprM pump provides a biofilm-specific defense mechanism against colistin (
      • Pamp S.J.
      • Gjermansen M.
      • Johansen H.K.
      • Tolker-Nielsen T.
      Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes.
      ). These initial studies targeting known RND-type efflux pumps already pointed to a role for efflux in biofilm resistance, and this was further confirmed in a large-scale screening study in which ∼4000 P. aeruginosa transposon mutants were investigated (
      • Zhang L.
      • Mah T.F.
      Involvement of a novel efflux system in biofilm-specific resistance to antibiotics.
      ). In this study, the PA1874–1877 operon was identified as a novel efflux system potentially involved in biofilm-specific resistance to antibiotics: the expression of PA1874 was found to be 10-fold higher in sessile than in planktonic cells, deletion of the operon only affected susceptibility to tobramycin in biofilms, and overexpression of the operon decreased the susceptibility toward selected aminoglycosides and fluoroquinolones in planktonic cells (
      • Zhang L.
      • Mah T.F.
      Involvement of a novel efflux system in biofilm-specific resistance to antibiotics.
      ). In addition, in Pseudomonas fluorescens, expression of two efflux systems was up-regulated in glutaraldehyde-treated biofilms, and efflux pump inhibitors increased sensitivity of P. aeruginosa and P. fluorescens biofilms to this disinfectant, confirming that not only antibiotic resistance is mediated by efflux (
      • Vikram A.
      • Bomberger J.M.
      • Bibby K.J.
      Efflux as a glutaraldehyde resistance mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms.
      ). Finally, it was observed that planktonic P. aeruginosa cells grown under hypoxic conditions showed an increased expression of MexEF-OprN (
      • Schaible B.
      • Taylor C.T.
      • Schaffer K.
      Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps.
      ). Although biofilm cells were not investigated in this particular study, the authors speculate that the hypoxia encountered by P. aeruginosa biofilms in the lungs of CF patients could contribute to their antibiotic resistance.
      So far, little is known about the regulation of efflux pump expression in biofilms. However, the work of Karin Sauer and co-workers on the P. aeruginosa biofilm-specific MerR-type transcriptional regulator BrlR points to a possible molecular link between efflux pump expression and the biofilm phenotype. BrlR is a transcription factor that responds to changes in the concentration of the secondary messenger c-di-GMP (see Ref.
      • Römling U.
      • Galperin M.Y.
      • Gomelsky M.
      Cyclic di-GMP: the first 25 years of a universal bacterial second messenger.
      for a recent review as well as Ref.
      • Valentini M.
      • Filloux A.
      Biofilms and c-di-GMP signaling: lessons from Pseudomonas aeruginosa and other bacteria.
      in this minireview series) and regulates the expression of several genes involved in biofilm tolerance, including ndvB (
      • Liao J.
      • Sauer K.
      The MerR-like transcriptional regulator BrlR contributes to Pseudomonas aeruginosa biofilm tolerance.
      ,
      • Chambers J.R.
      • Liao J.
      • Schurr M.J.
      • Sauer K.
      BrlR from Pseudomonas aeruginosa is a c-di-GMP-responsive transcription factor.
      ). Sauer and co-workers (
      • Liao J.
      • Schurr M.J.
      • Sauer K.
      The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug efflux pumps in Pseudomonas aeruginosa biofilms.
      ) also demonstrated that brlR is required for maximal expression of the MexAB-OprM and MexEF-OprN efflux pumps in P. aeruginosa biofilms, and demonstrated a direct regulation of these pumps by BrlR, with BrlR binding to the promotor regions of the mexAB-oprM and mexEF-oprN operons.
      In contrast to the work cited above, in two studies carried out by Stewart and co-workers (
      • Folsom J.P.
      • Richards L.
      • Pitts B.
      • Roe F.
      • Ehrlich G.D.
      • Parker A.
      • Mazurie A.
      • Stewart P.S.
      Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis.
      ,
      • Stewart P.S.
      • Franklin M.J.
      • Williamson K.S.
      • Folsom J.P.
      • Boegli L.
      • James G.A.
      Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.
      ), no evidence was found for a role of efflux pumps in tolerance of P. aeruginosa biofilms. These at first sight contradictory findings suggest that the role of efflux systems in protecting sessile cells against antibiotics may depend on the global biofilm physiology (and thus on the experimental conditions) and/or may point to the presence of specific subpopulations in biofilms that benefit from these efflux pumps, whereas others do not. In addition, the regulatory mechanisms linking efflux with the biofilm-phenotype may be strain- and condition-dependent.
      Most of the above mentioned studies used antibiotic-exposed P. aeruginosa mutant strains to confirm the role of efflux systems in biofilm tolerance, but the role of these systems in P. aeruginosa biofilm formation as such (in the absence of antibiotics) has not been investigated in great detail. However, using efflux pump inhibitors, including carbonyl cyanide m-chlorophenylhydrazone, chlorpromazine, and PAβN, it was shown that in static as well as in flow conditions, efflux pump inhibitors decreased biofilm formation, leading to the suggestion that efflux pump inhibitors could be used as anti-biofilm agents (
      • Baugh S.
      • Phillips C.R.
      • Ekanayaka A.S.
      • Piddock L.J.
      • Webber M.A.
      Inhibition of multidrug efflux as a strategy to prevent biofilm formation.
      ).

      Burkholderia cenocepacia

      The genome of another CF pathogen, B. cenocepacia, contains genes coding for 22 RND efflux systems (
      • Guglierame P.
      • Pasca M.R.
      • De Rossi E.
      • Buroni S.
      • Arrigo P.
      • Manina G.
      • Riccardi G.
      Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome.
      ,
      • Buroni S.
      • Pasca M.R.
      • Flannagan R.S.
      • Bazzini S.
      • Milano A.
      • Bertani I.
      • Venturi V.
      • Valvano M.A.
      • Riccardi G.
      Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance.
      ,
      • Bazzini S.
      • Udine C.
      • Sass A.
      • Pasca M.R.
      • Longo F.
      • Emiliani G.
      • Fondi M.
      • Perrin E.
      • Decorosi F.
      • Viti C.
      • Giovannetti L.
      • Leoni L.
      • Fani R.
      • Riccardi G.
      • Mahenthiralingam E.
      • Buroni S.
      Deciphering the role of RND efflux transporters in Burkholderia cenocepacia.
      ,
      • Rushton L.
      • Sass A.
      • Baldwin A.
      • Dowson C.G.
      • Donoghue D.
      • Mahenthiralingam E.
      Key role for efflux in the preservative susceptibility and adaptive resistance of Burkholderia cepacia complex bacteria.
      ), and high levels of expression of these efflux pumps are frequently observed in B. cepacia complex clinical isolates (
      • Tseng S.P.
      • Tsai W.C.
      • Liang C.Y.
      • Lin Y.S.
      • Huang J.W.
      • Chang C.Y.
      • Tyan Y.C.
      • Lu P.L.
      The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.
      ). Exposure of B. cenocepacia biofilms to the disinfectant chlorhexidine results in the up-regulation of eight RND family efflux pumps (
      • Coenye T.
      • Van Acker H.
      • Peeters E.
      • Sass A.
      • Buroni S.
      • Riccardi G.
      • Mahenthiralingam E.
      Molecular mechanisms of chlorhexidine tolerance in Burkholderia cenocepacia biofilms.
      ). Using mutants in which single RND efflux pumps were inactivated, it was shown that for some mutants (e.g. the ΔRND-9 mutant), biofilms were less tolerant than wild type biofilms (whereas planktonic susceptibility remaining unaltered). However, in other mutants (e.g. the ΔRND-4 mutant), planktonic cells were more susceptible, whereas the susceptibility of sessile cells remained unchanged, confirming that at least some of the RND efflux pumps in this organism are lifestyle-specific (
      • Coenye T.
      • Van Acker H.
      • Peeters E.
      • Sass A.
      • Buroni S.
      • Riccardi G.
      • Mahenthiralingam E.
      Molecular mechanisms of chlorhexidine tolerance in Burkholderia cenocepacia biofilms.
      ).
      Expanding on this work, the role of 16 B. cenocepacia efflux systems in resistance to various antibiotics, including tobramycin and ciprofloxacin, was subsequently investigated (
      • Buroni S.
      • Matthijs N.
      • Spadaro F.
      • Van Acker H.
      • Scoffone V.C.
      • Pasca M.R.
      • Riccardi G.
      • Coenye T.
      Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells.
      ). By measuring susceptibility in planktonic and sessile cultures, it was demonstrated that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia populations, whereas the RND-3, RND-8, and RND-9 efflux pumps are important for protecting sessile cells against tobramycin, again pointing toward the existence of life style-specific RND-type efflux pumps. Interestingly, in the wild type strain, little regulation of expression of these efflux pumps at the mRNA level was observed using RT-quantitative PCR, suggesting that the regulation occurs mainly at the posttranscriptional level and/or at the level of activation of particular efflux systems (
      • Buroni S.
      • Matthijs N.
      • Spadaro F.
      • Van Acker H.
      • Scoffone V.C.
      • Pasca M.R.
      • Riccardi G.
      • Coenye T.
      Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells.
      ).

      Physiological Adaptation in Microbial Biofilms Leads to Reduced Activity of Antimicrobial Agents: Dormancy and the Persister Phenomenon

      Bacterial populations are known to contain a subpopulation of cells tolerant to antimicrobial treatment (
      • Lewis K.
      Multidrug tolerance of biofilms and persister cells.
      ). These so-called persister cells are present in both sessile and planktonic cultures, but are especially problematic in a biofilm environment, where they are shielded from the immune system (
      • Lewis K.
      Persister cells, dormancy and infectious disease.
      ,
      • Jayaraman R.
      Bacterial persistence: some new insights into an old phenomenon.
      ). Persisters are not mutants, but phenotypic variants of the wild type that upon re-inoculation produce a culture that again contains both persister and non-persister bacteria like the original population (
      • Lewis K.
      Persister cells, dormancy and infectious disease.
      ). Unlike antibiotic-resistant bacteria, they do not grow in the presence of bactericidal agents, but resume growth after the antibiotics have been removed (
      • Lewis K.
      Persister cells.
      ). Because antibiotics kill cells by corrupting specific targets, dormant persisters, in which the antibiotic targets are inactive, escape killing (
      • Lewis K.
      Persister cells.
      ). Non-growing or slowly growing bacteria are generally less sensitive to antibiotics, a phenomenon called “drug indifference” (
      • Jayaraman R.
      Bacterial persistence: some new insights into an old phenomenon.
      ). However, persistence and drug indifference are different phenomena: the latter reflects the overall reduced sensitivity of dormant/slow-growing microbial populations without a specific mechanistic basis (e.g. stationary phase bacterial cultures surviving treatment with β-lactams), whereas persistence gives rise to a subpopulation with a different phenotype (
      • Jayaraman R.
      Bacterial persistence: some new insights into an old phenomenon.
      ). The mechanisms leading to persistence are still largely unknown. Screening knock-out libraries has not led to the identification of mutants completely lacking the ability to form persisters, indicating that the mechanisms involved are redundant (
      • Lewis K.
      Persister cells.
      ). However, various studies have identified putative persister genes (
      • Hansen S.
      • Lewis K.
      • Vulić M.
      Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli.
      ,
      • De Groote V.N.
      • Verstraeten N.
      • Fauvart M.
      • Kint C.I.
      • Verbeeck A.M.
      • Beullens S.
      • Cornelis P.
      • Michiels J.
      Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening.
      ), and a general picture starts to emerge in which multiple (often connected) mechanisms play a role (Fig. 1). Below we will discuss the role of stress responses and metabolism, and focus on the similarities and differences between biofilms and planktonic cultures.
      Figure thumbnail gr1
      FIGURE 1Schematic overview of physiological adaptations in biofilm tolerance.

      Oxidative Stress Response

      Because bactericidal antibiotics are known to induce oxidative stress (
      • Dwyer D.J.
      • Collins J.J.
      • Walker G.C.
      Unraveling the physiological complexities of antibiotic lethality.
      ), lowering cellular hydroxyl radical levels by decreasing their production or by increased detoxification could counteract bactericidal activity. Shatalin et al. (
      • Shatalin K.
      • Shatalina E.
      • Mironov A.
      • Nudler E.
      H2S: a universal defense against antibiotics in bacteria.
      ) showed in both biofilms and stationary phase cultures of various bacteria that H2S induced tolerance by reducing oxidative stress, through sequestration of ferrous iron and stimulation of catalases and superoxide dismutases. Similarly, it was observed that the active lowering of cellular hydroxyl levels played a role in planktonic and sessile P. aeruginosa persistence during starvation (
      • Nguyen D.
      • Joshi-Datar A.
      • Lepine F.
      • Bauerle E.
      • Olakanmi O.
      • Beer K.
      • McKay G.
      • Siehnel R.
      • Schafhauser J.
      • Wang Y.
      • Britigan B.E.
      • Singh P.K.
      Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria.
      ). Activation of the stringent response increased catalase and superoxide dismutase levels and repressed the production of hydroxy-alkylquinolines, intercellular signaling molecules with pro-oxidant properties (
      • Nguyen D.
      • Joshi-Datar A.
      • Lepine F.
      • Bauerle E.
      • Olakanmi O.
      • Beer K.
      • McKay G.
      • Siehnel R.
      • Schafhauser J.
      • Wang Y.
      • Britigan B.E.
      • Singh P.K.
      Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria.
      ). Data obtained for tobramycin-treated B. cenocepacia biofilms confirmed that avoiding exposure to antibiotic-induced reactive oxygen species (ROS) is a key factor in survival of persisters in these biofilms (
      • Van Acker H.
      • Sass A.
      • Bazzini S.
      • De Roy K.
      • Udine C.
      • Messiaen T.
      • Riccardi G.
      • Boon N.
      • Nelis H.J.
      • Mahenthiralingam E.
      • Coenye T.
      Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.
      ). Several genes encoding proteins involved in the generation of ROS, including a ferredoxin reductase (involved in recycling Fe3+ to Fe2+ and thus driving the Fenton reaction), were found to be down-regulated in persisters, whereas genes encoding proteins involved in ROS detoxification were up-regulated. These results suggest that persisters are to some extent protected against the detrimental effects of ROS produced upon antibiotic treatment.

      The Stringent Response

      Several studies have specified a role for guanosine tetra- or pentaphosphate, (p)ppGpp (known as the “alarmone”), the central mediator of the stringent response, in persistence (
      • Gerdes K.
      • Maisonneuve E.
      Bacterial persistence and toxin-antitoxin loci.
      ,
      • Germain E.
      • Castro-Roa D.
      • Zenkin N.
      • Gerdes K.
      Molecular mechanism of bacterial persistence by HipA.
      ,
      • Maisonneuve E.
      • Castro-Camargo M.
      • Gerdes K.
      (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity.
      ,
      • Ramisetty B.C.
      • Natarajan B.
      • Santhosh R.S.
      mazEF-mediated programmed cell death in bacteria: “What is this?”.
      ). The relA-spoT gene pair, involved in the synthesis of (p)ppGpp, was linked to persistence in various studies (
      • Viducic D.
      • Ono T.
      • Murakami K.
      • Susilowati H.
      • Kayama S.
      • Hirota K.
      • Miyake Y.
      Functional analysis of spoT, relA and dksA genes on quinolone tolerance in Pseudomonas aeruginosa under nongrowing condition.
      ,
      • Fauvart M.
      • De Groote V.N.
      • Michiels J.
      Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies.
      ,
      • Korch S.B.
      • Henderson T.A.
      • Hill T.M.
      Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis.
      ). For example, reduced levels of persisters were reported in P. aeruginosa biofilms deficient in (p)ppGpp synthesis (
      • Stewart P.S.
      • Franklin M.J.
      • Williamson K.S.
      • Folsom J.P.
      • Boegli L.
      • James G.A.
      Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.
      ). Gerdes et al. (
      • Gerdes K.
      • Maisonneuve E.
      Bacterial persistence and toxin-antitoxin loci.
      ) proposed a model in which (p)ppGpp induces persistence by activating toxin antitoxin (TA) modules via polyphosphate and Lon proteases. It was previously demonstrated that deletion of lon dramatically reduces persistence, whereas a moderate overproduction stimulates persistence by degrading antitoxins and hereby activating their cognate toxins (
      • Maisonneuve E.
      • Shakespeare L.J.
      • Jørgensen M.G.
      • Gerdes K.
      Bacterial persistence by RNA endonucleases.
      ). A link between (p)ppGpp and the transcriptional activation of the toxin HokB by Obg, a universally conserved GTPase, was recently identified (
      • Verstraeten N.
      • Knapen W.J.
      • Kint C.I.
      • Liebens V.
      • Van den Bergh B.
      • Dewachter L.
      • Michiels J.E.
      • Fu Q.
      • David C.C.
      • Fierro A.C.
      • Marchal K.
      • Beirlant J.
      • Versées W.
      • Hofkens J.
      • Jansen M.
      • et al.
      Obg and membrane depolarization are part of a microbial bet-hedging strategy that leads to antibiotic tolerance.
      ). Obg was shown to control persistence, in both planktonic cultures and P. aeruginosa biofilms, by inducing the expression of HokB, whereas elevated levels of HokB resulted in membrane depolarization and dormancy.

      TA Modules

      TA modules are thought to be a major player in the induction of persistence (
      • Gerdes K.
      • Maisonneuve E.
      Bacterial persistence and toxin-antitoxin loci.
      ) (for a review on TA modules, see Ref.
      • Goeders N.
      • Van Melderen L.
      Toxin-antitoxin systems as multilevel interaction systems.
      ). Type II TA modules usually consist of two proteins: a toxin that can inhibit an important cellular function and an antitoxin that can form a complex with the toxin and hence inactivates it (
      • Schuster C.F.
      • Bertram R.
      Toxin-antitoxin systems are ubiquitous and versatile modulators of prokaryotic cell fate.
      ) (Fig. 2) (for a schematic overview of the different types of TA modules, see supplemental Fig. S2). The antitoxin is typically proteolytically degraded during stress, and as a result, the free toxin can impede cellular processes such as DNA replication, translation, ATP, or cell wall synthesis (
      • Gerdes K.
      • Maisonneuve E.
      Bacterial persistence and toxin-antitoxin loci.
      ,
      • Yamaguchi Y.
      • Park J.H.
      • Inouye M.
      Toxin-antitoxin systems in bacteria and archaea.
      ). TA modules, although initially thought to be involved only in cell death, have been shown to play a role in various essential cellular processes, including biofilm formation and persistence (
      • Gerdes K.
      • Maisonneuve E.
      Bacterial persistence and toxin-antitoxin loci.
      ). Because bactericidal antibiotics kill cells by corrupting cellular functions, which are inhibited by toxins, the role of TA modules in persistence has been documented in various studies (
      • Gerdes K.
      • Maisonneuve E.
      Bacterial persistence and toxin-antitoxin loci.
      ). Toxin inhibition of cell wall synthesis, translation, or replication would prevent antibiotics from killing and give rise to persister cells. HipAB was the first TA module linked to persistence (
      • Keren I.
      • Shah D.
      • Spoering A.
      • Kaldalu N.
      • Lewis K.
      Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli.
      ), and many key insights on persistence were obtained while studying this particular module. Rotem et al. (
      • Rotem E.
      • Loinger A.
      • Ronin I.
      • Levin-Reisman I.
      • Gabay C.
      • Shoresh N.
      • Biham O.
      • Balaban N.Q.
      Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence.
      ) noticed that persistence occurred once the toxin HipA reached a certain threshold level and that the amount by which the threshold was exceeded determined the duration of dormancy. Since the identification of HipAB, various studies have reported an up-regulation of TA modules in persister cells (
      • Keren I.
      • Shah D.
      • Spoering A.
      • Kaldalu N.
      • Lewis K.
      Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli.
      ,
      • Shah D.
      • Zhang Z.
      • Khodursky A.
      • Kaldalu N.
      • Kurg K.
      • Lewis K.
      Persisters: a distinct physiological state of E. coli.
      ). For example, transcriptome analysis of dormant E. coli cells identified mqsR as the most highly induced gene in persister cells as compared with non-persisters (
      • Shah D.
      • Zhang Z.
      • Khodursky A.
      • Kaldalu N.
      • Kurg K.
      • Lewis K.
      Persisters: a distinct physiological state of E. coli.
      ). In B. cenocepacia, several toxins were found to be up-regulated in biofilms as compared with planktonic cells, and overexpression of these toxins contributed to persistence in biofilms after treatment with tobramycin or ciprofloxacin (
      • Van Acker H.
      • Sass A.
      • Dhondt I.
      • Nelis H.J.
      • Coenye T.
      Involvement of toxin-antitoxin modules in Burkholderia cenocepacia biofilm persistence.
      ). Although overproduction of almost any toxin may increase persistence, only two TA pairs have been shown to decrease persistence upon deletion (
      • Wang X.
      • Wood T.K.
      Toxin-antitoxin systems influence biofilm and persister cell formation and the general stress response.
      ,
      • Dörr T.
      • Vulić M.
      • Lewis K.
      Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli.
      ). Deletion of mqsRA significantly reduced persistence in E. coli biofilms (
      • Wang X.
      • Wood T.K.
      Toxin-antitoxin systems influence biofilm and persister cell formation and the general stress response.
      ), whereas deletion of tisAB/istr1 led to a decrease in survival in planktonic cultures (
      • Dörr T.
      • Vulić M.
      • Lewis K.
      Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli.
      ). Simultaneous deletion of multiple other TA systems also decreased the number of persisters in E. coli (
      • Maisonneuve E.
      • Shakespeare L.J.
      • Jørgensen M.G.
      • Gerdes K.
      Bacterial persistence by RNA endonucleases.
      ). The latter confirms the role of TA modules in persistence, but again suggests redundancy.
      Figure thumbnail gr2
      FIGURE 2Schematic overview of type II TA modules.
      Additionally, recent studies have indicated a role for TA systems in the switch from the planktonic to the sessile lifestyle. MqsRA was the first TA module linked to biofilm formation: in a transcriptome study comparing sessile and planktonic cells, mqsR was found to be induced in biofilms (
      • Ren D.
      • Bedzyk L.A.
      • Thomas S.M.
      • Ye R.W.
      • Wood T.K.
      Gene expression in Escherichia coli biofilms.
      ), and biofilm formation in E. coli was found to be mediated by MqsRA (
      • González Barrios A.F.
      • Zuo R.
      • Hashimoto Y.
      • Yang L.
      • Bentley W.E.
      • Wood T.K.
      Autoinducer 2 controls biofilm formation in Escherichia coli through a novel motility quorum-sensing regulator (MqsR, B3022).
      ). In support of these observations, the antitoxin MqsA was more recently found to repress rpoS, thereby reducing the c-di-GMP concentration, which leads to increased motility and decreased biofilm formation (
      • Wang X.
      • Wood T.K.
      Toxin-antitoxin systems influence biofilm and persister cell formation and the general stress response.
      ). Degradation of MqsA by Lon proteases in turn resulted in rpoS induction and a switch from the planktonic to the biofilm state. A model was proposed in which there is a spectrum of MqsR activities, and depending on the activity of MqsR, a cell would respond to stress by biofilm formation and the production of proteins to withstand stress, or alternatively, become dormant (
      • Wang X.
      • Wood T.K.
      Toxin-antitoxin systems influence biofilm and persister cell formation and the general stress response.
      ). In this regard, persister cell formation can be seen as an extreme example of the general stress response mediated by MqsR. Further evidence for the involvement of TA modules in E. coli biofilm formation was obtained by studying Δ5, a strain in which five different TA modules were deleted. Deletion of these systems reduced biofilm formation after 8 h but increased biofilm formation after 24 h (
      • Kim Y.
      • Wang X.
      • Ma Q.
      • Zhang X.S.
      • Wood T.K.
      Toxin-antitoxin systems in Escherichia coli influence biofilm formation through YjgK (TabA) and fimbriae.
      ). Transcriptome profiling revealed that deletion of these TA modules induced the expression of an uncharacterized gene, yjgK, which repressed fimbriae at 8 h (
      • Kim Y.
      • Wang X.
      • Ma Q.
      • Zhang X.S.
      • Wood T.K.
      Toxin-antitoxin systems in Escherichia coli influence biofilm formation through YjgK (TabA) and fimbriae.
      ). Although this repression of fimbriae may explain the decrease in biofilm formation at 8 h, a reduction in biofilm dispersal may explain the increase in biofilm formation at 24 h.

      The SOS Response

      The SOS response is triggered by DNA damage (
      • Michel B.
      After 30 years of study, the bacterial SOS response still surprises us.
      ). This damage is recognized by RecA, which causes self-cleavage of the LexA repressor, hereby activating SOS genes (
      • Michel B.
      After 30 years of study, the bacterial SOS response still surprises us.
      ). Based on transcriptome analyses and screening of mutant libraries, lexA and recA were found to be involved in E. coli persistence (
      • Fauvart M.
      • De Groote V.N.
      • Michiels J.
      Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies.
      ,
      • Keren I.
      • Shah D.
      • Spoering A.
      • Kaldalu N.
      • Lewis K.
      Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli.
      ). In line with this observation, deletion of the tisAB/istr1 TA module, which contains a Lex box, dramatically reduced the number of surviving persisters in exponentially growing cultures of E. coli treated with fluoroquinolones, but not after treatment with antibiotics that do not cause DNA damage (
      • Dörr T.
      • Vulić M.
      • Lewis K.
      Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli.
      ). Moreover, a functional RecA protein was needed for persistence, confirming dependence on the SOS pathway. The induction of persisters by the SOS-induced TisB toxin links two survival strategies, i.e. active repair on the one hand and shutdown of cellular metabolism on the other. This suggests that in the presence of DNA-damaging agents, the optimal strategy is to induce repair and at the same time increase the number of dormant cells that will survive when repair would fail (
      • Lewis K.
      Persister cells.
      ). Interestingly, although inactivation of genes involved in the SOS response also increased susceptibility to fluoroquinolones in stationary phase cultures, TisB was not found to be involved, suggesting that persisters form through other mechanisms in non-growing cultures (
      • Dörr T.
      • Vulić M.
      • Lewis K.
      Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli.
      ). Tolerance to ofloxacin significantly increased in E. coli biofilms upon starvation, whereas starvation did not have a significant effect on tolerance to ofloxacin in planktonic cultures (
      • Bernier S.P.
      • Lebeaux D.
      • DeFrancesco A.S.
      • Valomon A.
      • Soubigou G.
      • Coppée J.Y.
      • Ghigo J.M.
      • Beloin C.
      Starvation, together with the SOS response, mediates high biofilm-specific tolerance to the fluoroquinolone ofloxacin.
      ). Similarly, tolerance was dependent on the SOS response but independent of known SOS-induced TA modules. Although starvation also induces the stringent response, deletion of relA only partially increased sensitivity to ofloxacin and only upon leucine starvation, suggesting that the stringent response plays a minor role in tolerance to ofloxacin.

      Metabolism

      A regulator of phosphate metabolism, phoU, was identified as a persister gene as its deletion reduced persistence in planktonic cultures of E. coli and inactivation of phoU was shown to lead to a hyperactive metabolic state (
      • Li Y.
      • Zhang Y.
      PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli.
      ). However, mutations in sucB or ubiF, leading to reduced ATP synthesis, also negatively affected survival of persisters (
      • Ma C.
      • Sim S.
      • Shi W.
      • Du L.
      • Xing D.
      • Zhang Y.
      Energy production genes sucB and ubiF are involved in persister survival and tolerance to multiple antibiotics and stresses in Escherichia coli.
      ). SucB is a key enzyme of the TCA cycle, whereas UbiF is involved in the biosynthesis of ubiquinone, an acceptor of electrons in the respiratory electron transport chain. These at first sight contradictory results illustrate the critical role regulation of metabolism may play in persistence. Based on persistence assays and FACS sorting of E. coli cells fluorescently labeled according to their metabolic activity, Orman and Brynildsen (
      • Orman M.A.
      • Brynildsen M.P.
      Dormancy is not necessary or sufficient for bacterial persistence.
      ) suggested that a low metabolic activity prior to antibiotic exposure only increases the likelihood of a cell to become a persister. Additionally, they observed that inhibition of respiration decreased the number of persisters surviving treatment with ampicillin (
      • Orman M.A.
      • Brynildsen M.P.
      Inhibition of stationary phase respiration impairs persister formation in E. coli.
      ). Inhibition of respiration during stationary phase may prevent digestion of endogenous proteins and mRNA and thus allow translation and replication to proceed and render bacteria susceptible to antibiotics. In B. cenocepacia biofilms, a metabolic shift bypassing the TCA cycle was observed in persister cells (
      • Van Acker H.
      • Sass A.
      • Bazzini S.
      • De Roy K.
      • Udine C.
      • Messiaen T.
      • Riccardi G.
      • Boon N.
      • Nelis H.J.
      • Mahenthiralingam E.
      • Coenye T.
      Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.
      ). In cells surviving treatment with tobramycin, genes encoding proteins involved in the TCA cycle were down-regulated, and at the same time, the glyoxylate shunt was activated, most likely to sustain ATP production. Mok et al. (
      • Mok W.W.
      • Park J.O.
      • Rabinowitz J.D.
      • Brynildsen M.P.
      RNA futile cycling in model persisters derived from MazF accumulation.
      ) studied the metabolic aspects of persisters in an E. coli strain in which the antitoxin MazE and the toxin MazF were artificially and independently induced. Upon accumulation of MazF, an endoribonuclease, reversible stasis was achieved and populations were almost entirely tolerant to fluoroquinolones and β-lactams. Although these induced persisters were found to be non-replicative, they maintained oxygen and glucose consumption. Further analysis also indicated accumulation of all four ribonucleotide monophosphates, confirming futile cycling in these persisters. Energy derived from catabolism was used to continue transcription, but at the same time, the transcripts were degraded by MazF.
      An obvious question is whether or not persister cells can be considered as dormant, given the mechanisms involved. Based on studies indicating the role of different stress responses in persister cells, it was suggested that persistence is an actively maintained state (
      • Cohen N.R.
      • Lobritz M.A.
      • Collins J.J.
      Microbial persistence and the road to drug resistance.
      ). However, according to Wood et al. (
      • Wood T.K.
      • Knabel S.J.
      • Kwan B.W.
      Bacterial persister cell formation and dormancy.
      ), most evidence points to a role for dormancy, toxin induction, down-regulation of metabolic pathways, and shutdown of protein synthesis in persistence. Although both viewpoints seem contradictory, they could be reconciled as active responses to stress may play a role in inducing dormancy because most stress responses lead to slowing down or inhibition of cell growth (
      • Cohen N.R.
      • Lobritz M.A.
      • Collins J.J.
      Microbial persistence and the road to drug resistance.
      ,
      • Wood T.K.
      • Knabel S.J.
      • Kwan B.W.
      Bacterial persister cell formation and dormancy.
      ). Additionally, although shutdown of metabolic processes may be involved in the entry into a dormant antibiotic tolerant state, residual metabolic activity may be required to maintain viability, and reactivation is necessary to resume growth after removal of the antibiotics (for recent reviews on targeting metabolism, see Refs.
      • Amato S.M.
      • Fazen C.H.
      • Henry T.C.
      • Mok W.W.
      • Orman M.A.
      • Sandvik E.L.
      • Volzing K.G.
      • Brynildsen M.P.
      The role of metabolism in bacterial persistence.
      and
      • Prax M.
      • Bertram R.
      Metabolic aspects of bacterial persisters.
      ). For example, in stationary phase cultures of E. coli, there is no difference in DNA damage between persisters and non-persisters (
      • Völzing K.G.
      • Brynildsen M.P.
      Stationary-phase persisters to ofloxacin sustain DNA damage and require repair systems only during recovery.
      ). Moreover, neither the level of SOS machinery before nor during treatment impacted the level of persisters, but the cell's ability to repair ofloxacin-induced DNA damage during recovery when the antibiotic was removed was critical to maintain persistence.

      Concluding Remarks

      Many different mechanisms leading to biofilm resistance and tolerance have been described, including efflux-mediated removal of antimicrobial agents from the cytoplasm and the occurrence of tolerant and persistent subpopulations of cells. Although there is currently no evidence linking increased efflux in bacterial populations with cell density, it is clear that at least in some bacterial species particular efflux systems are involved in biofilm formation and/or resistance. The currently available evidence suggests that life style-specific efflux pumps (i.e. efflux systems that are preferentially used in either planktonic or sessile populations) exist, as well as general systems that are used by bacteria to get rid of unwanted compounds irrespective of the mode of growth. Little is known about the regulation of the expression of these efflux pumps and how that would be affected by growth in a biofilm, although work with the regulator BrlR in P. aeruginosa points to a role for c-di-GMP.
      Persister cells have been described in all bacteria examined so far, and persisters present in biofilms are thought to be an important reason for treatment failure. Persisters are generally considered as dormant cells in which the antibiotic targets are inactive. Recent research, however, suggest that persisters are not necessarily inactive but rather have a different metabolism. Several studies points to the importance of residual metabolic activity to maintain viability and the ability to resuscitate after removal of the antibiotics. Additionally, active responses to stress are thought to induce persistence, and different stress responses are likely involved depending on the conditions.

      References

        • Costerton J.W.
        The Biofilm Primer.
        Springer, Berlin2007
        • Hall-Stoodley L.
        • Costerton J.W.
        • Stoodley P.
        Bacterial biofilms: from the natural environment to infectious diseases.
        Nat. Rev. Microbiol. 2004; 2: 95-108
        • Wolcott R.D.
        • Rhoads D.D.
        • Bennett M.E.
        • Wolcott B.M.
        • Gogokhia L.
        • Costerton J.W.
        • Dowd S.E.
        Chronic wounds and the medical biofilm paradigm.
        J. Wound Care. 2010; 19: 45-53
        • Bjarnsholt T.
        The role of bacterial biofilms in chronic infections.
        APMIS. Suppl. 2013; : 1-51
        • Peeters E.
        • Nelis H.J.
        • Coenye T.
        In vitro activity of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole against planktonic and sessile Burkholderia cepacia complex bacteria.
        J. Antimicrob. Chemother. 2009; 64: 801-809
        • Lewis K.
        Multidrug tolerance of biofilms and persister cells.
        Curr. Top. Microbiol. Immunol. 2008; 322: 107-131
        • Lebeaux D.
        • Ghigo J.M.
        • Beloin C.
        Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics.
        Microbiol. Mol. Biol. Rev. 2014; 78: 510-543
        • Van Acker H.
        • Van Dijck P.
        • Coenye T.
        Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms.
        Trends Microbiol. 2014; 22: 326-333
        • Sheppard D.C.
        • Howell P.L.
        Biofilm exopolysaccharides of pathogenic fungi: lessons from bacteria.
        J. Biol. Chem. 2016; 291: 12529-12537
        • Gunn J.S.
        • Bakaletz L.O.
        • Wozniak D.J.
        What's on the outside matters: The role of the extracellular polymeric substance of Gram-negative biofilms in evading host immunity and as a target for therapeutic intervention.
        J. Biol. Chem. 2016; 291: 12538-12546
        • Routh M.D.
        • Zalucki Y.
        • Su C.C.
        • Zhang Q.
        • Shafer W.M.
        • Yu E.W.
        Efflux pumps of the resistance-nodulation-division family: a perspective of their structure, function, and regulation in Gram-negative bacteria.
        Adv. Enzymol. Relat. Areas Mol. Biol. 2011; 77: 109-146
        • Soto S.M.
        Role of efflux pumps in the antibiotic resistance of bacteria embedded in a biofilm.
        Virulence. 2013; 4: 223-229
        • Tseng T.T.
        • Gratwick K.S.
        • Kollman J.
        • Park D.
        • Nies D.H.
        • Goffeau A.
        • Saier Jr., M.H.
        The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins.
        J. Mol. Microbiol. Biotechnol. 1999; 1: 107-125
        • Nikaido H.
        Structure and mechanism of RND-type multidrug efflux pumps.
        Adv. Enzymol. Relat. Areas Mol. Biol. 2011; 77: 1-60
        • Kvist M.
        • Hancock V.
        • Klemm P.
        Inactivation of efflux pumps abolishes bacterial biofilm formation.
        Appl. Environ. Microbiol. 2008; 74: 7376-7382
        • Imuta N.
        • Nishi J.
        • Tokuda K.
        • Fujiyama R.
        • Manago K.
        • Iwashita M.
        • Sarantuya J.
        • Kawano Y.
        The Escherichia coli efflux pump TolC promotes aggregation of enteroaggregative E. coli 042.
        Infect. Immun. 2008; 76: 1247-1256
        • Baugh S.
        • Ekanayaka A.S.
        • Piddock L.J.
        • Webber M.A.
        Loss of or inhibition of all multidrug resistance efflux pumps of Salmonella enterica serovar Typhimurium results in impaired ability to form a biofilm.
        J. Antimicrob. Chemother. 2012; 67: 2409-2417
        • Barnhart M.M.
        • Chapman M.R.
        Curli biogenesis and function.
        Annu. Rev. Microbiol. 2006; 60: 131-147
        • Baugh S.
        • Phillips C.R.
        • Ekanayaka A.S.
        • Piddock L.J.
        • Webber M.A.
        Inhibition of multidrug efflux as a strategy to prevent biofilm formation.
        J. Antimicrob. Chemother. 2014; 69: 673-681
        • Kumar A.
        • Schweizer H.P.
        Bacterial resistance to antibiotics: active efflux and reduced uptake.
        Adv. Drug Deliv. Rev. 2005; 57: 1486-1513
        • Fernández L.
        • Hancock R.E.
        Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance.
        Clin. Microbiol. Rev. 2012; 25: 661-681
        • De Kievit T.R.
        • Parkins M.D.
        • Gillis R.J.
        • Srikumar R.
        • Ceri H.
        • Poole K.
        • Iglewski B.H.
        • Storey D.G.
        Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms.
        Antimicrob. Agents Chemother. 2001; 45: 1761-1770
        • Stewart P.S.
        • Franklin M.J.
        Physiological heterogeneity in biofilms.
        Nat. Rev. Microbiol. 2008; 6: 199-210
        • Coenye T.
        Response of sessile cells to stress: from changes in gene expression to phenotypic adaptation.
        FEMS Immunol. Med. Microbiol. 2010; 59: 239-252
        • Gillis R.J.
        • White K.G.
        • Choi K.H.
        • Wagner V.E.
        • Schweizer H.P.
        • Iglewski B.H.
        Molecular basis of azithromycin-resistant Pseudomonas aeruginosa biofilms.
        Antimicrob. Agents Chemother. 2005; 49: 3858-3867
        • Pamp S.J.
        • Gjermansen M.
        • Johansen H.K.
        • Tolker-Nielsen T.
        Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes.
        Mol. Microbiol. 2008; 68: 223-240
        • Zhang L.
        • Mah T.F.
        Involvement of a novel efflux system in biofilm-specific resistance to antibiotics.
        J. Bacteriol. 2008; 190: 4447-4452
        • Vikram A.
        • Bomberger J.M.
        • Bibby K.J.
        Efflux as a glutaraldehyde resistance mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms.
        Antimicrob. Agents Chemother. 2015; 59: 3433-3440
        • Schaible B.
        • Taylor C.T.
        • Schaffer K.
        Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps.
        Antimicrob. Agents Chemother. 2012; 56: 2114-2118
        • Römling U.
        • Galperin M.Y.
        • Gomelsky M.
        Cyclic di-GMP: the first 25 years of a universal bacterial second messenger.
        Microbiol. Mol. Biol. Rev.: MMBR. 2013; 77: 1-52
        • Valentini M.
        • Filloux A.
        Biofilms and c-di-GMP signaling: lessons from Pseudomonas aeruginosa and other bacteria.
        J. Biol. Chem. 2016; 291: 12547-12555
        • Liao J.
        • Sauer K.
        The MerR-like transcriptional regulator BrlR contributes to Pseudomonas aeruginosa biofilm tolerance.
        J. Bacteriol. 2012; 194: 4823-4836
        • Chambers J.R.
        • Liao J.
        • Schurr M.J.
        • Sauer K.
        BrlR from Pseudomonas aeruginosa is a c-di-GMP-responsive transcription factor.
        Mol. Microbiol. 2014; 92: 471-487
        • Liao J.
        • Schurr M.J.
        • Sauer K.
        The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug efflux pumps in Pseudomonas aeruginosa biofilms.
        J. Bacteriol. 2013; 195: 3352-3363
        • Folsom J.P.
        • Richards L.
        • Pitts B.
        • Roe F.
        • Ehrlich G.D.
        • Parker A.
        • Mazurie A.
        • Stewart P.S.
        Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis.
        BMC Microbiol. 2010; 10: 294
        • Stewart P.S.
        • Franklin M.J.
        • Williamson K.S.
        • Folsom J.P.
        • Boegli L.
        • James G.A.
        Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.
        Antimicrob. Agents Chemother. 2015; 59: 3838-3847
        • Guglierame P.
        • Pasca M.R.
        • De Rossi E.
        • Buroni S.
        • Arrigo P.
        • Manina G.
        • Riccardi G.
        Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome.
        BMC Microbiol. 2006; 6: 66
        • Buroni S.
        • Pasca M.R.
        • Flannagan R.S.
        • Bazzini S.
        • Milano A.
        • Bertani I.
        • Venturi V.
        • Valvano M.A.
        • Riccardi G.
        Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance.
        BMC Microbiol. 2009; 9: 200
        • Bazzini S.
        • Udine C.
        • Sass A.
        • Pasca M.R.
        • Longo F.
        • Emiliani G.
        • Fondi M.
        • Perrin E.
        • Decorosi F.
        • Viti C.
        • Giovannetti L.
        • Leoni L.
        • Fani R.
        • Riccardi G.
        • Mahenthiralingam E.
        • Buroni S.
        Deciphering the role of RND efflux transporters in Burkholderia cenocepacia.
        PLoS ONE. 2011; 6e18902
        • Rushton L.
        • Sass A.
        • Baldwin A.
        • Dowson C.G.
        • Donoghue D.
        • Mahenthiralingam E.
        Key role for efflux in the preservative susceptibility and adaptive resistance of Burkholderia cepacia complex bacteria.
        Antimicrob. Agents Chemother. 2013; 57: 2972-2980
        • Tseng S.P.
        • Tsai W.C.
        • Liang C.Y.
        • Lin Y.S.
        • Huang J.W.
        • Chang C.Y.
        • Tyan Y.C.
        • Lu P.L.
        The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.
        PLoS ONE. 2014; 9e104986
        • Coenye T.
        • Van Acker H.
        • Peeters E.
        • Sass A.
        • Buroni S.
        • Riccardi G.
        • Mahenthiralingam E.
        Molecular mechanisms of chlorhexidine tolerance in Burkholderia cenocepacia biofilms.
        Antimicrob. Agents Chemother. 2011; 55: 1912-1919
        • Buroni S.
        • Matthijs N.
        • Spadaro F.
        • Van Acker H.
        • Scoffone V.C.
        • Pasca M.R.
        • Riccardi G.
        • Coenye T.
        Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells.
        Antimicrob. Agents Chemother. 2014; 58: 7424-7429
        • Lewis K.
        Persister cells, dormancy and infectious disease.
        Nat. Rev. Microbiol. 2007; 5: 48-56
        • Jayaraman R.
        Bacterial persistence: some new insights into an old phenomenon.
        J. Biosci. 2008; 33: 795-805
        • Lewis K.
        Persister cells.
        Annu. Rev. Microbiol. 2010; 64: 357-372
        • Hansen S.
        • Lewis K.
        • Vulić M.
        Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli.
        Antimicrob. Agents Chemother. 2008; 52: 2718-2726
        • De Groote V.N.
        • Verstraeten N.
        • Fauvart M.
        • Kint C.I.
        • Verbeeck A.M.
        • Beullens S.
        • Cornelis P.
        • Michiels J.
        Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening.
        FEMS Microbiol. Lett. 2009; 297: 73-79
        • Dwyer D.J.
        • Collins J.J.
        • Walker G.C.
        Unraveling the physiological complexities of antibiotic lethality.
        Annu. Rev. Pharmacol. Toxicol. 2015; 55: 313-332
        • Shatalin K.
        • Shatalina E.
        • Mironov A.
        • Nudler E.
        H2S: a universal defense against antibiotics in bacteria.
        Science. 2011; 334: 986-990
        • Nguyen D.
        • Joshi-Datar A.
        • Lepine F.
        • Bauerle E.
        • Olakanmi O.
        • Beer K.
        • McKay G.
        • Siehnel R.
        • Schafhauser J.
        • Wang Y.
        • Britigan B.E.
        • Singh P.K.
        Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria.
        Science. 2011; 334: 982-986
        • Van Acker H.
        • Sass A.
        • Bazzini S.
        • De Roy K.
        • Udine C.
        • Messiaen T.
        • Riccardi G.
        • Boon N.
        • Nelis H.J.
        • Mahenthiralingam E.
        • Coenye T.
        Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.
        PLoS ONE. 2013; 8e58943
        • Gerdes K.
        • Maisonneuve E.
        Bacterial persistence and toxin-antitoxin loci.
        Annu. Rev. Microbiol. 2012; 66: 103-123
        • Germain E.
        • Castro-Roa D.
        • Zenkin N.
        • Gerdes K.
        Molecular mechanism of bacterial persistence by HipA.
        Mol. Cell. 2013; 52: 248-254
        • Maisonneuve E.
        • Castro-Camargo M.
        • Gerdes K.
        (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity.
        Cell. 2013; 154: 1140-1150
        • Ramisetty B.C.
        • Natarajan B.
        • Santhosh R.S.
        mazEF-mediated programmed cell death in bacteria: “What is this?”.
        Crit. Rev. Microbiol. 2015; 41: 89-100
        • Viducic D.
        • Ono T.
        • Murakami K.
        • Susilowati H.
        • Kayama S.
        • Hirota K.
        • Miyake Y.
        Functional analysis of spoT, relA and dksA genes on quinolone tolerance in Pseudomonas aeruginosa under nongrowing condition.
        Microbiol. Immunol. 2006; 50: 349-357
        • Fauvart M.
        • De Groote V.N.
        • Michiels J.
        Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies.
        J. Med. Microbiol. 2011; 60: 699-709
        • Korch S.B.
        • Henderson T.A.
        • Hill T.M.
        Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis.
        Mol. Microbiol. 2003; 50: 1199-1213
        • Maisonneuve E.
        • Shakespeare L.J.
        • Jørgensen M.G.
        • Gerdes K.
        Bacterial persistence by RNA endonucleases.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 13206-13211
        • Verstraeten N.
        • Knapen W.J.
        • Kint C.I.
        • Liebens V.
        • Van den Bergh B.
        • Dewachter L.
        • Michiels J.E.
        • Fu Q.
        • David C.C.
        • Fierro A.C.
        • Marchal K.
        • Beirlant J.
        • Versées W.
        • Hofkens J.
        • Jansen M.
        • et al.
        Obg and membrane depolarization are part of a microbial bet-hedging strategy that leads to antibiotic tolerance.
        Mol. Cell. 2015; 59: 9-21
        • Goeders N.
        • Van Melderen L.
        Toxin-antitoxin systems as multilevel interaction systems.
        Toxins. 2014; 6: 304-324
        • Schuster C.F.
        • Bertram R.
        Toxin-antitoxin systems are ubiquitous and versatile modulators of prokaryotic cell fate.
        FEMS Microbiol. Lett. 2013; 340: 73-85
        • Yamaguchi Y.
        • Park J.H.
        • Inouye M.
        Toxin-antitoxin systems in bacteria and archaea.
        Annu. Rev. Genet. 2011; 45: 61-79
        • Keren I.
        • Shah D.
        • Spoering A.
        • Kaldalu N.
        • Lewis K.
        Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli.
        J. Bacteriol. 2004; 186: 8172-8180
        • Rotem E.
        • Loinger A.
        • Ronin I.
        • Levin-Reisman I.
        • Gabay C.
        • Shoresh N.
        • Biham O.
        • Balaban N.Q.
        Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 12541-12546
        • Shah D.
        • Zhang Z.
        • Khodursky A.
        • Kaldalu N.
        • Kurg K.
        • Lewis K.
        Persisters: a distinct physiological state of E. coli.
        BMC Microbiol. 2006; 6: 53
        • Van Acker H.
        • Sass A.
        • Dhondt I.
        • Nelis H.J.
        • Coenye T.
        Involvement of toxin-antitoxin modules in Burkholderia cenocepacia biofilm persistence.
        Pathog. Dis. 2014; 71: 326-335
        • Wang X.
        • Wood T.K.
        Toxin-antitoxin systems influence biofilm and persister cell formation and the general stress response.
        Appl. Environ. Microbiol. 2011; 77: 5577-5583
        • Dörr T.
        • Vulić M.
        • Lewis K.
        Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli.
        PLos Biol. 2010; 8e1000317
        • Ren D.
        • Bedzyk L.A.
        • Thomas S.M.
        • Ye R.W.
        • Wood T.K.
        Gene expression in Escherichia coli biofilms.
        Appl. Microbiol. Biotechnol. 2004; 64: 515-524
        • González Barrios A.F.
        • Zuo R.
        • Hashimoto Y.
        • Yang L.
        • Bentley W.E.
        • Wood T.K.
        Autoinducer 2 controls biofilm formation in Escherichia coli through a novel motility quorum-sensing regulator (MqsR, B3022).
        J. Bacteriol. 2006; 188: 305-316
        • Kim Y.
        • Wang X.
        • Ma Q.
        • Zhang X.S.
        • Wood T.K.
        Toxin-antitoxin systems in Escherichia coli influence biofilm formation through YjgK (TabA) and fimbriae.
        J. Bacteriol. 2009; 191: 1258-1267
        • Michel B.
        After 30 years of study, the bacterial SOS response still surprises us.
        PLoS Biol. 2005; 3: e255
        • Bernier S.P.
        • Lebeaux D.
        • DeFrancesco A.S.
        • Valomon A.
        • Soubigou G.
        • Coppée J.Y.
        • Ghigo J.M.
        • Beloin C.
        Starvation, together with the SOS response, mediates high biofilm-specific tolerance to the fluoroquinolone ofloxacin.
        PLoS Genet. 2013; 9e1003144
        • Li Y.
        • Zhang Y.
        PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli.
        Antimicrob. Agents Chemother. 2007; 51: 2092-2099
        • Ma C.
        • Sim S.
        • Shi W.
        • Du L.
        • Xing D.
        • Zhang Y.
        Energy production genes sucB and ubiF are involved in persister survival and tolerance to multiple antibiotics and stresses in Escherichia coli.
        FEMS Microbiol. Lett. 2010; 303: 33-40
        • Orman M.A.
        • Brynildsen M.P.
        Dormancy is not necessary or sufficient for bacterial persistence.
        Antimicrob. Agents Chemother. 2013; 57: 3230-3239
        • Orman M.A.
        • Brynildsen M.P.
        Inhibition of stationary phase respiration impairs persister formation in E. coli.
        Nat. Commun. 2015; 6 (Correction (2016) Nat. Commun. 7, 10756)7983
        • Mok W.W.
        • Park J.O.
        • Rabinowitz J.D.
        • Brynildsen M.P.
        RNA futile cycling in model persisters derived from MazF accumulation.
        mBio. 2015; 6: e01588-15
        • Cohen N.R.
        • Lobritz M.A.
        • Collins J.J.
        Microbial persistence and the road to drug resistance.
        Cell Host Microbe. 2013; 13: 632-642
        • Wood T.K.
        • Knabel S.J.
        • Kwan B.W.
        Bacterial persister cell formation and dormancy.
        Appl. Environ. Microbiol. 2013; 79: 7116-7121
        • Amato S.M.
        • Fazen C.H.
        • Henry T.C.
        • Mok W.W.
        • Orman M.A.
        • Sandvik E.L.
        • Volzing K.G.
        • Brynildsen M.P.
        The role of metabolism in bacterial persistence.
        Front. Microbiol. 2014; 5: 70
        • Prax M.
        • Bertram R.
        Metabolic aspects of bacterial persisters.
        Front. Cell Infect. Microbiol. 2014; 4: 148
        • Völzing K.G.
        • Brynildsen M.P.
        Stationary-phase persisters to ofloxacin sustain DNA damage and require repair systems only during recovery.
        mBio. 2015; 6: e00731-00715