Conlon, Brian P.

O'Gara, James P.

Rowe, Sarah E.

Waters, Elaine M.

PLoS Pathogens

English

PubMed

Elaine M Waters

Sarah E Rowe

James P O'Gara

Brian P Conlon

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Convergence of Staphylococcus aureus Persister and Biofilm Research: Can Biofilms Be Defined as Communities of Adherent Persister Cells?

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PEARLSConvergence of Staphylococcus aureusPersister and Biofilm Research: Can BiofilmsBe Defined as Communities of AdherentPersister Cells?Elaine M. Waters1, Sarah E. Rowe2, James P. O’Gara1*, Brian P. Conlon2*1 Department of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland,2 Department of Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NorthCarolina, United States of America* jamesp.ogara@nuigalway.ie (JPO); bconlon@med.unc.edu (BPC)The remarkable tolerance of bacterial biofilms to antimicrobial drugs underpins their role inchronic and recurring infections. Staphylococcus aureus biofilms are embedded in an extracel-lular matrix composed of self-produced extracellular polysaccharides, DNA, and proteins orhost-derived matrices such as fibrin, prompting speculation that limited drug diffusion intobiofilms contributes to tolerance. However, the slow- and non-growing phenotypes of biofilmcells resemble those observed in the stationary growth phase, which is known to enrich for thehighly antibiotic-tolerant persister phenotype. Indeed, recent studies have revealed that theantibiotic tolerance phenotypes of S. aureus biofilm and persister cells are strikingly similar[1–5]. Here, we will explore the idea that biofilms are enriched with adherent persister cellsand that research into the biofilm and persister phenotypes has converged.Why Do Biofilms Exhibit High-Level Antibiotic Tolerance?The visible extracellular polysaccharide matrix encasing many bacterial biofilms, e.g., S. epider-midis slime on infected medical devices or mucoid Pseudomonas aeruginosa recovered fromthe lungs of patients with cystic fibrosis [6, 7], has prompted the hypothesis that impaired anti-biotic penetration is an important determinant in biofilm tolerance to drugs and other toxins.However, the relative contribution of impaired antibiotic penetration to biofilm toleranceremains questionable. It has been demonstrated that antibiotics do penetrate the biofilmmatrix and reach the cells embedded within [8, 9] without always killing the bacteria [10]. Fur-thermore, cells released from biofilms are more tolerant to antibiotics than planktonic cells[11, 12], which strongly suggests that overall biofilm tolerance is not primarily the result ofimpaired antibiotic penetration.Overall, it appears that the biofilm matrix does not significantly control antibiotic penetra-tion. Rather, these data indicate that the biofilm matrix is primarily a protective agent againstimmune defenses during infection [13]. The altered physiology of biofilm cells reflects theunique environmental milieu and high cell density, which is likely to limit nutrient and oxygenavailability and impact quorum sensing-regulated phenotypes [14]. These growth conditionsappear to force a subset of biofilm cells into a stationary and persister-like state. Cells in thisphysiological state are primed to survive wide-ranging environmental assaults, including anti-biotic challenge [2].PLOS Pathogens | DOI:10.1371/journal.ppat.1006012 December 29, 2016 1 / 5a11111OPENACCESSCitation: Waters EM, Rowe SE, O’Gara JP, ConlonBP (2016) Convergence of Staphylococcus aureusPersister and Biofilm Research: Can Biofilms BeDefined as Communities of Adherent PersisterCells? PLoS Pathog 12(12): e1006012.doi:10.1371/journal.ppat.1006012Editor: Kimberly A. Kline, Nanyang TechnologicalUniversity, SINGAPOREPublished: December 29, 2016Copyright: © 2016 Waters et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, whichpermits unrestricted use, distribution, andreproduction in any medium, provided the originalauthor and source are credited.Funding: The authors received no specific fundingfor this study.Competing Interests: The authors have declaredthat no competing interests exist.How Similar Are Biofilm Cells, Stationary Phase Cells, andPersisters?The physiology of S. aureus biofilm cells bears striking similarity to that of S. aureus persistercells. Persister cells are subpopulations of antibiotic-tolerant cells in an otherwise susceptiblepopulation. Persisters are transient phenotypic variants, which exhibit drug susceptibilityupon subculture [15]. Similarly, cells that detach from antibiotic-tolerant biofilms and growplanktonically also revert to a drug-susceptible state [11, 12].Stationary phase cultures of S. aureus demonstrate remarkable antibiotic tolerance [1, 2, 16,17]. By definition, stationary phase cells are slow- or non-growing, a characteristic shared bybiofilm cells and persister cells. Cells in such a metabolically inactive state are inherently moretolerant to antimicrobial drugs that target actively growing cells. For example, beta-lactamantibiotics are ineffective against cells that are not actively dividing and synthesizing new cellwall peptidoglycan [18]. Like biofilms, cells from stationary phase cultures also exist in a highcell density environment. At high cell densities, cells are likely to become starved of nutrients,oxygen, or both, resulting in a drop in intracellular ATP.Recently, we reported that intracellular ATP concentration appears to be the major deter-minant of survival to antibiotic challenge for both stationary phase cells and persister cells in S.aureus [17]. The same may also be true for biofilm-associated cells. The limited nutrient andoxygen availability within the biofilm presumably results in reduced metabolic activity and alower energy state, which is a hallmark of persister cells that can survive exposure to most bac-tericidal antibiotics.It may be that low cell energy levels are the major determinant of antibiotic tolerancein biofilm cells, persister cells, and stationary phase cells. For example, S. aureus initiatesexpression of biofilm adhesins in response to a variety of external stresses, including nutrientlimitation, pH stress, osmotic stress, and sublethal antibiotic challenge [6, 19, 20]. Thus, bio-film formation may also be viewed as a response by the bacteria to environmental stress thatnot only promotes intercellular adherence but also imposes a selective pressure for metaboli-cally inactive, energy-depleted cells that can survive hostile growth conditions, includingantibiotic challenge.However, this does not imply that all biofilm and persister cells are physiologically identical,but rather that the mechanism(s) underpinning the ability of otherwise susceptible S. aureuscells to tolerate and survive antibiotic challenge may be essentially the same (Fig 1).Does the Biofilm Mode of Growth Influence the Rate of PersisterCell Production in S. aureus Populations?Exposure of planktonic staphylococcal cells to subinhibitory antibiotic concentrations inducesbiofilm formation [21–23]. In addition, sub-MIC (minimum inhibitory concentration) antibi-otics and other external stresses enrich for drug-tolerant persister cells [15]. These observa-tions may be important in linking the phenotypes of antibiotic tolerance and biofilm matrixproduction. Although antibiotics penetrate and reach high concentrations within the biofilm,penetration dynamics are influenced by the matrix [8, 10]. Together with the limited nutrientand oxygen availability, the establishment of an antibiotic concentration gradient may add anadditional selective pressure for cells within the biofilm to enter a persister state upon encoun-tering sub-MIC levels of the antibiotic, allowing them to survive the subsequent lethal concen-tration. Relevant to this are studies showing that the extracellular matrix deployed by S. aureusbiofilms under different growth conditions, plus the age and cell density of the biofilm, signifi-cantly influences the ability of antibiotics such as rifampicin, daptomycin, vancomycin,gentamicin, and fosfomycin to kill biofilm cells [3, 4]. For instance, rifampicin can exhibitPLOS Pathogens | DOI:10.1371/journal.ppat.1006012 December 29, 2016 2 / 5significant activity against fibrin-dependent biofilms grown in Roswell Park Memorial Insti-tute (RPMI) media but not against FnBP/eDNA biofilms produced by the same strain in richculture media [4]. It is therefore conceivable that the relative numbers of persister cells vary indifferent types of biofilm. These observations support the idea that environmental conditions(nutrient availability, oxygen concentration, cell density, and sub-MIC antibiotic levels) regu-late expression of biofilm adhesins and the type of biofilm matrix produced, which influencesthe rate at which cells in the biofilm enter the persister state and, accordingly, the antibiotic tol-erance of the biofilm.Can the Shared Features of Persister and Biofilm Cells BeExploited to Better Treat Chronic Infections?The phenotypic heterogeneity of biofilm cells, as outlined above, suggests that new treatmentapproaches aimed at disrupting biofilms are not necessarily going to be effective against per-sister cells. By contrast, new therapeutic approaches targeting persister cells should also havepotential against biofilms. For example, persister cells can be killed by the acyldepsipeptideantibiotic ADEP4, which activates the nonspecific ClpP protease in an ATP-independent man-ner [1]. ADEP4 is active against persisters, stationary phase cells, and biofilms [1]. Similarly,the histidine kinase inhibitor NH125, which was shown to have significant antipersister activ-ity at low concentrations, was able to kill biofilm cells and disrupt the biofilm matrix at highconcentrations [24]. In S. epidermidis, the minimum bactericidal concentration of ciprofloxa-cin effective against biofilms can also kill most persister cells [25]. These data suggest thatFig 1. Role of persister cells in biofilm antibiotic tolerance. Antibiotic-tolerant persister cells (depicted in red) are enriched instationary phase planktonic cultures and biofilms compared to exponential phase planktonic cultures, which are primarily composed ofantibiotic-susceptible cells (depicted in green). The antibiotic susceptibility phenotype of exponential phase cells correlates with highlevels of metabolic activity and ATP associated with abundant nutrients and oxygen. As cells enter stationary phase or become encasedin a biofilm matrix, nutrients and oxygen are depleted and the level of intracellular ATP is reduced. Exposure to antibiotics, most of whichtarget ATP-dependent processes, has no significant effect on metabolically inactive persister cells with low intracellular ATP levels.doi:10.1371/journal.ppat.1006012.g001PLOS Pathogens | DOI:10.1371/journal.ppat.1006012 December 29, 2016 3 / 5persister cell populations can also be used as a model to evaluate antibiofilm therapeutics.Thus, recent reports that glucose can augment daptomycin-induced killing of S. aureus persist-ers [26] or that the anticancer drug cisplatin [27] and cis-2-decenoic acid [28] have activityagainst persisters may also be indicative of antibiofilm activity.Future research into how antibiotic-tolerant persister cells contribute to treatment failuresof biofilm-associated infections can exploit advances in techniques to better study persistercells. Reporter systems developed to label persister cells could be adapted and combined withmicroscopy to investigate the proportion, distribution, and metabolic niche of persister cellswithin heterogeneous biofilm populations, as well as their response to antibiotics. Like S.aureus, the Pseudomonas aeruginosa biofilm phenotype is associated with antibiotic tolerance[7]. Chronic biofilm-associated infections caused by P. aeruginosa are difficult to eradicatewith current antibiotic treatment regimens [7, 29], and it is feasible that such biofilms are alsoenriched with persister cells. Intriguingly, mannitol augments tobramycin-induced killing ofP. aeruginosa persisters and biofilm cells [29] in a manner comparable to glucose-enhanced,daptomycin-induced killing of S. aureus persisters [23]. Thus, despite differences in the mech-anisms of persister cell formation in gram-negative and gram-positive bacteria, the role of per-sister cells in biofilm tolerance may extend beyond S. aureus. 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Convergence of Staphylococcus aureus Persister and Biofilm Research: Can Biofilms Be Defined as Communities of Adherent Persister Cells?

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