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. 2014 Apr;58(4):2089–2097. doi: 10.1128/AAC.02552-13

Selective Target Inactivation Rather than Global Metabolic Dormancy Causes Antibiotic Tolerance in Uropathogens

Lee W Goneau a,b, Nigel S Yeoh c, Kyle W MacDonald a,b, Peter A Cadieux a,d, Jeremy P Burton a,b,e,f, Hassan Razvi e,f, Gregor Reid a,b,f,✉
PMCID: PMC4023725  PMID: 24449771

Abstract

Persister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed between Staphylococcus saprophyticus and Escherichia coli uropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancy per se cannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenic E. coli isolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure.

INTRODUCTION

Since their discovery by Joseph Bigger in 1944, persister cells have remained an elusive subpopulation of bacteria whose multidrug tolerance (MDT) may be an important contributing factor to antibiotic therapy failure (1). While antibiotic resistance is acquired through genetic changes (2), persister cells are typically thought to represent nongrowing, isogenic, physiological variants that gain MDT via the inactivation of antibiotic-targeted cellular processes through presumed metabolic dormancy (3, 4). Upon treatment cessation, these dormant cells reactivate and initiate a new infection cycle. This raises concerns over the effectiveness of standard antibiotic therapy, especially when infections fail to clear despite the absence of resistant organisms, such as in tuberculosis, cystic fibrosis, and recurrent urinary tract infection (RUTI) (5, 6, 7).

Persister subpopulations generally occur at a low frequency, with formation believed to be driven by two processes. Type I persisters are classified as slowly replicating variants which are enriched during late exponential and stationary phases (8), while type II persisters are those whose formation occurs stochastically during exponential phase and are believed to be derived from random fluctuations in transcriptional and translational noise (9–11). Persister subpopulations can also be enriched by various aversive conditions, including oxidative stress, nutrient deprivation, and hypoxia, and even by the addition of subinhibitory concentrations of antibiotics (12–17). This last point is of particular concern as continuous prophylactic antibiotics are often prescribed to manage chronic infections such as RUTIs, introducing subinhibitory antibiotic levels over the daily course of dosing (18). Such stressors generally result in MDT through the activation of various toxin/antitoxin (TA) systems, which are believed to provide a common “emergency stop” response mechanism to halt environmental stress (19–22). Activation of these systems is intimately connected to induction of the SOS response, which can occur via the application of fluoroquinolone and beta-lactam antibiotics (15, 19, 23). Importantly, a consensus regarding the nature of MDT has yet to be reached, with two scientific camps disputing whether persisters represent truly dormant bacterial variants or simply a slow-growing subpopulation of the greater culture (24–29). Identifying the mechanisms for persister onset and maintenance is essential for understanding the nature of chronic diseases and for the future development of persister-directed therapeutics.

The purpose of this study was to investigate the importance of persister states in RUTI and to understand the nature of MDT in uropathogens. We suggest two hypotheses to evaluate the contribution of dormancy toward persistence: (i) dormancy is necessary and sufficient to convey MDT such that persisters will respond to different antibiotics with the same levels of survival, and (ii) different antibiotics will induce global dormancy such that surviving persisters will demonstrate tolerance to both lethal levels of the inducing agent and different agents (cross-tolerance). Thus, we aimed to characterize the contribution of dormancy toward MDT in the uropathogens Escherichia coli and Staphylococcus saprophyticus by quantifying the surviving fraction following various antibiotic treatment conditions. These organisms represent the leading causes of both Gram-negative and Gram-positive bacterial UTIs, respectively, and are associated with recurrence (30–32). We considered the dynamics of persister cell induction in addition to maintenance by analyzing both type I and II persister fractions. We also compared the persister fractions of various uropathogenic E. coli (UPEC) isolates recovered from either same-strain recurrence (SSR) or acute infection (AI) sufferers, hypothesizing that SSR UPEC strains would demonstrate larger persister fractions than AI strains due to the selective pressure imparted by antibiotic therapy during RUTI treatment. Additionally, we used a panel of antibiotics with distinct cellular targets to assess the ability to induce tolerance. These results were extended using cross-tolerance to explore if antibiotic-induced persisters demonstrated MDT through the onset of global metabolic dormancy or if tolerance was specific and dependent on the mechanism of onset. Lastly, SOS-deficient UPEC strains were used to characterize the metabolic activity of type I persister cells. It was hypothesized that if complete metabolic dormancy is both necessary and sufficient for MDT, then established persisters should be refractory to antibiotics regardless of the functionality of their SOS systems.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Both E. coli CFT073 and S. saprophyticus 15305 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) while the UPEC clinical isolates E. coli TOP277, TOP263, TOP379, TOP344, TOP345, PUTS277, PUTS278, PUTS1127, PUTS1236, and UTI89 were kindly provided by Scott Hultgren (Washington University School of Medicine, St. Louis, MO, USA). Briefly, each strain was derived from a distinct patient presenting with RUTI. Patients were sampled over time, and organisms isolated from each infection episode were sequenced (33). If repeat recurrences were caused by the same strain, the isolate was assigned an SSR designation. Conversely, infections caused by a strain which varied from previous infections were designated AI. SOS-deficient strains E. coli PAS0209 (ΔrecA) and PAS0211 (lexAT355G) (which has a T-to-G change at position 355 encoded by lexA; also referred to as lexAG85D or Ind mutant) derived from the UTI89 background were provided by Sheryl Justice (The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA). MIC determination and antibiotic susceptibility experiments were performed at 37°C in cation-adjusted Mueller-Hinton (MH) II medium. All remaining persister cell experiments were conducted in Luria-Bertani (LB) medium, with shaking at 200 rpm (liquid cultures).

MIC determination.

The MICs of ciprofloxacin, ampicillin, and gentamicin were determined for all organisms using the broth microdilution technique outlined by the Clinical and Laboratory Standards Institute (CLSI) (34). Briefly, cultures were inoculated into medium containing various 2-fold dilutions of antibiotics and grown for 24 h in a 96-well plate. The MIC was regarded as the last well demonstrating growth based on turbidity. Experiments were carried out with at least three independent replicates. MIC quality control was performed using control organisms recommended by the CLSI with known MIC values for antibiotics used in this study.

Antibiotic susceptibility testing.

Susceptibility testing was performed using the Kirby-Bauer method as per standard CLSI guidelines (34). Briefly, strains were grown in MH broth overnight in a shaking incubator (200 rpm) at 37°C. Cultures were diluted 5-fold in phosphate-buffered saline (PBS) and streaked across the entire surface of an MH agar plate using a cotton-tipped swab. Ciprofloxacin (CIP; 5 μg), ampicillin (AMP; 10 μg), and gentamicin (GEN; 10 μg) test discs (VWR) were each added to one-third of the plate. Zones of growth inhibition (ZOIs; mm) around each disk were determined following overnight incubation. The ZOI for each strain was compared to reference standards to determine the clinically relevant category of susceptibility for each antibiotic.

Type I and type II persister cell assays.

Type I persisters were isolated by directly applying lethal concentrations of ciprofloxacin (5 μg/ml), ampicillin (100 μg/ml), or gentamicin (10 μg/ml) to stationary-phase (24 h) cultures for 3 h and enumerating the surviving fraction before and after treatment (35). Conversely, overnight cultures were first seeded at 1,000-fold into fresh LB medium and then grown for ∼3 or ∼7 h (for UPEC and S. saprophyticus, respectively) to deplete the type I persister population for type II persister assays (until early exponential phase; turbidity of 0.5 to 0.6 for S. saprophyticus and E. coli at 600 nm). A subsequent 1,000-fold seeding in fresh LB medium with antibiotics at 0.25× MIC and lasting ∼3 to 7 h (turbidity of 0.5 to 0.6 at 600 nm) was performed to enrich the type II persister fraction. Percent survival was determined through the enumeration of viable cells prior to and following treatment with lethal antibiotics. Colonies were isolated by serially diluting cultures in PBS, spot plating 10-μl drops on LB agar, and counting the resulting colonies. In cases where viability fell below 1,000 CFU/ml, 10- or 100-μl drops were applied directly from cultures reconstituted in PBS and spread over the entire surface of an LB plate. Survival due to phenotypic tolerance and not spontaneous resistance was determined by subculturing the surviving fraction in fresh LB medium and determining the MIC.

RESULTS

Antibiotics from distinct classes induce persister cell formation in both Gram-positive and -negative organisms.

We sought to illustrate the ubiquity of antibiotic-dependent persister induction using various antibiotics (ciprofloxacin, ampicillin, and gentamicin) targeting diverse cellular processes in both Gram-negative (E. coli) and Gram-positive (S. saprophyticus) organisms. A summary of the MICs and susceptibilities for strains used in this study is provided in Table 1. Although various sub-MICs induced survival, 0.25× MIC levels had the greatest effect without perturbing the bacterial growth rate (Fig. 1; see also Fig. S1 in the supplemental material). Both E. coli CFT073 and S. saprophyticus 15305 showed significant (P < 0.05 and P < 0.01, respectively) increases in percent survival following ciprofloxacin, ampicillin, and gentamicin pretreatment and challenge. This surviving fraction did not have any significant changes in MIC compared to the original starting culture, indicating spontaneous resistance developed during the previous challenge did not contribute to survival (data not shown).

TABLE 1.

Summary of strains used in this study and respective MICs and antibiotic susceptibilities

Strain (type) MIC (μg/ml)
 ZOI (mm [susceptibility])a
CIP AMP GEN CIP AMP GEN
S. saprophyticus 15305 0.25 0.125 0.0625 26.5 (S) 28.25 (S) 20 (S)
E. coli strains
    CFT073 0.015625 3 1 25.5 (S) 17 (S) 17 (S)
    TOP277 (SSR) 0.0125 6 0.75 22.75 (S) 19.75 (S) 16 (S)
    TOP263 (SSR) 0.0166 8 0.5625 23.5 (S) 16.25 (S) 16 (S)
    TOP379 (SSR) 0.020833 10 0.6875 23.25 (S) 14 (I) 15 (S)
    TOP344 (SSR) 0.0229166 10 0.875 21.75 (S) 14 (I) 15 (S)
    TOP345 (SSR) 0.0145833 8.5 0.875 26 (S) 19.75 (S) 16.25 (S)
    PUTS277 (AI) 0.020833 9 0.3125 26.25 (S) 17.5 (S) 17.25 (S)
    PUTS278 (AI) 0.0166 6 0.375 25 (S) 17.25 (S) 17.75 (S)
    PUTS1127 (AI) 0.0166 9.5 1 28 (S) 17.75 (S) 16 (S)
    PUTS1236 (AI) 0.0166 9 0.75 23.5 (S) 17.25 (S) 16.5 (S)
    UTI89 0.01875 9 1.25 26.5 (S) 16.75 (S) 15.75 (S)
    UTI89 ΔrecA 0.000977 8 1 43.75 (S) 25.75 (S) 25.25 (S)
    UTI89 lexAT355G 0.00391 8 1.25 35.5 (S) 22.75 (S) 21 (S)
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a

ZOI, zone of inhibition; S, susceptible; I, intermediately susceptible; R, resistant (per CLSI guidelines).

FIG 1.

FIG 1

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Persistence induction following a 0.25× MIC pretreatment with either CIP, AMP, or GEN for 3 h. Percent surviving fractions represent the persister subpopulation following 3 h of lethal treatment with either CIP, AMP, or GEN in E. coli CFT073 (A to C) or S. saprophyticus 15305 (D to F). Means from at least three independent experiments are shown with significance. Significance was determined using an unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Evaluating antibiotic susceptibility patterns in type I and II persister cells.

It was reasoned that persistent bacteria should share similar levels of dormancy, allowing the prediction that antibiotic susceptibility should be invariable across drug classes for this MDT population. The efficacy of ciprofloxacin, ampicillin, and gentamicin was surveyed against stationary-phase cultures (predominantly type I persister) (Fig. 2). S. saprophyticus demonstrated significantly greater antibiotic tolerance during stationary phase, with percent survival increasing 443.2-fold, 90,263.0-fold, and 14,682.4-fold (P < 0.001) compared to exponential-phase cultures for ciprofloxacin, ampicillin, and gentamicin, respectively. Similarly, E. coli survival increased 676.2-fold, 5,325.0-fold, and 7,782.1-fold (P < 0.001) following lethal ciprofloxacin, ampicillin, and gentamicin challenge, respectively. Heterogeneity in percent survival was observed across bacterial genera and antibiotic classes. S. saprophyticus was significantly more susceptible to gentamicin (33.5% survival) than to ciprofloxacin (80.9%) or ampicillin (80.7%) (P < 0.001). Conversely, E. coli demonstrated a high rate of survival against ampicillin (76.8% survival) and gentamicin (56.6%), while remaining significantly susceptible to ciprofloxacin (3.6%) (P < 0.001).

FIG 2.

FIG 2

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Fraction sizes of type I and type II persisters of exponential- and stationary-phase cultures. Percent survival was determined following a 3-h challenge with a lethal dose of CIP, AMP, or GEN for both E. coli CFT073 (A) and S. saprophyticus 15305 (B). Means from at least three independent experiments are shown with significance. Significance was determined using one-way analysis of variance and Bonferroni's multiple comparison test (***, P < 0.001).

The nature of the antibiotic-induced persister fraction was explored by challenge with the same agent that induced tolerance in addition to agents with different cellular targets. Cross-tolerance was detected for some antibiotic classes but varied with organism genus. Ciprofloxacin and gentamicin pretreatments most effectively enhanced survival of E. coli CFT073, while ampicillin pretreatment was favorable in S. saprophyticus 15305 (Fig. 3). Specifically, sub-MIC ciprofloxacin significantly enhanced percent survival 5.2-fold and 3.2-fold (P < 0.05) following lethal treatment with ampicillin and gentamicin, respectively, in E. coli while sub-MIC ampicillin significantly increased percent survival 4.1-fold and 34.9-fold following lethal ciprofloxacin and gentamicin challenge, respectively, in S. saprophyticus (P < 0.01).

FIG 3.

FIG 3

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Cross-tolerance susceptibility patterns of persisters induced with various antibiotic pretreatments. Percent surviving fractions represent the persister subpopulation following a 3-h lethal treatment with either CIP, AMP, or GEN, as indicated, for both E. coli CFT073 (A to C) and S. saprophyticus 15305 (D to F). Means from at least three independent experiments are shown with significance. Significance was determined using one-way analysis of variance and Bonferroni's multiple comparison test (*, P < 0.05; **, P < 0.01). Negative, no pretreatment.

Characterizing the persister fraction of UPEC strains isolated from same-strain recurrent and acute infections.

Persister frequency was determined for E. coli strains isolated from SSR infections and compared to those isolated from acute infections (AI strains). As growth phase influences the persister fraction size, we first confirmed that all UPEC strains demonstrated similar rates of growth (see Fig. S2 in the supplemental material). Thus, any change in the persister fraction size is due solely to variations in the organism's capacity to form persisters. Stationary-phase cultures were considered for type I persister frequency. Standard antibiotic susceptibility testing revealed all strains to be similarly susceptible to the antibiotics tested, with the exception of SSR organisms TOP379 and TOP344, which demonstrated intermediate susceptibility to ampicillin (Table 1). Similar to E. coli CFT073, all isolates within the type I stationary-phase fraction tolerated ampicillin and gentamicin while succumbing to ciprofloxacin treatment (Fig. 4). Unexpectedly, some SSR organisms revealed slight growth during lethal treatment. Although antibiotics were already provided at therapeutic levels, we observed the same result upon challenging with 10 times the previous lethal dose (data not shown). Viability of all AI strains declined as expected. When grouped, AI strains had significantly lower survival with ampicillin (69.15%) and gentamicin (77.93%) treatment than SSR organisms (118.0% and 151.2%, respectively), which had a higher persister fraction. Surviving organisms did not display any changes in MICs following the experiments, suggesting that spontaneous acquisition of resistance elements did not occur.

FIG 4.

FIG 4

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Percent survival of same-strain recurrent (SSR) and acute infection (AI) UPEC isolates challenged with a lethal dose of CIP (A), AMP (B), or GEN (C) during stationary-phase growth. Dashed lines indicate average SSR or AI group survival to the respective antibiotic. Means from at least four independent experiments are shown with significance. Significance between SSR and AI groups was determined by comparing average survival rates using an unpaired t test (ns, not significant; **, P < 0.01).

Cross-tolerance assays were also conducted to determine sub-MIC antibiotic effects on type I persister formation during exponential-phase growth and to permit assessment of the susceptibility of type I persister cells. Ciprofloxacin and gentamicin pretreatments induced persister states at the highest frequency in SSR organisms, while most AI strains failed to respond to antibiotics (Fig. 5). However, gentamicin pretreatment significantly (P < 0.01) increased the persister fraction in some AI strains. In almost all cases, pretreatment with ampicillin significantly (P < 0.05) improved the efficacy of gentamicin in both SSR and AI organisms. Some antibiotic-SSR organism combinations again demonstrated slight growth at lethal antibiotic concentrations. This was especially true in strains TOP379 and TOP344, which are intermediately susceptible to ampicillin, with growth enhanced in all pretreatment groups following lethal ampicillin challenge.

FIG 5.

FIG 5

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Cross-tolerance susceptibility patterns of SSR (A) and AI (B) UPEC persisters induced with various antibiotic pretreatments. Percent survival was determined after challenge with a lethal dose of either CIP, AMP, or GEN. Means from at least four independent experiments are shown with significance. Significance was determined using one-way analysis of variance and Bonferroni's multiple comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

SOS-deficient UPEC persister cells are not tolerant to ciprofloxacin.

E. coli UTI89 strains deficient in RecA production (ΔrecA strain) and LexA autoproteolysis (lexAT355G strain) were considered to compare the effects of insufficient double-strand break repair and SOS induction on persister cell maintenance and onset. ΔrecA and lexAT355G strains were significantly (P < 0.001) sensitized to ciprofloxacin challenge; thus, 0.25× MIC pretreatment values were adjusted accordingly (Table 1). We observed that ciprofloxacin pretreatment significantly (P < 0.01) induced persister cell production following subinhibitory pretreatment and subsequent lethal challenge in early-exponential-phase cultures. This effect was completely abrogated in both SOS-deficient strains (Fig. 6). Additionally, ampicillin and gentamicin significantly (P < 0.001) induced persister formation in wild-type UTI89 but not in the SOS-deficient strains (Fig. 6). Although less proficient in antibiotic-dependent persister induction, SOS-deficient strains did produce type II persisters comparable to wild-type UTI89 levels during normal growth, as demonstrated in the surviving ampicillin and gentamicin fractions.

FIG 6.

FIG 6

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Cross-tolerance assays comparing persister induction capacity of SOS-deficient strains following subinhibitory antibiotic therapy. Percent survival was determined after challenge with a lethal dose of either CIP (A), AMP (B), or GEN (C). Means from at least five independent experiments are shown with significance. Significance was determined using one-way analysis of variance and Bonferroni's multiple comparison test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). WT, wild type.

The contribution of active double-strand break repair and SOS response to antibiotic tolerance was investigated using the type I, stationary-phase persister fraction. The surviving fractions in both the ΔrecA and lexAT355G strains were significantly (P < 0.001) reduced (to the limit of detection) following ciprofloxacin challenge (Fig. 7). However, persister cell levels were unchanged following incubation with ampicillin and gentamicin compared to wild-type UTI89 levels, indicating that these strains were not deficient in persister cell development.

FIG 7.

FIG 7

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Sensitivity of type I UPEC persisters deficient in SOS to various antibiotics. Fraction size was determined following a 3-h challenge of stationary-phase cultures with a lethal dose of CIP, AMP, or GEN and enumeration of the surviving population. Means from at least four independent experiments are shown with significance. Significance was determined using one-way analysis of variance and Bonferroni's multiple comparison test (***, P < 0.001).

DISCUSSION

Using two population-based approaches, we have characterized the persister fractions of representative uropathogens. We conclude that observed variations in persister fraction sizes between type I stationary and type II exponential subpopulations indicate that global dormancy cannot solely account for MDT. Rather, we propose that persisters can be classified based on the metabolic activity of distinct cellular processes such as peptidoglycan cross-linking, transcription, and translation, with each demonstrating various degrees of activity and influencing a persister cell's susceptibility to antibiotics. In this way, MDT is a phenomenon only of the population as a whole and not of individual persister cells, which are heterogeneous in regard to their antibiotic sensitivity and tolerance. Furthermore, antibiotic-induced tolerance is not dependent on onset of global dormancy, with cross-tolerance results suggesting that an individual persister cell's susceptibility to different agents is contingent on its mechanism of onset.

Generally, most antibiotics appear to primarily influence their cognate targets, increasing tolerance against further challenge with the same agent rather than resulting in global downregulation of overall cellular activity, as hypothesized (36). However, there are notable exceptions, including the observation that ampicillin pretreatment results in heightened MDT against all agents in S. saprophyticus. The ability of this drug to halt replication in an SOS-dependent manner and induce beta-lactam tolerance has been demonstrated in E. coli but is perhaps more significant in Gram-positive bacteria (23, 37). Agents affecting transcription and translation may impart more broad-spectrum tolerance by influencing downstream processes and, therefore, the activity of other potential drug targets in a similar manner. SOS-deficient UPEC strains were deficient in antibiotic-induced persister formation against all agents tested. Surprisingly, gentamicin induction was also affected despite its reported inability to induce SOS responses in E. coli (38). This finding might reflect a novel SOS-dependent mechanism of persister induction which requires further exploration. Considering the heterogeneity in Gram-negative and Gram-positive responses, various antibiotics may demonstrate different persister-inducing potentials against different organisms. If true, this hypothesis may have predictive clinical value in the prescribing of antibiotics which effectively ameliorate disease without unnecessarily enriching persister fractions.

Enumeration of the type I and type II persister fractions of UPEC isolates revealed heterogeneity in both subpopulations' responses to antibiotics. Ciprofloxacin significantly reduced survival in stationary-phase cultures compared to ampicillin and gentamicin treatments, which were largely ineffective against both SSR and AI isolates, as demonstrated in other strains tested. The comparatively enhanced efficacy observed for the DNA gyrase inhibitor ciprofloxacin suggests significant activity in persisters. This may indicate that transcription and replication actively occur in persisters and that they are sensitive to changes affecting these processes (39). This hypothesis is further confirmed through the observation that SOS-deficient mutants are severely inhibited when challenged with ciprofloxacin while remaining refractory to ampicillin and gentamicin, agents which do not directly damage DNA. In addition to revealing DNA gyrase activity in persisters, this result also suggests that an active SOS response is required to abrogate ciprofloxacin-induced DNA damage (especially with respect to RecA-dependent double-strand break repair), which may be critical for fluoroquinolone tolerance in UPEC. The concurrent inhibition of the SOS response [such as with the application of the RecA inhibitor N6-(1-napthyl)-ADP] along with the application of fluoroquinolone antibiotics may be an effective means to clear persister-dependent chronic infections (40).

Type II persisters were induced by antibiotics at a high frequency in SSR compared to AI isolates, suggesting that these traits may be subject to selection and enriched over the course of RUTI and prophylaxis. Selection of genes influencing persistence has been previously demonstrated, where overproduction of the persister-inducing molecule indole corresponded with increased antibiotic levels over time and subsequent MDT (41). In addition, Mulcahy et al. observed that Pseudomonas aeruginosa persister fractions increased over a 96-month period in an individual suffering with cystic fibrosis and undergoing antibiotic therapy (7). These results are intriguing as antibiotics often increase the incidence of mutation within organisms, thereby increasing the frequency with which organisms gain persister traits during long-term prophylactic therapy (42). This observation may have severe clinical ramifications in suggesting that a patient's history of antibiotic use may increase the risk of the patient's suffering from recurrent infections. In some cases, SSR strains not only survived lethal antibiotic dosing but actually continued replicating in their presence despite organism susceptibility. This resistance-like phenotype has been observed previously in susceptible type II persister fractions but is often unreported. Notably, Balaban et al. (9) demonstrated that E. coli persisters with mutations in the TA hipQ gene could continue limited growth when exposed to lethal ampicillin amounts, corroborating our results. Other groups have noted periods of slight replication following lethal antibiotic dosing (6, 43). Wiuff et al. (43) concluded that this could not be explained alone by degeneration of the antibiotic in the growth medium over time. Maisonneuve et al. recently showed that slow-growing variants within an exponentially growing E. coli population demonstrate MDT in a (p)ppGpp-dependent manner (27). Admittedly, it is possible that the observed limited replication of persisters is a result of the categorical susceptibility cutoffs provided by the Kirby-Bauer method of testing. Although strains appeared as susceptible during this routine test, they were often highly tolerant, with low ZOIs (nearly intermediately susceptible to resistant). This is of clinical concern as antibiograms may inaccurately designate intermediately susceptible organisms as fully susceptible. However, the observed growth of persisters in this and other studies is supportive of the idea that these cells maintain at least some metabolic activity.

Further analysis and comparisons using cross-tolerance illustrated that SSR isolates have a greater capacity to form persisters following antibiotic pretreatment than AI strains. However, ampicillin often failed to induce persistence in the clinical isolates tested, with pretreatment resulting in greater susceptibility especially following gentamicin challenge. This finding supports the notion that persister cells demonstrate limited replication as ampicillin was capable of corrupting the still moderately active transpeptidase enzyme. The resulting leakiness likely improved the uptake of gentamicin, which in turn enhanced its efficacy against the still active ribosome once the gentamicin was available intracellularly (37). This observation is supported by the work of Allison et al., who demonstrated that increasing gentamicin uptake resulted in aminoglycoside sensitivity in E. coli (44). The observation that persister cells do not demonstrate global metabolic dormancy suggests that some combinatorial antibiotic therapies may be effective in treating persister-related chronic infections. Larger, high-throughput screens are recommended to validate this hypothesis.

In summary, we have demonstrated that persisters are differentially induced at subinhibitory levels by ampicillin and gentamicin in addition to ciprofloxacin in both Gram-negative and Gram-positive bacteria. Furthermore, type I persister fraction analysis indicates that global metabolic dormancy is not solely responsible for MDT. Rather, the metabolic activities of individual targets dictate overall bacterial responses to agents applied, with some antibiotics imparting a greater inhibitory effect on multiple cellular targets than others. This is supported in SOS-deficient strains which are unable to specifically tolerate ciprofloxacin. Lastly, persister traits are enriched in organisms with a history of antibiotic therapy.

The use of low-dose suppressive antibiotic therapy and antibiotic prophylaxis are commonly utilized regimens in clinical medicine that may, in fact, be playing a role in the development of MDT. The findings of this study raise concern that the incidence of chronic and recurrent bacterial infections may actually increase with the use of antibiotics for prophylaxis, corroborating a recent theory that modern RUTI frequency correlates with the widespread use of antibiotics (45). A greater understanding of these unique bacterial physiological states will be essential to improve management of these common debilitating infections. Specifically, we propose that treatment should focus not on the “waking” of persisters but, rather, on the characterization of cellular targets that are active and thus susceptible to antibiotic corruption.

Supplementary Material

Supplemental material
supp_58_4_2089__index.html (761B, html)

ACKNOWLEDGMENTS

We kindly thank Scott Hultgren for providing the RUTI clinical isolates and Sheryl Justice for the SOS-deficient strains.

L.W.G. is funded by a Frederick Banting and Charles Best Canadian Institutes of Health Research Doctoral Research Award.

Footnotes

Published ahead of print 21 January 2014

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.02552-13.

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