Treatment of Staphylococcus aureus in stationary growth phase with high doses of the antibiotic daptomycin (DAP) eradicates the vast majority of the culture and leaves persister cells behind. Despite resting in a drug-tolerant and dormant state, persister cells exhibit metabolic activity which might be exploited for their elimination. We here report that the addition of glucose to S. aureus persisters treated with DAP increased killing by up to five-fold within one hour. This glucose-DAP effect also occurred with strains less sensitive to the drug. The underlying mechanism is independent of the proton motive force and was not observed with non-metabolizable 2-deoxy-glucose. Our results are consistent with two hypotheses on the glucose-DAP interplay. The first is based upon glucose-induced carbohydrate transport proteins that may influence DAP and the second suggests that glucose may trigger the release or activity of cell-lytic proteins to augment DAP’s mode of action.
Citation: Prax M, Mechler L, Weidenmaier C, Bertram R (2016) Glucose Augments Killing Efficiency of Daptomycin Challenged Staphylococcus aureus Persisters. PLoS ONE 11(3): e0150907. doi:10.1371/journal.pone.0150907
Editor: Dirk-Jan Scheffers, University of Groningen, Groningen Institute for Biomolecular Sciences and Biotechnology, NETHERLANDS
Received: September 1, 2014; Accepted: February 21, 2016; Published: March 9, 2016
Copyright: © 2016 Prax et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by grant BE4038/2 within the priority programme 1316 “host adapted metabolism of bacterial pathogens” of the Deutsche Forschungsgemeinschaft (www.dfg.de). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Eradication of harmful bacteria in the human body is often cumbersome due to drug resistance and drug tolerance particularly in biofilm embedded cells [1–7]. Biofilms accommodate a high percentage of persister cells which are in a non-dividing and metabolically less active state . Persisters are regarded as genetically identical variants among a population of unicellular organisms that tolerate and survive high concentrations of antibiotics over extended periods of time [9–12]. This kind of phenotypic heterogeneity is a successful bet-hedging strategy to endure hostile conditions, such as antibiotic treatment or immune response and provides a rationale for recurrent or chronic bacterial infections [9,13,14]. The level of persister cells among a clonal bacterial culture is influenced by nutrient limitation, growth phase, various stresses, quorum sensing and other factors [15–17]. Compared to the identification of numerous persister-genes, information available on metabolic aspects of persisters is more limited . A change in carbon source utilization upon glucose limitation stimulates persister formation in E. coli  and accordingly, E. coli persisters maintain glycerol and glucose metabolism [20–22]. De novo synthesis of amino acids was observed with persister cells of the notorious pathogen Staphylococcus aureus , which is causative of skin infections, osteomyelitis, endocarditis, bacteremia and further illnesses [24–27]. Multiple antibiotic resistant S. aureus strains continue to pose a formidable challenge in hospitals and in the community . The bactericidal lipopeptide daptomycin (DAP) is one of few antibiotics that is generally effective against many S. aureus strains , as well as other Gram positive bacteria [30–32]. The amphiphilic character of DAP in combination with calcium cations facilitates the incorporation into the bacterial membrane . According to the current model, oligomerization of DAP leads to pore formation and increased permeability for ions resulting in perturbation of the proton motive force (PMF) and cell death . DAP is highly efficient also against S. aureus cells in stationary phase, which are tolerant towards a broad range of other antibiotics . As shown previously, the eradication efficiency of S. aureus by DAP is enhanced upon combination with other antibiotics [36,37] or D-cycloserine . First cases of DAP non-susceptible strains were documented in hospitals briefly after introduction of the drug . Such strains frequently exhibit changes in the cell envelope [40–42]. To prevent resistance formation and selection for non-susceptible strains due to prolonged drug-treatments , it is necessary to develop new efficient therapeutic strategies, with a special focus on targeting persister cells .
A new means for persister eradication in biofilms was achieved by a combination therapy with rifampicin and the acyldepsipeptide antibiotic ADEP4, leading to the permanent activation of protease ClpP . Furthermore, the administration of carbohydrates increases persister killing by aminoglycosides due to their dependency on the proton motive force (PMF) [45,46]. We here show that supplementing cultures of DAP challenged S. aureus cells with specific carbohydrates in vitro leads to accelerated killing, which intriguingly also pertains to strains less susceptible to this drug. According to our data, the underlying mechanism is not-dependent on the PMF but may be dependent on metabolization of glucose. Unraveling the molecular basis and exploiting this phenomenon provides perspectives for a powerful anti-persister therapy.
Materials and Methods
Bacteria, growth conditions, and working solutions
Bacterial strains used in this study are listed in Table 1. Unless stated otherwise, cultures were incubated at 37°C with aeration and shaking (150 rpm) in tryptic soy broth (TSB). To provide stationary growth phase cultures, incubations were performed overnight. In the course of this study, two different types of TSB were used, either composed of casein peptone (pancreatic) (17 g/L), soy peptone (A3SC) (3 g/L), NaCl (5 g/L), K2HPO4 (2.5 g/L) and glucose (2.5 g/L, sterile filtered and added after autoclaving, or a ready-to-use powder purchased from Sigma, both at an approximate 1:10 culture-to-flask volume ratio. Daptomycin (DAP, analytic grade powder; designated ‘Cubicin’, Novartis Pharma, Nuremberg, Germany) was prepared freshly prior to each application, filter sterilized (0.2 μM pore size, Whatman, Dassel, Germany) and used to challenge stationary-phase S. aureus cells. 100-fold the MIC of DAP solution had been determined as 400 mg/L for S. aureus SA113 before  and 150 mg/L corresponding to 250-fold the MIC was determined and used for NARSA strains . Strain HG003 D6  was treated with 400 mg/L corresponding to 100-fold the MIC and the clinical strains with 400 mg/L corresponding to 200-fold and 800-fold the MIC for strains 616/621 and 701/703 respectively. Ca2+ cations, required for DAP activity , were provided as CaCl2 (Merck, Darmstadt, Germany) at a final concentration of 50 μg/mL.
The MIC of all strains used in this study towards DAP was determined as described before  in triplicates with at least two independent cultures grown in TSB medium. The MIC was defined as the lowest antibiotic concentration that inhibited growth after 24 hours incubation at 37°C without shaking.
Glucose and acetate determination
Glucose and acetate concentrations in culture supernatants were both determined using enzymatic kit systems (R-Biopharm, Art. No. 10716251035, Art. No. 10148261035) according to the manufacturer’s instructions but with the following modifications: first, the supernatant of each sample was diluted 1:10 with water prior to the reaction. For the glucose amount determination, the assay was scaled down to one third of the final volume recommended in the manual. The extinction was measured photometrically at λ = 340 nm using a Hitachi spectrometer U-2900. The acetate measurement was performed using the plate reader SpectraMax 340 (Molecular Devices) and 96 well plates (Greiner) loaded with a final volume of 200 μl of the assay mix. Glucose (Roth, Karlsruhe, Germany) added to cultures was sterilized by filter of 0.2 μm pore size.
Overnight cultures of S. aureus SA113 were treated with 100-fold the MIC of DAP. Glucose was added at indicated time-points to final concentrations of 5 g/L (25 mM) unless stated otherwise. For the experiments with other compounds, glucose, fructose, ribose, xylose, glycerol, pyruvate, succinate, arginine or 2-deoxy-glucose were added at indicated time-points to final concentrations of 5 mM each. This concentration was chosen due to poorer aqueous solubility of some of the compounds compared to glucose. Viable counts were analyzed by CFU analysis on non-selective TSB agar plates. Cultures with viable counts below a threshold of 100 CFU/mL were judged as sterilized. All viable count experiments were conducted using at least three biological replicates.
Cells of stationary growth phase S. aureus SA113 cultures maintain an active amino acid anabolism during high-level DAP treatment for several hours . We assume that persister cells recalcitrant to eradication by this drug contribute significantly to this metabolic activity. Notably, a closer inspection of killing curves of our previous study suggested an increased killing efficiency by DAP after the addition of glucose. We here scrutinized this effect in more detail.
Addition of glucose accelerates DAP-dependent killing of stationary growth phase S. aureus in vitro
Three SA113 cultures were grown identically in TSB medium to stationary growth phase at which the medium is depleted for glucose . 100-fold the MIC of DAP was added at time point t = 0h to each of the cultures and at t = 3h, 5h or 7h, respectively, one culture each was supplemented with glucose at a final concentration of 5 g/L The viable counts indicated significantly enhanced killing when glucose was added at t = 3h compared to a culture challenged with DAP only (Fig 1). We now refer to the observation of enhanced DAP killing upon addition of glucose as the “Glc-DAP effect”. Between t = 0h and t = 1h, the number of CFU decreased to approximately 0.05% of the initial amount. Within the next two hours, the eradication slowed down, reflecting a typical biphasic killing behavior . Of note, the addition of glucose three or five hours after DAP challenge resulted in an eradication of persister cells in the culture within the next five hours. In comparison, an SA113 culture exposed to 100-fold the MIC of DAP but no glucose still contained more than 1.6x103 CFU/mL after ten hours. Cells residing in a DAP-tolerant state for a longer period (up to 7 hours after addition of the drug) also were susceptible to the Glc-DAP effect, albeit less pronounced. Notably, a comparable behavior was observed when stationary growth phase cells were washed and resuspended in PBS buffer, ruling out that other components in the spent medium are causative for the Glc-DAP effect (S1 Fig).
SA113 cells were treated with 100-fold the MIC of DAP starting at t = 0h. Glucose was added to cultures to final concentrations of 5 g/L each at time points 3h (triangles), 5h (circles), or 7h (squares), respectively, as indicated by arrows. CFU concentrations of a culture treated with DAP only (diamonds) were measured as a control. For statistical analysis area the delta of the CFU/mL was calculated 1 h and 4 h after glucose addition. Delta CFU/mL (n = 3) of glucose added after 3h (from 4h to 6h), 5h (6h to 8h) and 7h (8h to 10 h) and delta CFU/mL for the same time points of the control without Glc, were compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test. ***p<0.001. n.s.: not significant.
Upon supplementing the DAP-containing medium with lower concentrations of glucose (50 or 100 mg/L), killing of strain SA113 was only slightly affected, whereas higher glucose concentration markedly enhanced and killing efficiency (S2 Fig). The maximal effect was observed with 1 g/L glucose, whereas higher concentrations (up to 5 g/L) did not accelerate killing further. To account for glucose consumption we performed all further experiments using 5 g/L glucose.
Glucose-enhanced killing of stationary growth phase S. aureus is DAP-specific and independent from cell division
At this point, it was conceivable that glucose induced killing of DAP challenged cells was the result of nutrient-dependent induction of cell division, rendering the cells generally more vulnerable to antibiotics. We have previously shown that stationary growth phase S. aureus cultures are extremely tolerant to a number of antibiotics in vitro, even at elevated drug-concentrations . This was consistent with observations in the present study, in which drugs targeting the cell envelope (penicillin, 100-fold the MIC, 2 mg/L, or vancomycin, 100-fold the MIC, 400 mg/L) did not discernibly decrease the life count (S3 Fig). The addition of glucose did not induce killing by these antibiotics, which would be expected if the reversion to a replicating mode was responsible for the reinstated drug susceptibility. We therefore conclude that nutrient-dependent triggering of cell growth is not a reason for the Glc-DAP effect.
Enhanced killing by DAP is specific to selected carbohydrates
Are other metabolites also capable of accelerating DAP-dependent killing of stationary growth phase S. aureus? We chose fructose, ribose, glycerol, pyruvate, succinate and arginine, all of which are substrates or intermediates of catabolic pathways or anaplerotic reactions of S. aureus (Fig 2A) to test this hypothesis. The compounds were added to final concentrations of 5 mM each three hours after the onset of DAP treatment. Xylose and 2-deoxy-glucose, which are both transported into the cytoplasm of S. aureus but are not further metabolized [52,53], served as controls. Killing was enhanced with fructose, glycerol, succinate and arginine, but only glucose showed a significant effect compared to the DAP-only control. Eradication kinetics were unaffected with xylose or 2-deoxy-glucose (Fig 2B).
A) Schematic overview of tested metabolites and their entrance into the metabolism of S. aureus and genes responsible for selected metabolic reactions; fructose (fru), glyceraldehyde-3-phosphate (GAP), Dihydroxyacetone phosphate (DHAP), phosphoenolpyruvate (PEP). B) Selected compounds were added to final concentrations of 5 mM at t = 3h and the CFU values were determined after 24 hours. Groups were compared to the untreated control group by 1-way ANOVA with Dunnett's Multiple Comparison Test. *p<0.05.
As a further control, cultures were grown overnight in TSB-like medium in which glucose had been replaced by fructose. Also these were eradicated more efficiently by the addition of glucose, ruling out that an adaptation of metabolism to glucose during the cell cycle was mainly causative for the Glc-DAP effect (data not shown).
The Glc-DAP effect is independent of the proton motive force
The proton motive force (PMF) is generated by the electron transport chain and reduction equivalents originating from glycolysis and the TCA cycle. In order to examine a possible involvement of the PMF in the Glc-DAP effect, the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added to DAP challenged cells one hour before the addition of glucose. CCCP impedes energizing of membranes by scavenging protons, rapidly leading to a lack of ATP in the cell . As shown previously, the activity of DAP against exponential growth phase S. aureus is unaffected by CCCP . In our experiments, CCCP treatment of cultures during exponential growth phase ceased growth independent of DAP (S4 Fig). Treatment of cells with CCCP prior to the addition of DAP resulted in approximately 10-fold elevated persister levels compared to the untreated control (Fig 3), in agreement to results with CCCP-treated exponential phase E. coli cultures . The Glc-DAP effect, however, was still observed in the presence of CCCP, suggesting a PMF independent mechanism.
CCCP pretreatment of DAP challenged SA113 cultures. Stationary phase SA113 cells were treated with 50 μM CCCP (triangles) at t = -1h, corresponding to one hour before the addition of 100-fold the MIC of DAP. At t = 3h (arrows), glucose was added to cultures (filled symbols). Cultures without CCCP pretreatment (squares) were handled identically. For statistical analysis endpoint ODs after eight hours were compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test. *p<0.05.
Analysis of S. aureus strains defective in glucose transport and catabolism
We next inspected selected steps of glucose transport and catabolism by exploiting specific mutant strains of the S. aureus USA300 JE2 based NARSA Transposon Mutant Library . The first two chosen strains exhibited disrupted phosphotransferase (PTS) systems, namely the glucose-PTS specific IIABC component, SAUSA300_2476 (NE39) and the glucose-PTS specific IIBC component domain protein, SAUSA300_0191 (NE172). Compared to our PTS-positive control strain NE1046 (fdhD, formate dehydrogenase, SAUSA300_2231), both PTS mutants’ killing curves showed a less pronounced incongruity between challenge with Glc-DAP and DAP only (Fig 4). The reduced but still discernible Glc-DAP effect may be rationalized by redundant PTS systems or residual glucose transport due to secondary uptake systems of S. aureus . The results hitherto suggest the involvement of one or more metabolic pathway(s) in the Glc-DAP phenomenon. In order to take a deeper look into central carbon metabolism, further transposon mutants affected at certain branch points of glycolysis, or TCA cycle, respectively, were examined (Fig 2A). Results obtained with these mutants were inconclusive as DAP susceptibility among the strains varied considerably even without glucose. However, a trend towards an accelerated killing of all the tested strains upon the addition of glucose was observed (S5 Fig).
Transposon mutants were treated with 250-fold the MIC of DAP. Glucose was added to the culture at t = 3h (arrow). NE39 (glucose-PTS specific IIABC component, circle), NE172 (glucose-PTS specific IIBC component domain protein, square). Strain NE1046 (formate dehydrogenase, triangle) served as control. For statistical analysis, the area under curve (AUC) was calculated from the time point of glucose (Glc) addition (3h). AUCs (n = 3) of all groups where compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test, according to which the differences were not significant.
The Glc-DAP effect is not affected by physiologic changes in the pH
The interplay between oxygen supply and an excess of glucose can lead to an overload of metabolic pathways . In S. aureus this results in accumulation of acetate and lactate stemming from pyruvate and concomitant acidification of the medium . We determined both the pH value and the amount of acetate in DAP-containing cultures before and after the addition of glucose. The medium became slightly alkalized during the first three hours of DAP treatment (S6A Fig). After the addition of glucose the pH value rapidly decreased from 7.8 to about 7.4 and then leveled off, arguably due to glucose metabolism. The concentration of acetate was stable for the first three hours and rose upon glucose addition (S6B Fig). An artificial adjustment of the pH value in the medium, to resemble the glucose-dependent changes, did not influence the killing behavior by DAP only (data not shown). Thus, it is unlikely that acidification may be causative of the Glc-DAP effect.
Strains less susceptible to DAP are also subject to the Glc-DAP effect
We next investigated, whether the addition of glucose also increases DAP-dependent killing of strains with decreased susceptibility to this drug. Strain HG003 D6  had previously been isolated as a highly DAP-tolerant mutant generated by cyclic treatment with high doses of the drug. The addition of glucose and DAP led to a tremendous killing also of this strain (Fig 5A) whereas the culture treated with DAP only still contained more than 1x 108 CFU/mL seven hours after drug-addition. The parent strain HG003 was highly susceptible for the Glc-DAP effect reflecting a more pronounced killing behavior than SA113 (Fig 1). In addition, two DAP sensitive strains (616, 621; MIC = 0.5 mg/L) and two less DAP-susceptible strains (701, 703; MIC = 2 mg/L), all isolated from a patient with relapsing endocarditis during DAP therapy [42,58] were tested for the Glc-DAP effect. Intriguingly, the killing efficiency was increased about 100-fold (strain 703) and 600-fold (strain 701) compared to the treatment without glucose after 24 hours of incubation (Fig 5B). Similar results were obtained for the two less tolerant strains 616 and 621 (Fig 5C). While DAP is regarded as non-lytic against S. aureus , the experiments with strains 616, 621, 701 and 703 revealed a drastic decrease in OD578 values after a 24 hour period of Glc-DAP treatment (Fig 5D). The OD578 of the samples treated with Glc-DAP decreased by more than 75% compared to the cultures treated with DAP only. This apparent cell lysis was observed with all four strains, irrespective of their sensitivity towards the drug.
Stationary phase cultures of HG003, HG003 D6 and the clinical S. aureus strains 616, 621, 701 and 703 were treated with 100-fold, or 250-fold the MIC of DAP, respectively. At t = 3h, glucose was added (arrow) to the cultures (filled symbols) and CFU values were determined over time. For statistical analysis endpoint ODs after 24 hours for -Glc and +Glc for each strain were compared with unpaired t-test (with Welch’s correction for unequal variances if appropriate) ***p<0.001. A) Growth-phase-dependent DAP-tolerant strain HG003 D6 (square) and parent wild type strain HG003 (triangle). B) DAP-tolerant strains 701 (square) and 703 (triangle) (MIC = 2 mg/L). C) Sensitive strains 616 (square) and 621 (triangle) (MIC = 0.5 mg/L). D) Optical densities after 24h of incubation. Strains were challenged with 100-fold the MIC of DAP from t = 0h. Identically treated cultures were supplemented with glucose from t = 3h.
Metabolite induced killing of bacterial persisters has been associated with PMF generation and concomitant uptake of aminoglycoside compounds [20,45,46]. We here show that the lipopeptide antibiotic DAP also exhibits enhanced killing efficiency of S. aureus in the presence of glucose. Of note, this effect was also observed with a number of strains with low DAP-susceptibility. Our experiments with the uncoupler CCCP furthermore indicate that the Glc-DAP effect is not merely a consequence of PMF generation. Instead, the metabolism of glucose appears to be crucial for the Glc-DAP effect, which was neither prominent with low concentrations of glucose, nor with the non-metabolizable 2-deoxy-glucose. The observations of our study are consistent with two hypotheses for the mechanistic basis of the Glc-DAP effect. The first one suggests an influence of Glc-transport proteins on DAP’s mode of action, while the second is based upon Glc induced and DAP-specific lysis of cells. Based upon our observations with a number of S. aureus transposon-mutants, it is conceivable that components of the Glc PTS transport system may serve as receptors or targets of DAP. Accordingly, Glc-dependent induction of specific PTS transporters [60,61] will increase susceptibility to DAP. A similar effect was observed in Lactococcus lactis with bacteriocins that share biochemical features with DAP . Moreover, mutations in PTS proteins confer a high degree of DAP-non-susceptibility in Enterococcus faecium . Regarding the integrity of S. aureus cells upon DAP treatment, contradictory results have been reported. Electron microscopic images have shown tremendous morphological changes of S. aureus cells during DAP challenge, but not lysis , in agreement with another study also suggesting a lysis-independent mechanism of this drug . However, autolysis after DAP addition was observed, at least partially, in some S. aureus strains during exponential phase . Cell lysis may be augmented by intrinsic murein hydrolases. A potentiated lysis of exponential phase Staphylococcus cohnii upon addition of glucose was described with Pep 5, a cationic bactericidal peptide produced by Staphylococcus. epidermidis 5 . It is conceivable that carbohydrates in combination with specific drugs induce a suicidal mechanism in persister cells comparable to programmed cell death . Of note, carbohydrate metabolism influences murein hydrolase activity in S. aureus [68,69]. For example, the pleiotropic regulator CcpA activates transcription of the hydrolase activator cidA in the presence of glucose . cidA is part of the cidABC operon which together with lrgAB is involved in the regulation of murein hydrolase activity and autolysis [69,71,72]. According to our previous data, an upregulation of the TCA cycle activity may lead to an overflow metabolism  and acetate derived from pyruvate activates cidABC and lrgAB transcription. Further studies are required to verify these speculations in regard to the Glc-DAP effect.
Recently, a resuscitation promoting factor of S. aureus was postulated that is involved in shifting dormant cells back to a dividing state . This factor can be ruled out as responsible for the Glc-DAP effect which we also observed in buffered solution, devoid of components found in culture supernatants. Although the regulatory network of hydrolase activity is still not well understood, it should be considered as a potential target for the development of new anti-persister therapies of S. aureus. Artificial activation of peptidoglycan hydrolases could thereby lead to a random lysis process with fatal consequences for the cells independent of both the susceptibility towards antibiotics and their physiological state. It would be interesting to investigate the significance of the Glc-DAP effect in the treatment of staphylococcal infection. Notably, our experiments were based upon in vitro stationary growth phase cultures that were challenged with DAP concentrations that exceed serum concentrations in patients treated with this drug by more than tenfold [74,75]. Certainly, the systemic application of glucose to enhance DAP-dependent killing of S. aureus persisters in a patient is limited, as the blood sugar level in the human body is normally subject to homeostatic regulation. Of note, glycemia of non-diabetic humans is in a comparable range as the glucose concentrations that we determined to enhance DAP’s function. The importance of glucose as an adjuvant for DAP may thus have gone unnoticed in patients so far. It may be an option to improve DAP-efficiency in the treatment of non-invasive acute bacterial skin and skin-structure infections by increasing local concentrations of glucose. Hopefully, recent achievements regarding the eradication of persister cells will also aid in reducing the formation of drug resistant cells that pose an ever-growing issue in public and clinical health [4,39,76–78].
S1 Fig. Time-dependent killing of SA113 cells in PBS.
Stationary phase SA113 cells grown in TSB were harvested and resuspended in PBS. At t = 0h, 100-fold the MIC of DAP was added to the cell suspensions. At t = 3h, one cell suspension was supplemented with glucose (filled square), the other was left unaffected (open square) and CFU concentrations were determined over time.
S2 Fig. Influence of the glucose concentration on the efficiency of the Glc-DAP effect.
Stationary phase SA113 cells were treated with 100-fold the MIC of DAP at t = 0h. At t = 3h, different amounts of glucose were added and CFU values were determined after another four hours. Pearson’s r coefficient: r = -0,704.
S3 Fig. Penicillin and vancomycin treatment of SA113 ± glucose.
Stationary phase SA113 cells were treated with 100-fold the MIC of penicillin (square) or 100-fold the MIC of vancomycin (triangle) at t = 0h. Glucose was added at t = 3h (arrow and filled symbols).
S4 Fig. Effect of CCCP on growth of SA113.
SA113 was grown in TSB supplemented with glucose (t = 0h, squares), 100 μM CCCP (t = 0h, diamonds), glucose and CCCP (t = 0h, triangles), or glucose (t = 0h) and CCCP (t = 3h) (circles), respectively.
S5 Fig. Investigation of enzymatic branch points in glycolysis and TCA cycle.
Time dependent killing of stationary phase cultures with 250-fold the MIC of DAP. NE427 (fumC-, fumarate hydratase, diamonds), NE476 (fba-, fructose bisphosphate aldolase, squares), NE491 (icd-, isocitrate dehydrogenase, triangles), NE1003 (mqo-, malate-quinone oxidoreductase, x-mark), NE1046 (fdh-, formate dehydrogenase, circles), NE1407 (pyk-, pyruvate kinase, asterisks). For statistical analysis area under curve (AUC) was calculated from the time point of glucose (Glc) addition (3h). AUCs (n = 3) of all groups where compared to NE1046 fdh by 1-way ANOVA with Dunnett's Multiple Comparison Test.
S6 Fig. pH and acetate/glucose measurement.
Cultures were treated with 100-fold the MIC of DAP at t = 0h. A) Glucose was added (filled squares) at t = 3h (arrow) and pH values were determined over time. B) Acetate (triangle) and glucose (square) measurement of a culture with glucose added at t = 3h.
Conceived and designed the experiments: MP LM RB. Performed the experiments: MP LM. Analyzed the data: MP LM CW RB. Wrote the paper: MP RB.
- 1. Moy JA, Caldwell-Brown D, Lin AN, Pappa KA, Carter DM (1990) Mupirocin-resistant Staphylococcus aureus after long-term treatment of patients with epidermolysis bullosa. J Am Acad Dermatol 22: 893–895. pmid:2112168
- 2. Endimiani A, Blackford M, Dasenbrook EC, Reed MD, Bajaksouszian S, Hujer AM, et al. (2011) Emergence of linezolid-resistant Staphylococcus aureus after prolonged treatment of cystic fibrosis patients in Cleveland, Ohio. Antimicrob Agents Chemother 55: 1684–1692. doi: 10.1128/AAC.01308-10. pmid:21263048
- 3. Mariani PG, Sader HS, Jones RN (2006) Development of decreased susceptibility to daptomycin and vancomycin in a Staphylococcus aureus strain during prolonged therapy. J Antimicrob Chemother 58: 481–483. pmid:16847029
- 4. Skiest DJ (2006) Treatment failure resulting from resistance of Staphylococcus aureus to daptomycin. J Clin Microbiol 44: 655–656. pmid:16455939
- 5. Babra C, Tiwari J, Costantino P, Sunagar R, Isloor S, Hedge N, et al. (2013) Human methicillin-sensitive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens. J Basic Microbiol.
- 6. Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15: 167–193. pmid:11932229
- 7. Dortet L, Anguel N, Fortineau N, Richard C, Nordmann P (2013) In vivo acquired daptomycin resistance during treatment of methicillin-resistant Staphylococcus aureus endocarditis. Int J Infect Dis 17: e1076–1077. doi: 10.1016/j.ijid.2013.02.019. pmid:23578850
- 8. Wood TK, Knabel SJ, Kwan BW (2013) Bacterial persister cell formation and dormancy. Appl Environ Microbiol 79: 7116–7121. doi: 10.1128/AEM.02636-13. pmid:24038684
- 9. Lewis K (2010) Persister cells. Annu Rev Microbiol 64: 357–372. doi: 10.1146/annurev.micro.112408.134306. pmid:20528688
- 10. Balaban NQ, Gerdes K, Lewis K, McKinney JD (2013) A problem of persistence: still more questions than answers? Nat Rev Microbiol 11: 587–591. pmid:24020075
- 11. Casadesús J, Low DA (2013) Programmed heterogeneity: epigenetic mechanisms in bacteria. J Biol Chem 288: 13929–13935. doi: 10.1074/jbc.R113.472274. pmid:23592777
- 12. Helaine S, Kugelberg E (2014) Bacterial persisters: formation, eradication, and experimental systems. Trends Microbiol 22: 417–424. doi: 10.1016/j.tim.2014.03.008. pmid:24768561
- 13. Fauvart M, De Groote VN, Michiels J (2011) Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. J Med Microbiol 60: 699–709. doi: 10.1099/jmm.0.030932-0. pmid:21459912
- 14. Tuchscherr L, Medina E, Hussain M, Völker W, Heitmann V, Niemann S, et al. (2011) Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection. EMBO Mol Med 3: 129–141. doi: 10.1002/emmm.201000115. pmid:21268281
- 15. Que YA, Hazan R, Strobel B, Maura D, He J, Kesarwani M, et al. (2013) A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria. PLoS One 8: e80140. doi: 10.1371/journal.pone.0080140. pmid:24367477
- 16. Leung V, Levesque CM (2012) A stress-inducible quorum-sensing peptide mediates the formation of persister cells with noninherited multidrug tolerance. J Bacteriol 194: 2265–2274. doi: 10.1128/JB.06707-11. pmid:22366415
- 17. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K (2004) Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230: 13–18. pmid:14734160
- 18. Amato SM, Fazen CH, Henry TC, Mok WW, Orman MA, Sandvik EL, et al. (2014) The role of metabolism in bacterial persistence. Front Microbiol 5: 70. doi: 10.3389/fmicb.2014.00070. pmid:24624123
- 19. Amato SM, Orman MA, Brynildsen MP (2013) Metabolic control of persister formation in Escherichia coli. Mol Cell 50: 475–487. doi: 10.1016/j.molcel.2013.04.002. pmid:23665232
- 20. Orman MA, Brynildsen MP (2013) Establishment of a Method To Rapidly Assay Bacterial Persister Metabolism. Antimicrobial Agents and Chemotherapy 57: 4398–4409. doi: 10.1128/AAC.00372-13. pmid:23817376
- 21. Orman MA, Brynildsen MP (2013) Dormancy is not necessary or sufficient for bacterial persistence. Antimicrob Agents Chemother 57: 3230–3239. doi: 10.1128/AAC.00243-13. pmid:23629720
- 22. Bokinsky G, Baidoo EE, Akella S, Burd H, Weaver D, Alonso-Gutierrez J, et al. (2013) HipA-Triggered Growth Arrest and beta-Lactam Tolerance in Escherichia coli Are Mediated by RelA-Dependent ppGpp Synthesis. J Bacteriol 195: 3173–3182. doi: 10.1128/JB.02210-12. pmid:23667235
- 23. Lechner S, Prax M, Lange B, Huber C, Eisenreich W, Herbig A, et al. (2014) Metabolic and transcriptional activities of Staphylococcus aureus challenged with high-doses of daptomycin. Int J Med Microbiol 304: 931–940. doi: 10.1016/j.ijmm.2014.05.008. pmid:24980509
- 24. Gould IM (2009) Antibiotics, skin and soft tissue infection and meticillin-resistant Staphylococcus aureus: cause and effect. Int J Antimicrob Agents 34 Suppl 1: S8–11. doi: 10.1016/S0924-8579(09)70542-4. pmid:19560675
- 25. Brady RA, Leid JG, Calhoun JH, Costerton JW, Shirtliff ME (2008) Osteomyelitis and the role of biofilms in chronic infection. FEMS Immunol Med Microbiol 52: 13–22. pmid:18081847
- 26. Moore CL, Osaki-Kiyan P, Haque NZ, Perri MB, Donabedian S, Zervos MJ (2012) Daptomycin versus vancomycin for bloodstream infections due to methicillin-resistant Staphylococcus aureus with a high vancomycin minimum inhibitory concentration: a case-control study. Clin Infect Dis 54: 51–58. doi: 10.1093/cid/cir764. pmid:22109947
- 27. Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339: 520–532. pmid:9709046
- 28. DeLeo FR, Otto M, Kreiswirth BN, Chambers HF (2010) Community-associated meticillin-resistant Staphylococcus aureus. Lancet 375: 1557–1568. doi: 10.1016/S0140-6736(09)61999-1. pmid:20206987
- 29. French GL (2006) Bactericidal agents in the treatment of MRSA infections—the potential role of daptomycin. J Antimicrob Chemother 58: 1107–1117. pmid:17040922
- 30. Fuchs PC, Barry AL, Brown SD (2002) In vitro bactericidal activity of daptomycin against staphylococci. J Antimicrob Chemother 49: 467–470. pmid:11864946
- 31. Steenbergen JN, Alder J, Thorne GM, Tally FP (2005) Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. J Antimicrob Chemother 55: 283–288. pmid:15705644
- 32. Mascio CT, Alder JD, Silverman JA (2007) Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells. Antimicrob Agents Chemother 51: 4255–4260. pmid:17923487
- 33. Humphries RM, Pollett S, Sakoulas G (2013) A Current Perspective on Daptomycin for the Clinical Microbiologist. Clin Microbiol Rev 26: 759–780. doi: 10.1128/CMR.00030-13. pmid:24092854
- 34. Muraih JK, Pearson A, Silverman J, Palmer M (2011) Oligomerization of daptomycin on membranes. Biochim Biophys Acta 1808: 1154–1160. doi: 10.1016/j.bbamem.2011.01.001. pmid:21223947
- 35. Lechner S, Lewis K, Bertram R (2012) Staphylococcus aureus Persisters Tolerant to Bactericidal Antibiotics. J Mol Microbiol Biotechnol 22: 235–244. doi: 10.1159/000342449. pmid:22986269
- 36. Berti AD, Wergin JE, Girdaukas GG, Hetzel SJ, Sakoulas G, Rose WE (2012) Altering the proclivity towards daptomycin resistance in methicillin-resistant Staphylococcus aureus using combinations with other antibiotics. Antimicrob Agents Chemother 56: 5046–5053. doi: 10.1128/AAC.00502-12. pmid:22802248
- 37. Werth BJ, Sakoulas G, Rose WE, Pogliano J, Tewhey R, Rybak MJ (2013) Ceftaroline increases membrane binding and enhances the activity of daptomycin against daptomycin-nonsusceptible vancomycin-intermediate Staphylococcus aureus in a pharmacokinetic/pharmacodynamic model. Antimicrob Agents Chemother 57: 66–73. doi: 10.1128/AAC.01586-12. pmid:23070161
- 38. Gasch O, Pillai S, Dakos J, Miyakis S, Moellering R Jr., Eliopoulos G (2013) Daptomycin in vitro activity against methicillin-resistant Staphylococcus aureus is enhanced by D-cycloserine in a mechanism associated with a decrease in cell surface charge. Antimicrob Agents Chemother.
- 39. Hayden MK, Rezai K, Hayes RA, Lolans K, Quinn JP, Weinstein RA (2005) Development of Daptomycin resistance in vivo in methicillin-resistant Staphylococcus aureus. J Clin Microbiol 43: 5285–5287. pmid:16207998
- 40. Bayer AS, Schneider T, Sahl HG (2013) Mechanisms of daptomycin resistance in Staphylococcus aureus: role of the cell membrane and cell wall. Ann N Y Acad Sci 1277: 139–158. doi: 10.1111/j.1749-6632.2012.06819.x. pmid:23215859
- 41. Song Y, Rubio A, Jayaswal RK, Silverman JA, Wilkinson BJ (2013) Additional routes to Staphylococcus aureus daptomycin resistance as revealed by comparative genome sequencing, transcriptional profiling, and phenotypic studies. PLoS One 8: e58469. doi: 10.1371/journal.pone.0058469. pmid:23554895
- 42. Bertsche U, Weidenmaier C, Kuehner D, Yang SJ, Baur S, Wanner S, et al. (2011) Correlation of daptomycin resistance in a clinical Staphylococcus aureus strain with increased cell wall teichoic acid production and D-alanylation. Antimicrob Agents Chemother 55: 3922–3928. doi: 10.1128/AAC.01226-10. pmid:21606222
- 43. Cohen NR, Lobritz MA, Collins JJ (2013) Microbial persistence and the road to drug resistance. Cell Host Microbe 13: 632–642. doi: 10.1016/j.chom.2013.05.009. pmid:23768488
- 44. Conlon BP, Nakayasu ES, Fleck LE, LaFleur MD, Isabella VM, Coleman K, et al. (2013) Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503: 365–370. doi: 10.1038/nature12790. pmid:24226776
- 45. Allison KR, Brynildsen MP, Collins JJ (2011) Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473: 216–220. doi: 10.1038/nature10069. pmid:21562562
- 46. Barraud N, Buson A, Jarolimek W, Rice SA (2013) Mannitol Enhances Antibiotic Sensitivity of Persister Bacteria in Pseudomonas aeruginosa Biofilms. PLoS One 8: e84220. doi: 10.1371/journal.pone.0084220. pmid:24349568
- 47. Fey PD, Endres JL, Yajjala VK, Widhelm TJ, Boissy RJ, Bose JL, et al. (2013) A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio 4: e00537–00512. doi: 10.1128/mBio.00537-12. pmid:23404398
- 48. Mechler L, Herbig A, Paprotka K, Fraunholz M, Nieselt K, Bertram R (2015) A novel point mutation promotes growth phase-dependent daptomycin tolerance in Staphylococcus aureus. Antimicrob Agents Chemother 59: 5366–5376. doi: 10.1128/AAC.00643-15. pmid:26100694
- 49. Hanberger H, Nilsson LE, Maller R, Isaksson B (1991) Pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca2+ and albumin on these drugs. Antimicrob Agents Chemother 35: 1710–1716. pmid:1659305
- 50. Iordanescu S, Surdeanu M (1976) Two restriction and modification systems in Staphylococcus aureus NCTC8325. J Gen Microbiol 96: 277–281.
- 51. Herbert S, Ziebandt AK, Ohlsen K, Schäfer T, Hecker M, Albrecht D, et al. (2010) Repair of global regulators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates. Infect Immun 78: 2877–2889. doi: 10.1128/IAI.00088-10. pmid:20212089
- 52. Götz F, Bannerman T., Schleifer KH. (2006) The Genera Staphylococcus and Macrococcus. The Prokaryotes 4: 5–75.
- 53. Wick AN, Drury DR, Nakada HI, Wolfe JB (1957) Localization of the primary metabolic block produced by 2-deoxyglucose. J Biol Chem 224: 963–969. pmid:13405925
- 54. Cavari BZ, Avi-Dor Y (1967) Effect of carbonyl cyanide m-chlorophenylhydrazone on respiration and respiration-dependent phosphorylation in Escherichia coli. Biochem J 103: 601–608. pmid:4962086
- 55. Kwan BW, Valenta JA, Benedik MJ, Wood TK (2013) Arrested protein synthesis increases persister-like cell formation. Antimicrob Agents Chemother 57: 1468–1473. doi: 10.1128/AAC.02135-12. pmid:23295927
- 56. Paczia N, Nilgen A, Lehmann T, Gatgens J, Wiechert W, Noack S (2012) Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms. Microb Cell Fact 11: 122. doi: 10.1186/1475-2859-11-122. pmid:22963408
- 57. Somerville GA, Said-Salim B, Wickman JM, Raffel SJ, Kreiswirth BN, Musser JM (2003) Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for host-pathogen interactions. Infect Immun 71: 4724–4732. pmid:12874354
- 58. Fischer A, Yang SJ, Bayer AS, Vaezzadeh AR, Herzig S, Stenz L, et al. (2011) Daptomycin resistance mechanisms in clinically derived Staphylococcus aureus strains assessed by a combined transcriptomics and proteomics approach. J Antimicrob Chemother 66: 1696–1711. doi: 10.1093/jac/dkr195. pmid:21622973
- 59. Cotroneo N, Harris R, Perlmutter N, Beveridge T, Silverman JA (2008) Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus. Antimicrob Agents Chemother 52: 2223–2225. doi: 10.1128/AAC.01410-07. pmid:18378708
- 60. Jahreis K, Pimentel-Schmitt EF, Bruckner R, Titgemeyer F (2008) Ins and outs of glucose transport systems in eubacteria. FEMS Microbiol Rev 32: 891–907. doi: 10.1111/j.1574-6976.2008.00125.x. pmid:18647176
- 61. Stülke J, Martin-Verstraete I, Zagorec M, Rose M, Klier A, Rapoport G (1997) Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT. Mol Microbiol 25: 65–78. pmid:11902727
- 62. Diep DB, Skaugen M, Salehian Z, Holo H, Nes IF (2007) Common mechanisms of target cell recognition and immunity for class II bacteriocins. Proc Natl Acad Sci U S A 104: 2384–2389. pmid:17284603
- 63. Humphries RM, Kelesidis T, Tewhey R, Rose WE, Schork N, Nizet V, et al. (2012) Genotypic and phenotypic evaluation of the evolution of high-level daptomycin nonsusceptibility in vancomycin-resistant Enterococcus faecium. Antimicrob Agents Chemother 56: 6051–6053. doi: 10.1128/AAC.01318-12. pmid:22948885
- 64. Wale LJ, Shelton AP, Greenwood D (1989) Scanning electronmicroscopy of Staphylococcus aureus and Enterococcus faecalis exposed to daptomycin. J Med Microbiol 30: 45–49. pmid:2550648
- 65. Gustafson JE, Berger-Bachi B, Strassle A, Wilkinson BJ (1992) Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrob Agents Chemother 36: 566–572. pmid:1320363
- 66. Bierbaum G, Sahl HG (1985) Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes. Arch Microbiol 141: 249–254. pmid:4004448
- 67. Bayles KW (2014) Bacterial programmed cell death: making sense of a paradox. Nat Rev Microbiol 12: 63–69. doi: 10.1038/nrmicro3136. pmid:24336185
- 68. Rice KC, Nelson JB, Patton TG, Yang SJ, Bayles KW (2005) Acetic acid induces expression of the Staphylococcus aureus cidABC and lrgAB murein hydrolase regulator operons. Journal of Bacteriology 187: 813–821. pmid:15659658
- 69. Yang SJ, Rice KC, Brown RJ, Patton TG, Liou LE, Park YH, et al. (2005) A LysR-type regulator, CidR, is required for induction of the Staphylococcus aureus cidABC operon. Journal of Bacteriology 187: 5893–5900. pmid:16109930
- 70. Seidl K, Muller S, Francois P, Kriebitzsch C, Schrenzel J, Engelmann S, et al. (2009) Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus. BMC Microbiol 9: 95. doi: 10.1186/1471-2180-9-95. pmid:19450265
- 71. Rice KC, Bayles KW (2003) Death's toolbox: examining the molecular components of bacterial programmed cell death. Molecular Microbiology 50: 729–738. pmid:14617136
- 72. Groicher KH, Firek BA, Fujimoto DF, Bayles KW (2000) The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance. Journal of Bacteriology 182: 1794–1801. pmid:10714982
- 73. Pascoe B, Dams L, Wilkinson TS, Harris LG, Bodger O, Mack D, et al. (2014) Dormant Cells of Staphylococcus aureus Are Resuscitated by Spent Culture Supernatant. PLoS One 9: e85998. doi: 10.1371/journal.pone.0085998. pmid:24523858
- 74. Van der Auwera P (1989) Ex vivo study of serum bactericidal titers and killing rates of daptomycin (LY146032) combined or not combined with amikacin compared with those of vancomycin. Antimicrob Agents Chemother 33: 1783–1790. pmid:2556079
- 75. Kullar R, Chin JN, Edwards DJ, Parker D, Coplin WM, Rybak MJ (2011) Pharmacokinetics of single-dose daptomycin in patients with suspected or confirmed neurological infections. Antimicrob Agents Chemother 55: 3505–3509. doi: 10.1128/AAC.01741-10. pmid:21502620
- 76. Friedman L, Alder JD, Silverman JA (2006) Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob Agents Chemother 50: 2137–2145. pmid:16723576
- 77. Peleg AY, Miyakis S, Ward DV, Earl AM, Rubio A, Cameron DR, et al. (2012) Whole genome characterization of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staphylococcus aureus. PLoS One 7: e28316. doi: 10.1371/journal.pone.0028316. pmid:22238576
- 78. Gasch O, Camoez M, Dominguez MA, Padilla B, Pintado V, Almirante B, et al. (2013) Emergence of resistance to daptomycin in a cohort of patients with methicillin-resistant Staphylococcus aureus persistent bacteraemia treated with daptomycin. J Antimicrob Chemother.