Metabolite-enabled eradication of bacterial persisters by aminoglycosides.

Bacterial persistence is a state in which a sub-population of dormant cells, or 'persisters', tolerates antibiotic treatment. Bacterial persisters have been implicated in biofilms and in chronic and recurrent infections. Despite this clinical relevance, there are currently no viable means for eradicating persisters. Here we show that specific metabolic stimuli enable the killing of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) persisters with aminoglycosides. This potentiation is aminoglycoside-specific, it does not rely on growth resumption and it is effective in both aerobic and anaerobic conditions. It proceeds by the generation of a proton-motive force which facilitates aminoglycoside uptake. Our results demonstrate that persisters, although dormant, are primed for metabolite uptake, central metabolism and respiration. We show that aminoglycosides can be used in combination with specific metabolites to treat E. coli and S. aureus biofilms. Furthermore, we demonstrate that this approach can improve the treatment of chronic infections in a mouse urinary tract infection model. This work establishes a strategy for eradicating bacterial persisters that is based on metabolism, and highlights the importance of the metabolic environment to antibiotic treatment.

Researchers have shown that translation occurs at a reduced rate in persisters[2,8], suggesting that persisters should be susceptible to the ribosome-targeting bactericidal aminoglycoside antibiotics[9-13]. However, despite continued translation, aminoglycosides have weak activity against dormant bacteria[14, 15]. Given the dormancy of persisters and the known energy requirement for aminoglycoside activity[16], we reasoned that metabolic stimulation might potentiate aminoglycosides against bacterial persisters.

To test this, we screened metabolites for their ability to potentiate aminoglycosides against Escherichia coli persisters. We selected carbon sources to maximize coverage of glycolysis, the pentose-phosphate pathway (PPP) and the entner-douderoff pathway (EDP) (Fig. 1a, b). Persisters were isolated (Supplementary Information), re-suspended in minimal media supplemented with individual metabolites, and treated with aminoglycoside gentamicin for two hours.

Figure 1

Specific metabolites enable aminoglycoside killing of E. coli persisters

a, Survival of persisters after 2-hour treatment with gentamicin and respective metabolite. b, Metabolite-induced persister elimination superimposed on metabolic network. c, Survival of persisters after the following treatments: no treatment (black squares), mannitol (black triangles), gentamicin (red squares), gentamicin and mannitol (red triangles), ofloxacin (blue squares), ofloxacin and mannitol (blue triangles), ampicllin (green squares), or ampicillin and mannitol (green triangles). d, Metabolite-induced Gent-TR uptake by stationary phase cells superimposed on metabolic network (see also Supplementary Fig. 10). Mean ± s.e.m. are presented (n ≥ 3).

We found that gentamicin was greatly potentiated by specific metabolic stimuli against persisters (Fig. 1a, b). Metabolites entering upper glycolysis (glucose, mannitol, and fructose) and pyruvate induced rapid gentamicin killing of persisters, reducing persister viability by three orders of magnitude. In contrast, metabolites that entered lower glycolysis (excepting pyruvate) caused little potentiation. Metabolites entering metabolism via the PPP or EDP (arabinose, ribose, and gluconate) also showed low potentiation. No killing was observed in the control, demonstrating that treated cells were persistent to gentamicin, in the absence of added metabolite. We verified that metabolite-enabled persister eradication was general to the aminoglycoside class by testing kanamycin and streptomycin (Supplementary Fig. 2).

We considered that potentiating metabolites might be reverting persisters to normally growing cells, which would render them susceptible to quinolone (DNA-damage) and β-lactam (cell-wall inhibition) antibiotics. To test this, we treated persisters in the presence and absence of mannitol with a member of each of the three major classes of bactericidal antibiotics: aminoglycosides, quinolones, and β-lactams. As seen in the metabolite screen, gentamicin rapidly eliminated metabolically-stimulated persisters (Fig. 1c). However, neither the β-lactam ampicillin nor quinolone ofloxacin showed appreciable killing of persisters in the presence or absence of mannitol. This result demonstrates that potentiation is aminoglycoside-specific and that cells were persistent to quinolones and β-lactams. It further suggests that metabolic stimuli under these conditions do not rapidly revert persisters to a growth state in which cell-wall and DNA synthesis are active. To further explore this, we tested growth of persisters on the metabolites used for aminoglycoside potentiation, and observed negligible growth of persisters eight hours after metabolite addition (Supplementary Figs 3 and 4). Taken together, these data suggest that the metabolic stimuli bolster a process specific to aminoglycosides, and do not revert persisters to normally growing cells.

Given the energy dependence of aminoglycoside uptake[16], we investigated if the metabolic stimuli screened were increasing aminoglycoside uptake. We measured uptake by fluorescently labeling gentamicin with Texas Red and analyzing by FACS. Cells were pre-incubated with metabolites for 30 minutes, prior to five-minute treatment with Gentamicin-Texas Red (Gent-TR) to determine uptake (Fig. 1d and Supplementary Fig. 10). Metabolites that induced substantial aminoglycoside killing were observed to induce high levels of aminoglycoside uptake, implying that increased uptake induced by these metabolites was responsible for aminoglycoside killing. Further, metabolites that caused low potentiation did not significantly increase aminoglycoside uptake.

The requirement of proton-motive force (PMF) for aminoglycoside uptake in exponentially growing bacteria has been studied extensively[16]. Though the complete mechanism of aminoglycoside uptake is unclear, it is known that a threshold PMF is required. We reasoned that, though metabolic stimuli are not rapidly stimulating growth of persisters, they may be promoting PMF, thereby facilitating uptake of and killing by aminoglycosides. To test this hypothesis, we pre-incubated persisters with the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which inhibits PMF, before treating them with metabolites in conjunction with gentamicin. Treatment with CCCP was found to abolish aminoglycoside potentiation by all of the carbon sources, demonstrating that PMF, induced by metabolites, is required for persister elimination (Fig. 2a and Supplementary Fig. 12). We next verified that the requirement for PMF was due to aminoglycoside uptake. We pre-incubated samples with CCCP and performed Gent-TR uptake experiments, and found that inhibiting PMF suppressed metabolite-induced uptake of aminoglycoside (Fig. 2b and Supplementary Fig. 13). Further, using the DiOC2(3) membrane stain, we verified that metabolites that induce aminoglycoside uptake and killing were also the ones that elevate PMF (Supplementary Figs 14 and 15). These results demonstrate that specific metabolites induce PMF in persisters, thereby facilitating aminoglycoside uptake and killing.

Figure 2

Metabolite-enabled aminoglycoside uptake and killing requires PMF produced by oxidative electron transport chain

a, Survival of persisters after treatment with gentamicin plus uptake-potentiating metabolites with (dark grey bars) and without CCCP (light grey bars). b, Representative uptake measurement of Gent-TR by stationary phase cells after incubation with no sugar (black lines), mannitol (red lines), or mannitol and CCCP (blue lines) (see also Supplementary Fig. 13). c, Survival of persisters in cytochrome-inactivated strains after treatment with gentamicin plus mannitol (see also Supplementary Fig. 16). d, Percent survival of persisters in NADH-dehydrogenase-inactivated strains after treatment with gentamicin plus mannitol (see also Supplementary Fig. 19). Presence (green checks) and absence (red X’s) of functional complexes is indicated below test conditions. Mean ± s.e.m. are presented (n ≥ 3).

From these results, we hypothesized that aerobic respiration is primed in persisters and facilitates metabolic potentiation of aminoglycosides. We tested this using genetic knockout strains inactivated for each of the E. coli cytochrome quinol oxidases (bo, ΔcyoA; bd-I, ΔcydB; bd-II, ΔappB), as well as potassium cyanide (KCN) to inhibit all cytochromes simultaneously. Wild-type persisters, with and without KCN, and enzymatically-inactivated persisters, were treated for two hours with gentamicin plus metabolites (Fig. 2c and Supplemental Fig. 16). Treatment with KCN abolished killing, consistent with work in rapidly growing bacteria[17], demonstrating the necessity of aerobic respiration for aminoglycoside elimination of persisters under these conditions. The ΔcydB strain, which lacks activity of the microaerobic cytochrome bd-I[18, 19], suppressed killing by over two orders of magnitude, possibly as a result of its use in the oxygen-depleted and alkaline stationary phase cultures. Neither ΔcyoA nor ΔappB showed a significant effect. Though we found aerobic respiration was required for eradication in aerated conditions, we also found that metabolite-enabled eradication occurs anaerobically in conditions that support PMF (Supplementary Figs 17 and 18).

As aerobic respiration in E. coli is driven by NADH oxidation, we investigated the role of NADH utilization in this phenotype. Persister cells inactivated for NADH dehydrogenase I (ΔnuoI), NADH dehydrogenase II (Δndh), and both NADH dehydrogenases (ΔndhΔnuoI), were treated for two hours with gentamicin plus metabolites (Fig. 2d and Supplementary Fig. 19). We found that NADH dehydrogenase activity was important to this phenotype as gentamicin activity against the ΔndhΔnuoI strain was not potentiated by mannitol, fructose, or pyruvate, though there was slight potentiation by glucose (Supplementary Fig. 19a). Given that NADH drives electron transport, this requirement for NADH is not surprising though we found it is not essential for killing under all conditions (Supplementary Figs 18 and 20). Though both Δndh and ΔnuoI suppressed killing, the ΔnuoI strain had a greater effect, possibly reflecting its direct contribution to PMF. Using a series of genetic knockouts, we further determined that the enzyme pyruvate dehydrogenase was necessary for the observed phenotype, due to its NADH generation, whereas the PPP, EDP, and TCA cycle were not found to be necessary (Supplementary Figs 21-24).

These results demonstrate that persisters are primed for specific biochemical processes, including central metabolism, that allow PMF induction. This resumption of central metabolism and respiration in persisters, however, is not sufficient in the time-scales examined to support other processes necessary for cellular growth, such as cell-wall biogenesis and DNA replication. Thus, persisters treated with specific metabolites appear to be in an energized but non-dividing state that facilitates their elimination by aminoglycosides. On the basis of these findings, we propose the following mechanism for metabolite-enabled eradication of persisters by aminoglycosides (Fig. 3a). Certain metabolites—glucose, mannitol, fructose, and pyruvate—are transported to the cytoplasm, some by their specific PTS enzymes, and enter glycolysis, where their catabolism generates NADH. NADH is oxidized by enzymes in the electron transport chain, which in turn contribute to PMF. The elevated PMF facilitates the uptake of aminoglycosides which bind to the ribosome causing mistranslation-induced cell death.

Figure 3

Mechanism for metabolite-enabled eradication of persisters and clinically relevant experiments

a, Metabolite-enabled persister eradication proceeds through catabolism of carbon sources thereby generating NADH, the production of which does not require the PPP, EDP, or TCA cycle. The electron transport chain oxidizes NADH and contributes to PMF, which facilitates aminoglycoside uptake and killing of persisters. b, Survival of E. coli biofilms after treatment with ofloxacin, mannitol, gentamicin, or mannitol plus gentamicin. As quinolones have high efficacy against Gram-negative biofilms compared to other antibiotics[15, 20], ofloxacin was used as a benchmark for high biofilm killing. c, Survival of E. coli biofilms after treatment with ofloxacin, fructose, gentamicin, or fructose plus gentamicin. d, Schematic of in vivo experiments in mice (left). Survival of E. coli biofilms on urinary-tract-inserted catheters after treatment with gentamicin (1 mg/Kg) or mannitol (1.5 g/Kg) and gentamicin (1 mg/Kg) (right). Mean ± s.e.m. are presented (n ≥ 3).

We next investigated if this mechanism was applicable to clinically relevant cases, such as bacterial biofilms. We reasoned that metabolic stimulation might facilitate aminoglycoside elimination of biofilm persisters. To test this hypothesis, we grew E. coli biofilms, and treated them for four hours with ofloxacin, mannitol, gentamicin, and mannitol plus gentamicin (Fig. 3b). Ofloxacin (which is efficient against Gram-negative biofilms[15, 20]) reduced biofilm viability by almost two orders of magnitude, suggesting that greater than 1% of the biofilms were persisters. Mannitol and gentamicin in combination reduced biofilm viability by over 4 orders of magnitude, demonstrating a reduction of biofilm persisters by 2.5 orders of magnitude. We also tested the ability of fructose to induce biofilm elimination and observed similar results (Fig. 3c).

To determine the clinical relevance of metabolic potentiation of aminoglycosides in vivo, we tested the ability of mannitol in combination with gentamicin to treat chronic, biofilm-associated infection in a mouse model. Mice had catheters colonized with uropathogenic E. coli biofilms implanted in their urinary tracts (Fig. 3d). Two days after surgery, mice received no treatment or intravenous treatment with gentamicin or gentamicin and mannitol for three days, after which the catheters were removed and biofilm viability was determined. Gentamicin alone had no effect, whereas gentamicin in combination with mannitol reduced the viability of the catheter biofilms by nearly 1.5 orders of magnitude (Fig. 3d). We also found that treatment with gentamicin and mannitol inhibited the spread of bacterial infection to the kidneys, as compared to treatment with gentamicin alone and the no treatment control (Supplementary Fig. 27). These in vivo results demonstrate the feasibility of our approach for clinical use.

Having demonstrated that certain metabolites can enable aminoglycoside activity in Gram-negative (E. coli) bacterial persisters and biofilms, we sought to determine whether a similar phenomenon existed in Gram-positive bacteria. Persisters of the Gram-positive pathogen Staphylococcus aureus were treated with gentamicin in conjunction with metabolites. After an initial hour of no killing, gentamicin with fructose rapidly eliminated persistent S. aureus (Fig. 4a). Curiously, mannitol, glucose, and pyruvate, which showed strong potentiation against E. coli persisters, showed little potentiation in S. aureus. Using expression analysis of S. aureus microarrays, we present data suggesting this lack of potentiation results from differential expression of metabolite transporters (Supplementary Table 3). We next tested whether fructose-enabled killing of S. aureus was unique to aminoglycosides or general to other classes of bactericidal antibiotics. As with E. coli, we found that metabolite-enabled killing of S. aureus persisters was aminoglycoside-specific (Fig. 4b), suggesting that S. aureus persisters were not reverting to normally growing cells.

Figure 4

Fructose induces PMF-dependent aminoglycoside killing of S. aureus persisters

a, Survival of S. aureus persisters after treatment with gentamicin plus no metabolite (black squares), glucose (blue squares), mannitol (red squares), fructose (green squares), or pyruvate (orange squares). b, Survival of S. aureus persisters after 4-hour treatment with gentamicin, gentamicin and fructose, ofloxacin, ofloxacin and fructose, ampicillin, or ampicillin and fructose. c, Survival of S. aureus persisters after 4-hour treatment with gentamicin and fructose with (dark grey bars) or without CCCP (light grey bars). d, Survival of S. aureus biofilms after 4-hour treatment with ofloxacin, fructose, gentamicin, or fructose plus gentamicin. Mean ± s.e.m. are presented (n ≥ 3).

Given that aminoglycoside activity in growing S. aureus is dependent on PMF[21,22], we tested whether persister elimination mediated by fructose required PMF. We found that the potentiation of aminoglycoside by fructose in S. aureus, as in E. coli, requires PMF generation (Fig. 4c), suggesting that the PMF-requiring mechanism of aminoglycoside persister elimination exists in both Gram-negative and Gram-positive bacteria. We also investigated if gentamicin with fructose could be used to treat S. aureus biofilms. We found that the viability of S. aureus biofilms was reduced by nearly 1.5 orders of magnitude when treated for four hours with fructose and gentamicin (Fig. 4d).

Here we established a metabolic-based approach for eradicating persisters, one effective against both Gram-negative and Gram-positive bacteria. The metabolite-mediated potentiation proceeds by PMF generation, which we found is necessary for aminoglycoside uptake and killing in persisters. This work adds to a growing understanding of the role played by metabolism in killing by bactericidal antibiotics[13,23,24] and broadens our understanding of persister physiology. Moreover, our findings imply the benefit of delivering PMF-stimulating metabolites as adjuvants to aminoglycosides in the treatment of chronic bacterial infections.

Methods Summary

In all experiments, bacterial cells were cultured in 25mL Luria-Bertani broth (LB) for 16 hours at 37°C, 300RPM, and 80% humidity in 250mL flasks. Unless otherwise noted, the following concentrations were used: 10 μg/mL gentamicin, 100 μg/mL ampicillin, 5 μg/mL ofloxacin, 20 μM CCCP, 1 mM KCN. The concentration of all carbon sources added to potentiate aminoglycosides was normalized to deliver 60 mM carbon (e.g., 10 mM glucose, 20 mM pyruvate, etc.). E. coli (K12 EMG2) and S. aureus (ATCC 25923) were the two parent strains used in this study. Knockouts (Supplementary Table 1 and 2) were constructed by P1-phage transduction from the Keio knockout collection. In E. coli, non-persister stationary phase cells were killed by treatment with 5 μg/mL ofloxacin for 4 hours[25, 26]. Samples were then washed with phosphate buffered saline (PBS) and suspended in M9 salts with carbon source and antibiotic to determine metabolite-enabled killing of persisters. At specified time points, 10 μL aliquots of samples were removed, serially diluted, and spot-plated onto LB agar plates to determine colony forming units/mL (CFU/mL) and survival. Gent-TR was made as previously described[27]. Aminoglycoside uptake was measured by incubating stationary phase samples with 10 μg/mL Gent-TR for 5 minutes at 37°C, 300RPM, and 80% humidity. 100 μL of each sample was then washed and resuspended in PBS and analyzed on a BD FACS Aria II flow cytometer. Biofilm survival assays were performed as previously described[28]. Raw microarray data for S. aureus were downloaded from the Gene Expression Omnibus (GEO) series GSE20973[29] and processed with RMA express using background adjustment, quantile normalization, and median polish summarization to compute RMA expression values[30]. Mouse experiments were performed with female Charles River Balb/C mice in collaboration with ViviSource Laboratories and conformed to the ViviSource IACUC policies and Procedural Guidelines.