Researchers have shown that translation occurs at a reduced rate in persisters2,8
, suggesting that persisters should be susceptible to the ribosome-targeting bactericidal aminoglycoside antibiotics9-13
. However, despite continued translation, aminoglycosides have weak activity against dormant bacteria14, 15
. Given the dormancy of persisters and the known energy requirement for aminoglycoside activity16
, 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) (). Persisters were isolated (Supplementary Information
), re-suspended in minimal media supplemented with individual metabolites, and treated with aminoglycoside gentamicin for two hours.
Specific metabolites enable aminoglycoside killing of E. coli persisters
We found that gentamicin was greatly potentiated by specific metabolic stimuli against persisters (). 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 (). 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 uptake16
, 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 ( 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 extensively16
. 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 ( 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 ( 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.
Metabolite-enabled aminoglycoside uptake and killing requires PMF produced by oxidative electron transport chain
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 ( and Supplemental Fig. 16
). Treatment with KCN abolished killing, consistent with work in rapidly growing bacteria17
, 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-I18, 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
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 (Δndh
), were treated for two hours with gentamicin plus metabolites ( and Supplementary Fig. 19
). We found that NADH dehydrogenase activity was important to this phenotype as gentamicin activity against the Δndh
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
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 (). 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.
Mechanism for metabolite-enabled eradication of persisters and clinically relevant experiments
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 (). Ofloxacin (which is efficient against Gram-negative biofilms15, 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 ().
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 (). 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 (). 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
(). 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 (), suggesting that S. aureus
persisters were not reverting to normally growing cells.
Fructose induces PMF-dependent aminoglycoside killing of S. aureus persisters
Given that aminoglycoside activity in growing S. aureus
is dependent on PMF21,22
, 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 (), 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 ().
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 antibiotics13,23,24
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.