Metabolite-enabled eradication of bacterial persisters by aminoglycosides

Abstract

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.

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).

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).

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^, , 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).

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).