Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance

Abstract

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer's and Huntington's disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, "On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you."

Keywords: Enterococcus faecium; Staphylococcus aureus; Streptococcus pyogenes; antibiotic resistance.

FIG 1

Synergistic antimicrobial activity of PBT2 and zinc. (a) Growth of GAS, MRSA, and VRE serially diluted on THY agar in the presence or absence of PBT2 (1.5 μM) and/or zinc (400 μM) or vancomycin (20 μg/ml). Dilution values are indicated on the left of each figure panel. (b) Time-kill curves of GAS, MRSA, and VRE in THY broth with or without PBT2 (2 μM for GAS or 6 μM for MRSA and VRE) and/or ZnSO₄ (400 μM for GAS and 600 μM for MRSA and VRE) or vancomycin (bactericidal; 4 μg/ml for MRSA) or tetracycline (bacteriostatic; 1 μg/ml for MRSA). Error bars indicate standard deviations from 2 biological replicates. (c) Development of resistance during serial passage in the presence of subinhibitory concentrations of antimicrobial compounds in CAMHB. Data represent means for 3 biological replicates. (d) CFU recovered from a murine wound infection model 4 days after challenge with GAS (1.1 × 10⁷ CFU), MRSA (6 × 10⁵ CFU), or VRE (8.8 × 10⁵ CFU). Mice were treated twice daily with carrier ointment only or ointment with 5 mM PBT2 and/or 50 mM Zn (ZnSO₄ for MRSA or ZnCl₂ for GAS) or 2.75 mM PBT2 and/or 75 mM ZnSO₄ for VRE. Values for individual mice are plotted, and black lines are representative of group geometric mean (*, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001, one-way ANOVA).

FIG 2

PBT2 and zinc affect heavy metal homeostasis, metabolism and virulence. (a) Intracellular zinc concentrations as determined by ICP-MS for GAS, MRSA, and VRE grown in THY with or without PBT2 and zinc (GAS, 0.3 μM PBT2 + 50 μM ZnSO₄; MRSA, 1 μM PBT2 + 200 μM ZnSO₄; and VRE, 1 μM PBT2 + 150 μM ZnSO₄) (error bars indicate standard error of the mean from at least 3 biological replicates; *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001, one-way ANOVA). (b) RNASeq transcriptome analysis of bacteria treated with PBT2 and ZnSO₄ for GAS (4.75 μM PBT2 + 128 μM ZnSO₄), MRSA (2 μM PBT2 + 50 μM ZnSO₄), and VRE (1.75 μM PBT2 + 128 μM ZnSO₄) in CAMHB. Genes with log₂ fold changes of >1/<1 and P < 0.05 are shown in blue, and selected genes of interest are shown in red. Data were collected from 3 biological replicates. (c) Transcript levels for selected genes measured by real-time PCR. Log₂ fold changes were calculated relative to untreated controls and normalized to a reference gene using the ΔΔC_T method (reference genes were proS for GAS, rrsA for MRSA, and 23S for VRE). Error bars represent standard deviations from 3 biological replicates.

FIG 3

PBT2 and zinc reverse antibiotic resistance in a murine wound infection model. CFU recovered 4 days after wound infection with GAS (3.8 × 10⁶ CFU), MRSA (5.3 × 10⁵ CFU), or VRE (9.4 × 10⁵ CFU). Mice were treated twice daily with ointment only or ointment containing PBT2, Zn, and/or antibiotic (2 mM PBT2, 25 mM ZnSO₄, and/or 15 μg/ml tetracycline for GAS; 3 mM PBT2, 30 mM ZnSO₄, and/or 4 μg/ml erythromycin for MRSA; and 2 mM PBT2, 30 mM ZnSO₄, and/or 20 μg/ml vancomycin for VRE). Values for individual mice are plotted, and black lines are representative of group geometric mean (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001, one-way ANOVA). Abbreviations: Tet, tetracycline; Erm, erythromycin; Van, vancomycin.