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Pentamidine sensitizes Gram-negative pathogens to antibiotics and overcomes acquired colistin resistance

Abstract

The increasing use of polymyxins1 in addition to the dissemination of plasmid-borne colistin resistance threatens to cause a serious breach in our last line of defence against multidrug-resistant Gram-negative pathogens, and heralds the emergence of truly pan-resistant infections. Colistin resistance often arises through covalent modification of lipid A with cationic residues such as phosphoethanolamine—as is mediated by Mcr-1 (ref. 2)—which reduce the affinity of polymyxins for lipopolysaccharide3. Thus, new strategies are needed to address the rapidly diminishing number of treatment options for Gram-negative infections4. The difficulty in eradicating Gram-negative bacteria is largely due to their highly impermeable outer membrane, which serves as a barrier to many otherwise effective antibiotics5. Here, we describe an unconventional screening platform designed to enrich for non-lethal, outer-membrane-active compounds with potential as adjuvants for conventional antibiotics. This approach identified the antiprotozoal drug pentamidine6 as an effective perturbant of the Gram-negative outer membrane through its interaction with lipopolysaccharide. Pentamidine displayed synergy with antibiotics typically restricted to Gram-positive bacteria, yielding effective drug combinations with activity against a wide range of Gram-negative pathogens in vitro, and against systemic Acinetobacter baumannii infections in mice. Notably, the adjuvant activity of pentamidine persisted in polymyxin-resistant bacteria in vitro and in vivo. Overall, pentamidine and its structural analogues represent unexploited molecules for the treatment of Gram-negative infections, particularly those having acquired polymyxin resistance determinants.

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Figure 1: A vancomycin antagonism screening platform identifies pentamidine.
Figure 2: Pentamidine potentiates Gram-positive antibiotics in Gram-negative pathogens.
Figure 3: Pentamidine is an effective adjuvant against Gram-negative bacteria expressing mcr-1.
Figure 4: Pentamidine potentiates Gram-positive antibiotics against colistin-sensitive and -resistant A. baumannii in systemic murine infection models.

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Acknowledgements

The authors thank K. Iyer and L. Carfrae for assistance with mouse infection experiments, and M. Mulvey from the University of Manitoba for providing the environmental ­mcr-1-positive E. coli isolates. This work was supported by Discovery and Foundation grants from the Natural Sciences and Engineering Research Council and the Canadian Institutes of Health Research (FDN-143215) to E.D.B., by grants from Cystic Fibrosis Canada and the Ontario Research Fund to E.D.B., by a grant from the Michael G. DeGroote Institute for Infectious Disease Research to E.D.B. and B.K.C., by an operating grant from the Canadian Institutes of Health Research to B.K.C. (MOP-82704), by a Foundation grant from the Canadian Institutes of Health Research to C.W. (FDN-CEHA-26119), by salary awards to E.D.B., B.K.C. and C.W. from the Canada Research Chairs Program, by a fellowship from the Fonds de reserche en santé du Québec to J.-P.C., by a fellowship from the Canadian Institutes of Health Research DSECT Program to S.F., by a scholarship from the Ontario Graduate Scholarships Program to C.R.M. and by scholarships to J.M.S. from the Canadian Institutes of Health Research and the Ontario Graduate Scholarships Program.

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Authors

Contributions

J.M.S., C.R.M., B.I., S.F., J.-P.C., C.B., C.W., B.K.C. and E.D.B. designed the experiments. J.M.S. designed the vancomycin suppression screening platform. S.F., J.-P.C. and J.M.S. performed genetic screens. J.M.S. performed the chemical screen. S.F. performed atomic force microscopy. With input from C.W. and J.M.S., C.B. performed core OS, LPS shedding and periplasmic leaking assays. J.M.S. performed in vitro antibiotic susceptibility assays. B.I. performed qRT–PCR experiments. A.O.S. designed the pGDP2:mcr-1 plasmid. J.M.S. engineered the mcr-1-positive strains of E. coli BW25113 and K. pneumoniae ATCC 43816, and generated the colistin-resistant variant of A. baumannii ATCC 17978. C.R.M., B.I. and B.K.C. designed in vivo infection model experiments. C.R.M., B.I. and J.M.S. performed in vivo infection model experiments. J.M.S., M.A.F. and E.D.B. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Eric D. Brown.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–7. (PDF 479 kb)

Supplementary Table 1

E. coli Keio collection gene deletion mutants that displayed sensitivity to novobiocin, rifampicin, and/or erythromycin at 37 °C, and/or resistance to vancomycin at 15 °C. (XLSX 49 kb)

Supplementary Table 2

Gene ontology, biosynthetic pathway andpromoter activation enrichment by the vancomycin suppression screen of the E. coli Keio collection at 15 °C. (XLSX 42 kb)

Supplementary Table 3

Screen of 1,440 previously approved drugs against E. coli BW25113 at 15 °C in the presence of 16 μg/ml vancomycin. (XLSX 60 kb)

Supplementary Table 4

FIC indices of pentamidine/rifampicin combinations against Gram-negative clinical isolates from the Wright Clinical Collection. (XLSX 51 kb)

Supplementary Table 5

Activity of polymyxin B against naturally resistant clinical isolates. (XLSX 48 kb)

Supplementary Table 6

Characterization of spontaneous pentamidine/rifampicin suppressor mutants. (XLSX 35 kb)

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Stokes, J., MacNair, C., Ilyas, B. et al. Pentamidine sensitizes Gram-negative pathogens to antibiotics and overcomes acquired colistin resistance. Nat Microbiol 2, 17028 (2017). https://doi.org/10.1038/nmicrobiol.2017.28

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