Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Nation, R. L. & Li, J. Colistin in the 21st century. Curr. Opin. Infect. Dis. 22, 535–543 (2009).

    Article  CAS  Google Scholar 

  2. Liu, Y. Y. et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect. Dis. 16, 161–168 (2016).

    Article  Google Scholar 

  3. Needham, B. D. & Trent, M. S. Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis. Nat. Rev. Microbiol. 11, 467–481 (2013).

    Article  CAS  Google Scholar 

  4. Brown, E. D. & Wright, G. D. Antibacterial drug discovery in the resistance era. Nature 529, 336–343 (2016).

    Article  CAS  Google Scholar 

  5. Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).

    Article  CAS  Google Scholar 

  6. Sands, M., Kron, M. A. & Brown, R. B. Pentamidine: a review. Rev. Infect. Dis. 7, 625–634 (1985).

    Article  CAS  Google Scholar 

  7. Stokes, J. M., Davis, J. H., Mangat, C. S., Williamson, J. R. & Brown, E. D. Discovery of a small molecule that inhibits bacterial ribosome biogenesis. eLife 3, e03574 (2014).

    Article  Google Scholar 

  8. Stokes, J. M. et al. Cold stress makes Escherichia coli susceptible to glycopeptide antibiotics by altering outer membrane integrity. Cell Chem. Biol. 23, 267–277 (2016).

    Article  CAS  Google Scholar 

  9. Delcour, A. H. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta 1794, 808–816 (2009).

    Article  CAS  Google Scholar 

  10. Nikaido, H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264, 382–388 (1994).

    Article  CAS  Google Scholar 

  11. Gill, E. E., Franco, O. L. & Hancock, R. E. Antibiotic adjuvants: diverse strategies for controlling drug-resistant pathogens. Chem. Biol. Drug Des. 85, 56–78 (2015).

    Article  CAS  Google Scholar 

  12. Band, V. I. & Weiss, D. S. Mechanisms of antimicrobial peptide resistance in Gram-negative bacteria. Antibiotics 4, 18–41 (2015).

    Article  CAS  Google Scholar 

  13. MacDonald, I. A. & Kuehn, M. J. Offense and defense: microbial membrane vesicles play both ways. Res. Microbiol. 163, 607–618 (2012).

    Article  CAS  Google Scholar 

  14. Vaara, M. Agents that increase the permeability of the outer membrane. Microbiol. Rev. 56, 395–411 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Vaara, M. & Vaara, T. Polycations as outer membrane-disorganizing agents. Antimicrob. Agents Chemother. 24, 114–122 (1983).

    Article  CAS  Google Scholar 

  16. David, S. A. Towards a rational development of anti-endotoxin agents: novel approaches to sequestration of bacterial endotoxins with small molecules. J. Mol. Recognit. 14, 370–387 (2001).

    Article  CAS  Google Scholar 

  17. Sun, T. & Zhang, Y. Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation. Nucleic Acids Res. 36, 1654–1664 (2008).

    Article  CAS  Google Scholar 

  18. Miletti, K. E. & Leibowitz, M. J. Pentamidine inhibition of group I intron splicing in Candida albicans correlates with growth inhibition. Antimicrob. Agents Chemother. 44, 958–966 (2000).

    Article  CAS  Google Scholar 

  19. Zhu, W. et al. Antibacterial drug leads: DNA and enzyme multitargeting. J. Med. Chem. 58, 1215–1227 (2015).

    Article  CAS  Google Scholar 

  20. Ofek, I. et al. Antibacterial synergism of polymyxin B nonapeptide and hydrophobic antibiotics in experimental Gram-negative infections in mice. Antimicrob. Agents Chemother. 38, 374–377 (1994).

    Article  CAS  Google Scholar 

  21. Clifton, L. A. et al. Effect of divalent cation removal on the structure of Gram-negative bacterial outer membrane models. Langmuir 31, 404–412 (2014).

    Article  Google Scholar 

  22. Olaitan, A. O., Morand, S. & Rolain, J. M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front. Microbiol. 5, 643 (2014).

    Article  Google Scholar 

  23. Bystrova, O. V. et al. Structural studies on the core and the O-polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide. Eur. J. Biochem. 269, 2194–2203 (2002).

    Article  CAS  Google Scholar 

  24. Schoenbach, E. B. & Greenspan, E. M. The pharmacology, mode of action and therapeutic potentialities of stilbamidine, pentamidine, propamidine and other aromatic diamidines: a review. Medicine (Baltimore) 27, 327–377 (1948).

    Article  CAS  Google Scholar 

  25. Amos, H. & Vollmayer, E. Effect of pentamidine on the growth of Escherichia coli. J. Bacteriol. 73, 172–177 (1957).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Ando, M. et al. In situ potentiometric method to evaluate bacterial outer membrane-permeabilizing ability of drugs: example using antiprotozoal diamidines. J. Microbiol. Methods 91, 497–500 (2012).

    Article  CAS  Google Scholar 

  27. Yeung, K. T., Chan, M. & Chan, C. K. The safety of i.v. pentamidine administered in an ambulatory setting. Chest 110, 136–140 (1996).

    Article  CAS  Google Scholar 

  28. Ejim, L. et al. Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat. Chem. Biol. 7, 348–350 (2011).

    Article  CAS  Google Scholar 

  29. Dijkshoorn, L., Nemec, A. & Seifert, H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5, 939–951 (2007).

    Article  CAS  Google Scholar 

  30. Qureshi, Z. A. et al. Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance. Clin. Infect. Dis. 60, 1295–1303 (2015).

    Article  Google Scholar 

  31. Napier, B. A. et al. Clinical use of colistin induces cross-resistance to host antimicrobials in Acinetobacter baumannii. mBio 4, e00021–13 (2013).

    Article  CAS  Google Scholar 

  32. Du, H., Chen, L., Tang, Y. W. & Kreiswirth, B. N. Emergence of the mcr-1 colistin resistance gene in carbapenem-resistant enterobacteriaceae. Lancet Infect. Dis. 16, 287–288 (2016).

    Article  CAS  Google Scholar 

  33. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

  34. Mangat, C. S., Bharat, A., Gehrke, S. S. & Brown, E. D. Rank ordering plate data facilitates data visualization and normalization in high-throughput screening. J. Biomol. Screen. 19, 1314–1320 (2014).

    Article  Google Scholar 

  35. French, S. et al. A robust platform for chemical genomics in bacterial systems. Mol. Biol. Cell. 27, 1015–1025 (2016).

    Article  CAS  Google Scholar 

  36. Ihaka, R. & Gentleman, R. R: a language for data analysis and graphics. J. Comp. Graph. Stat. 5, 299–314 (1996).

    Google Scholar 

  37. Keseler, I. M. et al. Ecocyc: fusing model organism databases with systems biology. Nucleic Acids Res. 41, D605–D612 (2013).

    Article  CAS  Google Scholar 

  38. Karp, P. D. Pathway databases: a case study in computational symbolic theories. Science 293, 2040–2044 (2001).

    Article  CAS  Google Scholar 

  39. Karp, P. D. et al. Pathway Tools version 19.0 update: software for pathway/genome informatics and systems biology. Brief. Bioinform. 17, 877–890 (2016).

    Article  CAS  Google Scholar 

  40. Odds, F. C. Synergy, antagonism, and what the chequerboard puts between them. J. Antimicrob. Chemother. 52, 1 (2003).

    Article  CAS  Google Scholar 

  41. Hasman, H. et al. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015. Euro Surveill. 20, 30085 (2015).

    Article  Google Scholar 

  42. Groisman, E. A. The pleiotropic two-component regulatory system PhoP-PhoQ. J. Bacteriol. 183, 1835–1842 (2001).

    Article  CAS  Google Scholar 

  43. O'Neill, A. J., Cove, J. H. & Chopra, I. Mutation frequencies for resistance to fusidic acid and rifampicin in Staphylococcus aureus. J. Antimicrob. Chemother. 47, 647–650 (2001).

    Article  CAS  Google Scholar 

  44. Mariam, D. H., Mengistu, Y., Hoffner, S. E. & Andersson, D. I. Effect of rpoB mutations conferring rifampin resistance on fitness of Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 48, 1289–1294 (2004).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

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.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2017.28

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing