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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Nation, R. L. & Li, J. Colistin in the 21st century. Curr. Opin. Infect. Dis. 22, 535–543 (2009).
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).
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).
Brown, E. D. & Wright, G. D. Antibacterial drug discovery in the resistance era. Nature 529, 336–343 (2016).
Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).
Sands, M., Kron, M. A. & Brown, R. B. Pentamidine: a review. Rev. Infect. Dis. 7, 625–634 (1985).
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).
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).
Delcour, A. H. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta 1794, 808–816 (2009).
Nikaido, H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264, 382–388 (1994).
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).
Band, V. I. & Weiss, D. S. Mechanisms of antimicrobial peptide resistance in Gram-negative bacteria. Antibiotics 4, 18–41 (2015).
MacDonald, I. A. & Kuehn, M. J. Offense and defense: microbial membrane vesicles play both ways. Res. Microbiol. 163, 607–618 (2012).
Vaara, M. Agents that increase the permeability of the outer membrane. Microbiol. Rev. 56, 395–411 (1992).
Vaara, M. & Vaara, T. Polycations as outer membrane-disorganizing agents. Antimicrob. Agents Chemother. 24, 114–122 (1983).
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).
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).
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).
Zhu, W. et al. Antibacterial drug leads: DNA and enzyme multitargeting. J. Med. Chem. 58, 1215–1227 (2015).
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).
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).
Olaitan, A. O., Morand, S. & Rolain, J. M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front. Microbiol. 5, 643 (2014).
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).
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).
Amos, H. & Vollmayer, E. Effect of pentamidine on the growth of Escherichia coli. J. Bacteriol. 73, 172–177 (1957).
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).
Yeung, K. T., Chan, M. & Chan, C. K. The safety of i.v. pentamidine administered in an ambulatory setting. Chest 110, 136–140 (1996).
Ejim, L. et al. Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat. Chem. Biol. 7, 348–350 (2011).
Dijkshoorn, L., Nemec, A. & Seifert, H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5, 939–951 (2007).
Qureshi, Z. A. et al. Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance. Clin. Infect. Dis. 60, 1295–1303 (2015).
Napier, B. A. et al. Clinical use of colistin induces cross-resistance to host antimicrobials in Acinetobacter baumannii. mBio 4, e00021–13 (2013).
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).
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).
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).
French, S. et al. A robust platform for chemical genomics in bacterial systems. Mol. Biol. Cell. 27, 1015–1025 (2016).
Ihaka, R. & Gentleman, R. R: a language for data analysis and graphics. J. Comp. Graph. Stat. 5, 299–314 (1996).
Keseler, I. M. et al. Ecocyc: fusing model organism databases with systems biology. Nucleic Acids Res. 41, D605–D612 (2013).
Karp, P. D. Pathway databases: a case study in computational symbolic theories. Science 293, 2040–2044 (2001).
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).
Odds, F. C. Synergy, antagonism, and what the chequerboard puts between them. J. Antimicrob. Chemother. 52, 1 (2003).
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).
Groisman, E. A. The pleiotropic two-component regulatory system PhoP-PhoQ. J. Bacteriol. 183, 1835–1842 (2001).
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).
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).
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.
The authors declare no competing financial interests.
Supplementary Figures 1–7. (PDF 479 kb)
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)
Gene ontology, biosynthetic pathway andpromoter activation enrichment by the vancomycin suppression screen of the E. coli Keio collection at 15 °C. (XLSX 42 kb)
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)
FIC indices of pentamidine/rifampicin combinations against Gram-negative clinical isolates from the Wright Clinical Collection. (XLSX 51 kb)
Activity of polymyxin B against naturally resistant clinical isolates. (XLSX 48 kb)
Characterization of spontaneous pentamidine/rifampicin suppressor mutants. (XLSX 35 kb)
About this article
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
Pentamidine enhances photosensitization of Acinetobacter baumannii using diode lasers with emission of light at wavelength of ʎ = 405 nm and ʎ = 635 nm
Photodiagnosis and Photodynamic Therapy (2021)
Annals of the New York Academy of Sciences (2021)
In Vitro Activity of Ceftazidime-Avibactam Alone and in Combination with Amikacin Against Colistin-Resistant Gram-Negative Pathogens
Microbial Drug Resistance (2021)
A polymeric approach toward resistance-resistant antimicrobial agent with dual-selective mechanisms of action
Science Advances (2021)
Thrombin-Derived Peptides Potentiate the Activity of Gram-Positive-Specific Antibiotics against Gram-Negative Bacteria