Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae

Abstract

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.

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Figure 1: A colistin-resistant subpopulation increases in frequency during in vivo infection.
Figure 2: Innate immune host defences are required for the increased frequency of the colistin-resistant subpopulation during infection.
Figure 3: R/S is refractory to colistin during infection and leads to colistin treatment failure.
Figure 4: PhoQ is required for the presence of the colistin-resistant subpopulation.
Figure 5: Clinical isolate harbouring an undetected colistin-resistant subpopulation causes a lethal, antibiotic-resistant infection.

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Acknowledgements

The authors thank S. Satola, M. Farley and the Georgia Emerging Infections Program for providing Enterobacter cloacae strains Mu117, Mu819 and R/S-lo, P. Rather for providing plasmid pMQ310, the Yerkes Nonhuman Primate Genomics Core for help with DNA sequencing and analysis, and C.-Y. Chin and D. Bonenberger for breeding and genotyping knockout mice. The authors also thank R. Ahmed, A. Grakoui and W. Shafer for comments and revisions of the manuscript. D.S.W. is supported by a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award, VA Merit Award I01 BX002788 and National Institutes of Health (NIH) grant AI098800. E.K.C. is supported by the National Institute of Allergy and Infectious Diseases of the NIH under award no. T32AI106699. M.S.T. is supported by the NIH (grants nos RO1AI064184, RO1AI76322 and R21AI11987) and the Army Research Office (grant no. 61789-MA-MUR). T.D.R. and K.V. were supported by 1U01CI000906-05 ‘Emerging Infections Program (EIP) PPACA: Enhancing Epidemiology and Laboratory Capacity’ funding from the Emory Public Health Bioinformatics Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH, the Department of Veterans Affairs or the Centers for Disease Control and Prevention.

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Experiments were conducted by V.I.B., E.K.C. and B.A.N. The manuscript was prepared by V.I.B., E.K.C. and D.S.W. Clinical microbiological assays were conducted by E.M.B., who also provided the R/S strain. Lipid A analysis was performed by C.M.H. and M.S.T. Sequence analysis was performed by E.K.C., G.K.T., K.V., T.D.R. and S.E.B. J.P. synthesized and purified host antimicrobials. The study was planned and directed by D.S.W.

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Correspondence to David S. Weiss.

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Band, V., Crispell, E., Napier, B. et al. Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae. Nat Microbiol 1, 16053 (2016). https://doi.org/10.1038/nmicrobiol.2016.53

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