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Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection


Antibiotic-resistant bacteria are a significant threat to human health, with one estimate suggesting they will cause 10 million worldwide deaths per year by 2050, surpassing deaths due to cancer1. Because new antibiotic development can take a decade or longer, it is imperative to effectively use currently available drugs. Antibiotic combination therapy offers promise for treating highly resistant bacterial infections, but the factors governing the sporadic efficacy of such regimens have remained unclear. Dogma suggests that antibiotics ineffective as monotherapy can be effective in combination2. Here, using carbapenem-resistant Enterobacteriaceae (CRE) clinical isolates, we reveal the underlying basis for the majority of effective combinations to be heteroresistance. Heteroresistance is a poorly understood mechanism of resistance reported for different classes of antibiotics3,4,5,6 in which only a subset of cells are phenotypically resistant7. Within an isolate, the subpopulations resistant to different antibiotics were distinct, and over 88% of CRE isolates exhibited heteroresistance to multiple antibiotics (‘multiple heteroresistance’). Combinations targeting multiple heteroresistance were efficacious, whereas those targeting homogenous resistance were ineffective. Two pan-resistant Klebsiella isolates were eradicated by combinations targeting multiple heteroresistance, highlighting a rational strategy to identify effective combinations that employs existing antibiotics and could be clinically implemented immediately.

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Fig. 1: Enterobacter clinical isolate Mu208 is heteroresistant to multiple antibiotics but killed by their combinations.
Fig. 2: Multiple heteroresistance is common in CRE.
Fig. 3: Efficacy of antibiotic combinations is largely dependent on multiple heteroresistance.
Fig. 4: Eradication of pan-resistant Klebsiella by antibiotic combinations targeting multiple heteroresistance.

Data availability

All data needed to evaluate the conclusions in this Article are presented in the paper or the Supplementary Information. Any additional data can be requested from the corresponding author.


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The authors thank A. Grakoui, H. Ratner and W. Shafer for critical reading of the manuscript, and C. Bower and the GA EIP/MuGSI staff for providing CRE isolates. D.A.H. is supported by a postdoctoral research fellowship from the Cystic Fibrosis Foundation. E.X.S. is supported by T32 training grant AI106699 from the National Institutes of Health (NIH). D.S.W. is supported by a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award and NIH grant AI141883. The GA EIP is funded by the Centers for Disease Control and Prevention. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the CDC.

Author information




V.I.B. and D.A.H. performed the majority of the experiments. S.J., E.X.S. and J.E.W. contributed to the studies quantifying the prevalence of heteroresistance. E.M.B. performed clinical susceptibility testing. S.W.S., M.M.F. and J.J. contributed to CRE isolate collection, identification and characterization. V.I.B., D.A.H. and D.S.W. conceived of the experiments and wrote the manuscript, which all authors reviewed and edited.

Corresponding author

Correspondence to David S. Weiss.

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Competing interests

V.I.B., D.A.H. and D.S.W. are listed authors on a provisional patent that has been filed related to the work described here.

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Supplementary Figs. 1–12, Supplementary Tables 1–2, Supplementary File 1

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Band, V.I., Hufnagel, D.A., Jaggavarapu, S. et al. Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection. Nat Microbiol 4, 1627–1635 (2019).

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