@article{ec6383ce11a64fe6a1738fd085b7ad5f,
title = "Discovery and Characterization of a Nitroreductase Capable of Conferring Bacterial Resistance to Chloramphenicol",
abstract = "Widespread antibiotic resistance has led to the reappraisal of abandoned antibiotics including chloramphenicol. However, enzyme(s) underlying one form of chloramphenicol resistance, nitroreduction, have eluded identification. Here we demonstrate that expression of the Haemophilus influenzae nitroreductase gene nfsB confers chloramphenicol resistance in Escherichia coli. We characterized the enzymatic product of H. influenzae NfsB acting on chloramphenicol and found it to be amino-chloramphenicol. Kinetic analysis revealed reduction of diverse substrates including the incomplete reduction of 5-nitro antibiotics metronidazole and nitrofurantoin, likely resulting in activation of these antibiotic pro-drugs to their cytotoxic forms. We observed that expression of the H. influenzae nfsB gene in E. coli results in significantly increased susceptibility to metronidazole. Finally, we found that in this strain metronidazole attenuates chloramphenicol resistance synergistically, and in vitro metronidazole weakly inhibits chloramphenicol reduction by NfsB. Our findings reveal the underpinnings of a chloramphenicol resistance mechanism nearly 70 years after its description.",
keywords = "E. cloacae, H. influenzae, amphenicols, antibiotics, chloramphenicol, metronidazole, nfsB, nitroreductase, resistance, synergy",
author = "Crofts, {Terence Spencer} and Pratyush Sontha and King, {Amber O.} and Bin Wang and Biddy, {Brent A.} and Nicole Zanolli and John Gaumnitz and Gautam Dantas",
note = "Funding Information: Support for this work came in part by awards to G.D. through the Edward Mallinckrodt, Jr. Foundation (Scholar Award), the NIH Director's New Innovator Award (http://commonfund.nih.gov/newinnovator/), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK: http://www.niddk.nih.gov/), the National Institute of General Medical Sciences (NIGMS: http://www.nigms.nih.gov/), and the National Institute of Allergy and Infectious Diseases (NIAID: https://www.niaid.nih.gov/) of the National Institutes of Health (NIH) under award numbers DP2DK098089, R01GM099538, and R01AI123394, respectively. T.S.C. was supported by the http://www2.niddk.nih.gov/ National Institute of Diabetes and Digestive and Kidney Diseases training grant through award number T32 DK077653 (Phillip I. Tarr, Principal Investigator) and the National Institute of Child Health and Development training grant through award number T32 HD049305 (Kelle H. Moley, Principal Investigator). Mass spectrometry was performed in the Metabolomics Facility at Washington University (P30 DK020579). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. We are thankful to the Caparon lab for supplying S. pyogenes genomic DNA, to Dr. Tim Wencewicz for his insightful discussions regarding enzymology and biochemistry, and to Drew Gasparrini and the rest of the Dantas lab for many helpful discussions of the work and manuscript. Conceptualization, T.S.C. and G.D.; Methodology, T.S.C. and G.D.; Investigation, T.S.C. P.S. A.O.K. B.W. B.A.B. N.Z. and J.G.; Writing – Original Draft, T.S.C.; Writing – Review & Editing, T.S.C. P.S. and G.D.; Supervision, T.S.C. and G.D. Mass spectrometry was performed in the Metabolomics Facility at Washington University. The authors declare no competing interests. Funding Information: Support for this work came in part by awards to G.D. through the Edward Mallinckrodt, Jr. Foundation (Scholar Award), the NIH Director's New Innovator Award ( http://commonfund.nih.gov/newinnovator/ ), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK: http://www.niddk.nih.gov/ ), the National Institute of General Medical Sciences (NIGMS: http://www.nigms.nih.gov/ ), and the National Institute of Allergy and Infectious Diseases (NIAID: https://www.niaid.nih.gov/ ) of the National Institutes of Health (NIH) under award numbers DP2DK098089, R01GM099538, and R01AI123394, respectively. T.S.C. was supported by the http://www2.niddk.nih.gov/ National Institute of Diabetes and Digestive and Kidney Diseases training grant through award number T32 DK077653 (Phillip I. Tarr, Principal Investigator) and the National Institute of Child Health and Development training grant through award number T32 HD049305 (Kelle H. Moley, Principal Investigator). Mass spectrometry was performed in the Metabolomics Facility at Washington University (P30 DK020579). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. We are thankful to the Caparon lab for supplying S. pyogenes genomic DNA, to Dr. Tim Wencewicz for his insightful discussions regarding enzymology and biochemistry, and to Drew Gasparrini and the rest of the Dantas lab for many helpful discussions of the work and manuscript. Publisher Copyright: {\textcopyright} 2019 Elsevier Ltd",
year = "2019",
month = apr,
day = "18",
doi = "10.1016/j.chembiol.2019.01.007",
language = "English (US)",
volume = "26",
pages = "559--570.e6",
journal = "Cell Chemical Biology",
issn = "2451-9448",
publisher = "Elsevier Inc.",
number = "4",
}