title: An in vitro deletion in ribE encoding lumazine synthase contributes to nitrofurantoin resistance in Escherichia coli.
abstract: Nitrofurantoin has been used for decades for the treatment of urinary tract infections (UTIs), but clinically significant resistance in Escherichia coli is uncommon. Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two nitrofurantoin-susceptible intestinal E. coli strains (ST540-p and ST2747-p) to increasing nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole-genome sequencing was performed for both susceptible isolates and selected mutants that exhibited the highest nitrofurantoin resistance levels aerobically (ST540-a and ST2747-a) and anaerobically (ST540-an and ST2747-an). ST540-a/ST540-an and ST2747-a (aerobic MICs of >64 μg/ml) harbored mutations in the known nitrofurantoin resistance determinants nfsA and/or nfsB, which encode oxygen-insensitive nitroreductases. ST2747-an showed reduced nitrofurantoin susceptibility (aerobic MIC of 32 μg/ml) and exhibited remarkable growth deficits but did not harbor nfsA/nfsB mutations. We identified a 12-nucleotide deletion in ribE, encoding lumazine synthase, an essential enzyme involved in the biosynthesis of flavin mononucleotide (FMN), which is an important cofactor for NfsA and NfsB. Complementing ST2747-an with a functional wild-type lumazine synthase restored nitrofurantoin susceptibility. Six NIT-R E. coli isolates (NRCI-1 to NRCI-6) from stools of UTI patients treated with nitrofurantoin, cefuroxime, or a fluoroquinolone harbored mutations in nfsA and/or nfsB but not ribE. Sequencing of the ribE gene in six intestinal and three urinary E. coli strains showing reduced nitrofurantoin susceptibility (MICs of 16 to 48 μg/ml) also did not identify any relevant mutations. NRCI-1, NRCI-2, and NRCI-5 exhibited up to 4-fold higher anaerobic MICs, compared to the mutants generated in vitro, presumably because of additional mutations in oxygen-sensitive nitroreductases.
authors.affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.
authors.forename: Jascha
authors.initials: J
authors.lastname: Vervoort
authors.affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.
authors.forename: Basil Britto
authors.initials: BB
authors.lastname: Xavier
authors.affiliation: Infection Control Program, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Andrew
authors.initials: A
authors.lastname: Stewardson
authors.affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.
authors.forename: Samuel
authors.initials: S
authors.lastname: Coenen
authors.affiliation: Centre for Family and Community Medicine, Medical University of Lodz, Lodz, Poland.
authors.forename: Maciek
authors.initials: M
authors.lastname: Godycki-Cwirko
authors.affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.
authors.forename: Niels
authors.initials: N
authors.lastname: Adriaenssens
authors.affiliation: Centre for Family and Community Medicine, Medical University of Lodz, Lodz, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kowalczyk
authors.affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.
authors.forename: Christine
authors.initials: C
authors.lastname: Lammens
authors.affiliation: Infection Control Program, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland.
authors.forename: Stephan
authors.initials: S
authors.lastname: Harbarth
authors.affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.
authors.forename: Herman
authors.initials: H
authors.lastname: Goossens
authors.affiliation: Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium surbhi.malhotra@uantwerpen.be.
authors.forename: Surbhi
authors.initials: S
authors.lastname: Malhotra-Kumar
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002444:Cefuroxime / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000188:drug therapy / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D024841:Fluoroquinolones / Q000494:pharmacology; D005816:Genetic Complementation Test; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009582:Nitrofurantoin / Q000494:pharmacology; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism; D012258:Riboflavin Synthase / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D014552:Urinary Tract Infections / Q000188:drug therapy / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D024841:Fluoroquinolones; D009097:Multienzyme Complexes; C044822:6,7-dimethyl-8-ribityllumazine synthase; D009582:Nitrofurantoin; C116053:NfsA protein, E coli; C116054:NfsB protein, E coli; D009601:Nitroreductases; D012258:Riboflavin Synthase; D002444:Cefuroxime