An In Vitro Deletion in ribE Encoding Lumazine Synthase Contributes to Nitrofurantoin Resistance in Escherichia coli

Abstract

e 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 g/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 g/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 g/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.

Genomic Insights Into Nitrofurantoin Resistance Mechanisms and Epidemiology in Clinical Enterobacteriaceae

“…Interestingly, for E. coli ST167 (10_S4), T117I and D187G mutations in nfsA (deletions of WVF at positions 77–79 were found in this study's strain while W77*stop was detected by Sandegren et al. [2008]), and G66D in nfsB has already been reported in different clinical clones (ST540 and ST2747) from Europe and Canada, as responsible for high-level resistance to NFT in vitro [ 44 , 48 ]. This confirms the resistance mechanism underlying NFT resistance in this E. coli isolate and suggests that this mutation is common among E. coli strains of different clonality and geographical sources.…”

“…Moreover, it underscores the need to undertake periodic surveillance of hospitals and invasive medical devices, as well as moderate the use of invasive procedures to prevent reinfection of patients [ 41 ]. Such surveillance should not involve the use of rectal swabs as the low-level concentrations of NFT in the rectum have been cited as a reason for the higher NFT-R recorded among intestinal bacteria [ 44 , 45 ].…”

“…The species-specific and clonal-specific nature of the mutations suggest that the Enterobacter spp. were either exposed to different environments (aerobic or anaerobic) with varying NFT selection pressures or mutated according to clonal/species serendipities [ 44 , 45 ].…”

“…Furthermore, deletion(s) in the ribE gene, which encodes lumazine synthase that is needed for riboflavin biosynthesis, has been shown to increase MIC levels in laboratory mutants, although these mutations have so far not been described in clinical isolates. Deletions in ribE thus leads to NFT resistance by inhibiting the synthesis of riboflavin/flavin mononucleotide, an important cofactor of nfsA and nfsB [ 14 , 15 ]. Recently, plasmid-mediated efflux genes, oqxAB , have also been associated with clinically relevant levels of NFT-R, implicating the dissemination of these MDR efflux pumps in NFT-R [ 16 ].…”

Contemporary resistance trends and mechanisms for the old antibiotics colistin, temocillin, fosfomycin, mecillinam and nitrofurantoin

“…199The mechanism of resistance seems to be mainly mutations in nfsA or nfsB, both of which are 200 encoding oxygen insensitive nitroreductases[45]. The lack of reduction of the substance prevents 201 the formation of toxic intermediate compounds[48]. Recently, deletion in ribE, encoding lumazine 202 synthase needed for riboflavin biosynthesis, has also been shown to increase MIC-levels in laboratory mecillinam, nitofurantoin data from European outpatients infected by E. coli have generally 205…”