Plasmid-Mediated OqxAB Is an Important Mechanism for Nitrofurantoin Resistance in Escherichia coli

Abstract

Increasing consumption of nitrofurantoin (NIT) for treatment of acute uncomplicated urinary tract infections (UTI) highlights the need to monitor emerging NIT resistance mechanisms. This study investigated the molecular epidemiology of the multidrug-resistant efflux gene oqxAB and its contribution to nitrofurantoin resistance by using Escherichia coli isolates originating from patients with UTI (n = 205; collected in 2004 to 2013) and food-producing animals (n = 136; collected in 2012 to 2013) in Hong Kong. The oqxAB gene was highly prevalent among NIT-intermediate (11.5% to 45.5%) and -resistant (39.2% to 65.5%) isolates but rare (0% to 1.7%) among NIT-susceptible (NIT-S) isolates. In our isolates, the oqxAB gene was associated with IS26 and was carried by plasmids of diverse replicon types. Multilocus sequence typing revealed that the clones of oqxAB-positive E. coli were diverse. The combination of oqxAB and nfsA mutations was found to be sufficient for high-level NIT resistance. Curing of oqxAB-carrying plasmids from 20 NIT-intermediate/resistant UTI isolates markedly reduced the geometric mean MIC of NIT from 168.9 μg/ml to 34.3 μg/ml. In the plasmid-cured variants, 20% (1/5) of isolates with nfsA mutations were NIT-S, while 80% (12/15) of isolates without nfsA mutations were NIT-S (P = 0.015). The presence of plasmid-based oqxAB increased the mutation prevention concentration of NIT from 128 μg/ml to 256 μg/ml and facilitated the development of clinically important levels of nitrofurantoin resistance. In conclusion, plasmid-mediated oqxAB is an important nitrofurantoin resistance mechanism. There is a great need to monitor the dissemination of this transferable multidrug-resistant efflux pump.

FIG 1

Prevalence of oqxAB stratified by host source and nitrofurantoin susceptibility, Hong Kong. The number of isolates is shown in parentheses. NIT, nitrofurantoin; S, susceptible; I, intermediate; R, resistant.

FIG 2

Mutation prevention concentration (MPC) assay. Approximately 10¹⁰ organisms were applied to Mueller-Hinton agar plates containing 0 to 512 μg/ml of nitrofurantoin. Surviving colonies were counted after a 72-h incubation at 37°C. All bacterial strains were tested in duplicate in two independent experiments. The mean recovery fraction and standard deviations are shown. Recovery fractions of J53, J53/pLOW2, and J53/pLOW2::oqxAB strains were compared by ANOVA. *, P < 0.05; **, P < 0.001.

FIG 3

Comparison of nitrofurantoin geometric mean MICs for parent strains and plasmid-cured variants. The scatterplot shows MIC data for the parent strains and plasmid-cured variants following aerobic and anaerobic incubation. Solid triangles and open circles were used to represent the presence and absence of nfsA mutations, respectively. No strain had an nfsB mutation. Geometric means and 95% confidence intervals were indicated by horizontal lines in the groups. The geometric mean MICs for parent strains and plasmid-cured variants were compared by Wilcoxon signed-rank test.