Improving Nitrofurantoin Resistance Prediction inEscherichia colifrom Whole Genome Sequence by Integrating NfsA/B Enzyme Assays

Abstract

AbstractNitrofurantoin resistance inEscherichia coliis primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift innfsAandnfsBmaking it extremely difficult to predict nitrofurantoin resistance from whole genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here we report a WGS survey of 200E. colifrom community urine samples, of which 34 were nitrofurantoin resistant. We characterised individual non-synonymous mutations seen innfsAandnfsBamong this collection using complementation cloning and assays of NfsA/B enzyme activity in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, A112T and A122T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D and A188V in NfsA and G66D, M75I, V93A and A174E in NfsB, are functionally silent in this context. We identified that 9/166 (5.4%) of nitrofurantoin susceptible isolates were “pre-resistant”, defined as having loss of function mutations innfsAornfsB. Finally, using NfsA/B enzyme activity assay and proteomics we demonstrated that 9/34 (26.5%) of nitrofurantoin resistant isolates carried functionally wild-typenfsBornfsB/nfsA. In these cases, enzyme activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis, but is still insufficient to allow its reliable prediction from WGS data.