Selection of mutants with resistance or diminished susceptibility to ceftazidime/avibactam from ESBL- and AmpC- producing Enterobacteriaceae

Introduction. Difficult Gram-negative infections are increasingly treated with new ß-lactamase inhibitor combinations e.g. ceftazidime/avibactam. Disturbingly, mutations in KPC carbapenemases can confer ceftazidime/avibactam resistance, sometimes selected in therapy. We explored whether this risk extended to AmpC- and ESBL- enzymes.Materials and Methods. Mutants were selected by plating AmpC-derepressed strains, ESBL producers and ceftazidime-susceptible controls on agar containing ceftazidime + avibactam, 1 or 4 mg/L. MICs were determined by CLSI agar dilution; WGS was by Illumina methodology. Results Using 2x MIC of ceftazidime + 1 mg/L avibactam, mutants were selected from all strain types at frequencies of 10-7 - 10-9. Rates diminished to <10-9 with 4 mg/L avibactam or higher MIC multiples, except with AmpC-derepressed Enterobacteriaceae. Characterised mutants (n=10, MICs 4-64 mg/L) of AmpC-derepressed strains had modifications in ampC, giving Arg168Pro/HIs, Gly176Arg/Asp, Asn366Tyr or small deletions around positions 309-314. Mutants of ESBL producers (n=20; MICs 0.5-16 mg/L) mostly had changes affecting permeability, efflux or ß-lactamase quantity; only one had an altered ß-lactamase, with an Asp182Tyr substitution in CTX-M-15, raising the ceftazidime/avibactam MIC but abrogating other cephalosporin resistance. Mutants of ceftazidime-susceptible strains were not sequenced, but phenotypes suggested altered drug accumulation or, for Enterobacter cloacae only, AmpC derepression. In further experiments, avibactam reduced, but did not abolish, selection of AmpC-derepressed Enterobacteriaceae by ceftazidime.Conclusions. Most mutants of AmpC-derepressed Enterobacteriaceae had structural mutations in ampC; those of ESBL producers mostly had genetic modifications outside ß-lactamase genes, commonly affecting uptake efflux or ß-lactamase quantity. The clinical significance of these observations remains to be determined.