Characterization of b-lactamase and porin mutants of Enterobacteriaceae selected with ceftaroline1avibactam (NXL104)

Results: Overgrowth occurred on agar with low MIC multiples of ceftaroline+avibactam, but frequencies for single-step mutants were ,10. Most mutants were unstable, with only three remaining resistant on subculture. For one, from an CTX-M-15-positive Escherichia coli, the ceftaroline+avibactam MIC was raised, but the organism had reduced resistance to ceftaroline and lost resistance to other oxyimino-cephalosporins, with this profile retained when the mutant blaCTX-M-15 was cloned into E. coli DH5a. Sequencing identified a Lys237Gln substitution in the CTX-M-15 variant. The other two stable single-step mutants were from an AmpC-derepressed Enterobacter cloacae strain; these had unaltered or slightly reduced resistance to other b-lactams. Both had amino acids 213–226 deleted from the V loop of AmpC. Further stable mutants were obtained from AmpC-inducible and -derepressed E. cloacae in multi-step selection, and these variously had reduced expression of OmpC and OmpF, and/or Asn366His/Ile substitutions in AmpC.