Characterization of β-lactamase and porin mutants of enterobacteriaceae selected with ceftaroline+avibactam (NXL104)

Objectives: Ceftaroline+avibactam (NXL104) is a novel inhibitor combination active against Enterobacteriaceae with class A and C β-lactamases. We investigated its risk of mutational resistance. Methods: Single- and multi-step mutants were sought and characterized from Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases and KPC β-lactamases. Results: Overgrowth occurred on agar with low MIC multiples of ceftaroline+avibactam, but frequencies for single-step mutants were <10 ^-9. 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 bla _CTX-M-15 was cloned into E. coli DH5α. 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 β-lactams. Both had amino acids 213-226 deleted from the ω 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. Conclusions: Stable resistant mutants were difficult to select. Those from AmpC-derepressed E. cloacae had porin loss or AmpC changes, including ω loop deletions. A Lys237Gln substitution in CTX-M-15 conferred resistance, but largely abolished ESBL activity.