title: Characterization of β-lactamase and porin mutants of Enterobacteriaceae selected with ceftaroline + avibactam (NXL104).
abstract: OBJECTIVE
  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.
authors.affiliation: Antibiotic Resistance Monitoring & Reference Laboratory, Health Protection Agency Microbiology Services, Colindale, London NW9 5EQ, UK. david.livermore@hpa.org.uk
authors.forename: David M
authors.initials: DM
authors.lastname: Livermore
authors.forename: Shazad
authors.initials: S
authors.lastname: Mushtaq
authors.forename: Kevin
authors.initials: K
authors.lastname: Barker
authors.forename: Russell
authors.initials: R
authors.lastname: Hope
authors.forename: Marina
authors.initials: M
authors.lastname: Warner
authors.forename: Neil
authors.initials: N
authors.lastname: Woodford
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D002511:Cephalosporins / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D004755:Enterobacteriaceae / Q000187:drug effects* / Q000302:isolation & purification; D042822:Genomic Instability; D008826:Microbial Sensitivity Tests; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D018272:Porins / Q000235:genetics* / Q000378:metabolism; D012641:Selection, Genetic; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism; D000097583:Ceftaroline
chemical_list: D000900:Anti-Bacterial Agents; D053961:Azabicyclo Compounds; D002511:Cephalosporins; D050505:Mutant Proteins; D018272:Porins; D001618:beta-Lactamases; D000097583:Ceftaroline; C543519:avibactam