title: In vitro selection of ceftazidime-avibactam resistance in Enterobacteriaceae with KPC-3 carbapenemase.
abstract: Ceftazidime-avibactam is active against most Enterobacteriaceae isolates with KPC carbapenemases. We investigated whether this activity could be compromised by mutation. Single-step and multistep selections were attempted using ceftazidime-avibactam (avibactam fixed at 1 or 4 μg/ml) versus two strains each of Enterobacter cloacae and Klebsiella pneumoniae, all with the KPC-3 enzyme. Mutant bla KPC alleles were sequenced, and their parentage was confirmed by typing. Ceftazidime-avibactam selected mutants at up to 16× MIC, with frequencies of ca. 10(-9). This contrasted with previous experience for ceftaroline-avibactam, where mutant frequencies under similar conditions were <10(-9). The MICs of ceftazidime with 1 μg/ml avibactam for the ceftazidime-avibactam-selected mutants rose from 1 to 8 μg/ml to 16 to >256 μg/ml and those of ceftazidime with 4 μg/ml avibactam from 0.25 to 1 μg/ml to 4 to 128 μg/ml; ceftaroline-avibactam MICs rose less, typically from 0.5 to 1 μg/ml to 1 to 8 μg/ml. The MICs of carbapenems and cephalosporins except ceftazidime and piperacillin-tazobactam were reduced for many mutants. Sequencing of blaKPC revealed point and insertion changes in 12/13 mutants investigated, representing all four parents; one mutant lacked bla KPC changes and possibly had reduced permeability. Amino acid changes commonly involved Ω loop alterations or 1 to 6 amino acid insertions immediately C-terminal to this loop. The most frequent change, seen in four mutants from three strains, was Asp179Tyr, replacing a residue that ordinarily forms a salt bridge to stabilize the Ω loop. Since ceftaroline-avibactam was less affected than ceftazidime-avibactam, we postulate that these mutations increase ceftazidimase specificity rather than conferring avibactam resistance. The clinical relevance remains uncertain.
authors.affiliation: Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, London, United Kingdom Norwich Medical School, University of East Anglia, Norwich, United Kingdom d.livermore@uea.ac.uk.
authors.forename: David M
authors.initials: DM
authors.lastname: Livermore
authors.affiliation: Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, London, United Kingdom.
authors.forename: Marina
authors.initials: M
authors.lastname: Warner
authors.affiliation: Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, London, United Kingdom.
authors.forename: Dorota
authors.initials: D
authors.lastname: Jamrozy
authors.affiliation: Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, London, United Kingdom.
authors.forename: Shazad
authors.initials: S
authors.lastname: Mushtaq
authors.affiliation: AstraZeneca Pharmaceuticals LP, Boston, Massachusetts, USA.
authors.forename: Wright W
authors.initials: WW
authors.lastname: Nichols
authors.affiliation: Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, London, United Kingdom.
authors.forename: Nazim
authors.initials: N
authors.lastname: Mustafa
authors.affiliation: Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Public Health England, London, United Kingdom.
authors.forename: Neil
authors.initials: N
authors.lastname: Woodford
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002442:Ceftazidime / Q000494:pharmacology*; D004338:Drug Combinations; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D004755:Enterobacteriaceae / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D053961:Azabicyclo Compounds; D001426:Bacterial Proteins; D004338:Drug Combinations; C000595613:avibactam, ceftazidime drug combination; D002442:Ceftazidime; D001618:beta-Lactamases; C063912:carbapenemase