Analyses of a Ceftazidime-Avibactam-Resistant Citrobacter freundii Isolate Carrying bla KPC-2 Reveals a Heterogenous Population and Reversible Genotype

• Published in: mSphere 2018-09, Vol.3 (5)
• Main Authors: Castanheira, Mariana, Arends, S J Ryan, Davis, Andrew P, Woosley, Leah N, Bhalodi, Amira A, MacVane, Shawn H
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; Azabicyclo Compounds - pharmacology; Bacterial Proteins - genetics; beta-Lactamases - genetics; Ceftazidime - pharmacology; Citrobacter freundii - drug effects; Citrobacter freundii - genetics; Citrobacter freundii - isolation & purification; Drug Combinations; Drug Resistance, Multiple, Bacterial - genetics; Humans; Microbial Sensitivity Tests; Plasmids - genetics
• ISSN: 2379-5042; 2379-5042
• DOI: 10.1128/mSphere.00408-18

A -carrying isolate developed ceftazidime-avibactam resistance during treatment with this agent. The initial and follow-up isolates exhibited ceftazidime-avibactam MICs of 4 and 64 µg/ml, respectively. Overexpression of AcrAB-TolC and porin alterations were detected in both isolates, but no other resistance mechanism was observed. After passaging the initial clinical isolate in ceftazidime-avibactam at a fixed concentration of 4 µg/ml and a 4:1 ratio, resistance to all β-lactams was noted, and a percentage of the sequencing reads had mutations leading to the alterations D176Y ( -D176Y [78%]) or R164S plus P147L ( -R164S + P147L [82%]). Further investigation of the follow-up isolate showed that 11% of the reads had mutations leading to D179Y substitution ( -D179Y). In the absence of selective pressure, ceftazidime-avibactam MICs of the passaged and follow-up isolates revealed that 7 or 8 out of 20 screened colonies reverted to susceptible and possessed wild-type sequences. Recombinant plasmids carrying the alterations observed were transformed in , and MIC values for ceftazidime ± avibactam were elevated. Lower MICs for ceftriaxone, cefepime, aztreonam, meropenem, and imipenem for the mutated KPC-2-producing isolates were observed compared to those of the isolates producing a wild-type KPC-2. Avibactam at a fixed concentration of 4 µg/ml restored the activity of all β-lactams tested for the recombinant strains. The heterogenous population of wild-type and mutated and the reversibility of the genotypes observed suggest a significant challenge for managing KPC-producing isolates that develop ceftazidime-avibactam resistance during therapy. The development of ceftazidime-avibactam resistance among KPC-producing isolates during treatment with this agent has been reported. Usually isolates that become resistant have a mutated gene that confers resistance to ceftazidime-avibactam and susceptibility to meropenem. We report a isolate that developed ceftazidime-avibactam resistance due to mutations within the coding region of the Ω-loop previously reported; however, in this case, only 11% of the whole-genome sequencing reads had mutations, making this alteration difficult to detect and the treatment of these isolates more challenging. In addition to , the initial and the follow-up patient isolates displayed hyperexpression of the AcrAB-TolC efflux system and disruption of the outer membrane protein (OMP) OmpF, which contribute to carbapenem resistance. Experiments p