AUTHOR=García Patricia , Brito Bárbara , Alcalde-Rico Manuel , Munita José M. , Martínez Jose R. W. , Olivares-Pacheco Jorge , Quiroz Valeria , Wozniak Aniela 

TITLE=Acquisition of resistance to ceftazidime-avibactam during infection treatment in Pseudomonas aeruginosa through D179Y mutation in one of two blaKPC-2 gene copies without losing carbapenem resistance

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2022.981792

DOI=10.3389/fcimb.2022.981792

ISSN=2235-2988

ABSTRACT=<p>Ceftazidime/Avibactam (CAZ/AVI) is frequently used to treat KPC-producing <italic>Pseudomonas aeruginosa</italic> (KPC-PA) and <italic>Enterobacterales</italic>. CAZ/AVI resistance is driven by several mechanisms. In <italic>P. aeruginosa</italic> this mainly occurs through alteration of AmpC, porins, and/or efflux pump overexpression, whereas in <italic>Enterobacterales</italic> it frequently occurs through D179Y substitution in the active site of KPC enzyme. This aminoacid change abolishes AVI binding to the KPC active site, hence inhibition is impaired. However, this substitution also decreases KPC-mediated resistance to carbapenems (“see-saw” effect). The goal of this work was to characterize the <italic>in vivo</italic> acquisition of CAZ/AVI resistance through D179Y substitution in a KPC-PA isolated from a hospitalized patient after CAZ/AVI treatment. Two KPC-PA isolates were obtained. The first isolate, PA-1, was obtained before CAZ/AVI treatment and was susceptible to CAZ/AVI. The second isolate, PA-2, was obtained after CAZ/AVI treatment and exhibited high-level CAZ/AVI resistance. Characterization of isolates PA-1 and PA-2 was performed through short and long-read whole genome sequencing analysis. The hybrid assembly showed that PA-1 and PA-2A had a single plasmid of 54,030 bp, named pPA-1 and pPA-2 respectively. Each plasmid harbored two copies of the <italic>bla</italic><sub>KPC</sub>-containing Tn<italic>4401b</italic> transposon. However, while pPA-1 carried two copies of <italic>bla</italic><sub>KPC-2</sub>, pPA-2 had one copy of <italic>bla</italic><sub>KPC-2</sub> and one copy of <italic>bla</italic><sub>KPC-33</sub>, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the “see-saw” effect. The <italic>bla</italic><sub>KPC-33</sub> allele was detected only through hybrid assembly using a long-read-first approach. The present work describes a KPC-PA isolate harboring a plasmid-borne CAZ/AVI resistance mechanism based on two copies of <italic>bla</italic><sub>KPC-2</sub>-Tn<italic>4401b</italic> and D179Y mutation in one of them, that is not associated with loss of resistance to carbapenems. These findings highlight the usefulness of a fine-tuned combined analysis of short and long-read data to detect similar emerging resistance mechanisms.</p>