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

• Published in: Frontiers in cellular and infection microbiology Vol. 12; p. 981792
• Main Authors: García, Patricia, Brito, Bárbara, Alcalde-Rico, Manuel, Munita, José M., Martínez, Jose R. W., Olivares-Pacheco, Jorge, Quiroz, Valeria, Wozniak, Aniela
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 02-09-2022
• Subjects: blaKPC-2 gene; ceftazidime/avibactam resistance; Cellular and Infection Microbiology; D179Y substitution; pseudomonas aeruginosa; Tn4401b transposon
• ISSN: 2235-2988; 2235-2988
• DOI: 10.3389/fcimb.2022.981792

Ceftazidime/Avibactam (CAZ/AVI) is frequently used to treat KPC-producing
Pseudomonas aeruginosa
(KPC-PA) and
Enterobacterales
. CAZ/AVI resistance is driven by several mechanisms. In
P. aeruginosa
this mainly occurs through alteration of AmpC, porins, and/or efflux pump overexpression, whereas in
Enterobacterales
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
in vivo
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
bla
KPC
-containing Tn
4401b
transposon. However, while pPA-1 carried two copies of
bla
KPC-2
, pPA-2 had one copy of
bla
KPC-2
and one copy of
bla
KPC-33
, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the “see-saw” effect. The
bla
KPC-33
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
bla
KPC-2
-Tn
4401b
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.