Genomic Surveillance of Clinical Pseudomonas aeruginosa Isolates Reveals an Additive Effect of Carbapenemase Production on Carbapenem Resistance

Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the mechanisms of such resistance in low- and middle-income countries is limited. This study establishes the genotypic mechanisms of b-lactam resistance by whole-genome sequencing (WGS) in 142 P. aeruginosa clinical isolates recovered from three hospitals in Islamabad and Rawalpindi, Pakistan between 2016 and 2017. Isolates were subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion, and their genomes were assembled from Illumina sequencing data. b-lactam resistance was high, with 46% of isolates resistant to piperacillin-tazobactam, 42% to cefepime, 48% to ceftolozane-tazobactam, and 65% to at least one carbapenem. Twenty-two percent of isolates were resistant to all b-lactams tested. WGS revealed that carbapenem resistance was associated with the acquisition of metallo-b-lactamases (MBLs) or extended-spectrum b-lactamases (ESBLs) in the bla_GES, bla_VIM, and bla_NDM families, and mutations in the porin gene oprD. These resistance determinants were found in globally distributed lineages, including ST235 and ST664, as well as multiple novel STs which have been described in a separate investigation. Analysis of AST results revealed that acquisition of MBLs/ESBLs on top of porin mutations had an additive effect on imipenem resistance, suggesting that there is a selective benefit for clinical isolates to encode multiple resistance determinants to the same drugs. The strong association of these resistance determinants with phylogenetic background displays the utility of WGS for monitoring carbapenem resistance in P. aeruginosa, while the presence of these determinants throughout the phylogenetic tree shows that knowledge of the local epidemiology is crucial for guiding potential treatment of multidrug-resistant P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is associated with serious infections, and treatment can be challenging. Because of this, carbapenems and b-lactam/b-lactamase inhibitor combinations have become critical tools in treating multidrug-resistant (MDR) P. aeruginosa infections, but increasing resistance threatens their efficacy. Here, we used WGS to study the genotypic and phylogenomic patterns of 142 P. aeruginosa isolates from the Potohar region of Pakistan. We sequenced both MDR and antimicrobial susceptible isolates and found that while genotypic and phenotypic patterns of antibiotic resistance correlated with phylogenomic background, populations of MDR P. aeruginosa were found in all major phylogroups. We also found that isolates possessing multiple resistance mechanisms had significantly higher levels of imipenem resistance compared to the isolates with a single resistance mechanism. This study demonstrates the utility of WGS for monitoring patterns of antibiotic resistance in P. aeruginosa and potentially guiding treatment choices based on the local spread of b-lactamase genes.