Genomic and Antimicrobial Resistance Landscape of Pseudomonas aeruginosa Clinical Isolates from Pakistan

Abstract

Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the resistance mechanism in low and middle-income countries is limited. This study establishes the genotypic mechanisms of β-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 draft genomes were assembled from Illumina shotgun sequencing data. The β-lactam resistance was highest, with 46% of isolates resistant to piperacillin-tazobactam, 42% to cefepime, 48% to ceftolozane-tazobactam, and 65% to at least one carbapenem. 22% of isolates were resistant to all β-lactams tested. Most of these isolates were also resistant to ciprofloxacin (60%) and levofloxacin (66%). WGS revealed that carbapenem resistance was largely mediated by acquisition of metallo-β-lactamases (MBLs) or extended spectrum β-lactamases (ESBLs) in the blaGES, blaVIM, and blaNDM 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 new STs (ST3493, ST3494, ST3472, ST3489, ST3491 and ST3492) discovered in this cohort. The different genotypic features of the new STs revealed the adaptation of these new clones to a variety of infections by various mutations in genes affecting antimicrobial resistance, quorum sensing and biofilm formation. Close monitoring of antibiotic-resistant pathogens and surveillance mechanisms need to be adopted to reduce the further spread of these antibiotic resistant pathogens in the hospital settings of Pakistan. We believe that these new ST strains can be Abstract xvii used as reference strains for future comparative analysis of isolates belonging to the same STs. Moreover, the comparison of the inhibition zone sizes from AST results revealed that acquisition of multiple resistance mechanisms (porin gene mutations and horizontally acquired β-lactamases) 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 multi-drug resistant P. aeruginosa infections. A comprehensive strategy was also devised to design a B- and T-cell multi-epitope vaccine against P. aeruginosa using a subtractive proteomics and immuno-informatics approach. A stable structure of the designed construct was obtained through molecular dynamic simulations. The designed B- and T-cell multi-epitope vaccine candidate was predicted immunogenic in nature and showed strong interactions with TLR-4. Immune simulation predicted high-level production of B- and T-cell population and maximal expression was ensured in E. coli strain K12. The designed vaccine could be cloned in pET28a vector using designed primers and probably it could potentially provide both humoral and cellular immunity against P. aeruginosa in experimental studies.