High-resolution long-read sequencing maps genomic Island-associated gene duplication in South African carbapenem non-susceptible Pseudomonas aeruginosa.

INTRODUCTION: Carbapenem non-susceptible Pseudomonas aeruginosa is a major global health threat driven by the spread of high-risk lineages, but the genomic architecture of resistance evolution is often unresolved by short-read sequencing.

METHODS: Here, we used long-read whole-genome sequencing to characterize 20 clinical P. aeruginosa isolates collected sporadically during routine clinical care as part of the ERACE-PA surveillance programme from a single tertiary hospital in Cape Town, South Africa, between December 2015 and March 2016.

RESULTS: Of these isolates, 18 (90%) were resistant or intermediate resistant to imipenem and/or meropenem. Resistance determinants, mobile genetic elements, and virulence factors were strongly lineage structured. In the high-risk ST233 lineage, resistance was marked by extensive duplication of antimicrobial resistance genes across non-contiguous chromosomal loci. Genes including dfrB5, floR2, tet(G), arr-5, and sul1 occurred in up to three copies (sul1) or two copies (dfrB5, floR2, tet(G), arr-5) across all seven ST233 isolates, distributed on distinct genomic islands, revealing a dispersed amplification architecture that is invisible to short-read approaches because repetitive IS elements and integron boundaries flanking each copy preclude unambiguous mapping and phase-resolved assembly. These loci were linked by shared insertion sequences, class 1 integrons, and transposon-associated segments, consistent with modular transposition and co-integration driving intrachromosomal spread. In contrast, ST273 showed a more plasmid-associated resistance strategy, whereas ST235 displayed more limited duplication despite carrying related mobile resistance modules. Virulence repertoires also differed by lineage: ST233 and ST260 were predominantly ExoS positive, whereas ST273 was ExoU positive.

DISCUSSION: Together, these findings show that carbapenem non-susceptible in South African P. aeruginosa is shaped not only by gene acquisition but also by genomic structural redistribution and duplication across separate chromosomal sites, highlighting the value of long-read genomics for resolving genomic structural resistance mechanisms in P. aeruginosa surveillance.