Inactivation of the oprD porin gene by a novel insertion sequence ISPa195 associated with large deletion in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.

Objectives: Alteration of the porin-encoding gene oprD by insertion sequences (ISs) is one mechanism conferring carbapenem resistance in Pseudomonas aeruginosa. Here we describe a carbapenem-resistant clinical P. aeruginosa isolate 36-989 harbouring a novel IS (ISPa195) in oprD.

Methods: Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the broth microdilution method. Carbapenemase activity was assessed using a MALDI-TOF/MS-based assay of meropenem hydrolysis. Efflux-dependent carbapenem resistance was evaluated using an assay with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The oprD gene and IS sequence were analysed by the Sanger method. Whole-genome sequencing was performed on an Illumina HiSeq 2500 platform.

Results: Antimicrobial susceptibility testing demonstrated that P. aeruginosa 36-989 was resistant to imipenem (MIC=32mg/L) and meropenem (MIC=16mg/L). No carbapenemase activity was detected, however an efflux-mediated component of carbapenem resistance was revealed. A new IS element (ISPa195) was found in the oprD gene of P. aeruginosa 36-989. ISPa195 was 1190bp in length, belonging to the IS3 family, and contained two open reading frames that overlapped through a ribosomal slippage to translate the full-size transposase enzyme. There was an IS-associated 284-bp deletion in the oprD gene; no direct repeats at flanking regions of the IS were detected.

Conclusion: The absence of direct repeats at flanking regions in combination with the IS-associated deletion distinguished ISPa195 from other ISs previously detected in oprD. Carbapenem resistance in P. aeruginosa 36-989 was conferred by a combination of oprD alteration and carbapenem efflux.