Distribution of carbapenem resistance mechanisms in Pseudomonas aeruginosa isolates among hospitalised children in Poland: Characterisation of two novel insertion sequences disrupting the oprD gene.

This study aimed to analyse the distribution of carbapenem resistance mechanisms among Pseudomonas aeruginosa clinical isolates. Fifty-five P. aeruginosa isolates, resistant both to imipenem and meropenem, from children hospitalised in 2009-2010 were studied. All strains were genotyped by pulsed-field gel electrophoresis (PFGE). Mutations in the oprD gene and the occurrence of insertion sequences (ISs) were determined by DNA sequencing. Mex efflux systems were determined by analysis using the efflux pump inhibitor Phe-Arg β-naphthylamide. Metallo-β-lactamase (MBL) production was determined with Etest MBL strips and PCR for bla and bla. PFGE show high genetic diversity among the isolates. Mutations inactivating the oprD gene were detected in 44 strains (80%). Frameshift mutations detected in 20 isolates were the most common cause of inactivation of the oprD gene. Point mutations leading to premature stop codons were found in 12 isolates, and various substitutions were found in 6 isolates. Disruption of the coding sequence of oprD by ISs was found in six isolates. Two novel ISs (ISPa51 and ISPa52) were detected. Increased activity of different Mex systems was observed in 27 isolates (49%). Ten isolates simultaneously overexpressed two (n=3) or three (n=7) types of Mex efflux system. Seven (13%) P. aeruginosa strains were found to have minimum inhibitory concentrations (MICs) of >64mg/L both for imipenem and meropenem (two VIM-4, four VIM-2 and one IMP-1). These results show a significant diversity of P. aeruginosa strategies for resistance development. Noteworthy, a variety of ISs were found to disrupt the oprD gene.