Interplay between mutational and horizontally acquired resistance mechanisms and its association with carbapenem resistance amongst extensively drug-resistant Pseudomonas aeruginosa (XDR-PA)

Between 2003 and 2009, the prevalence of extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) increased significantly in northern Taiwan from 1.0% to 2.1%. Molecular methods were used to investigate the genetic relatedness and carbapenem resistance mechanisms of a collection of 203 non-repetitive XDR-PA isolates available for study. Using pulsed-field gel electrophoresis (PFGE), 52 genotypes were observed; one predominant genotype (pulsotype 1) was found in 57.6% of the isolates. Polymerase chain reaction (PCR), sequencing and quantitative reverse-transcriptase PCR analyses demonstrated that one horizontally acquired mechanism [metallo-β-lactamase (MBL) genes] and two mutational mechanisms (efflux and porins) accounted for the carbapenem resistance. The most predominant horizontally acquired mechanism was carriage of bla(VIM-3), which was found in 61.1% of isolates. Decreased expression of oprD was the most prevalent mutational mechanism and was found in 70.0% of the XDR-PA isolates, whereas overexpression of mexA was found in 27.6% of the isolates. The highlight of this study was the discovery of statistically significant relationships between certain horizontally acquired and mutational resistance mechanisms and their contribution to carbapenem susceptibility. MBL-producers expressed significantly lower MexAB and higher OprD than non-MBL-producers. Amongst isolates without an acquired β-lactamase gene, oprD expression was significantly reduced, whilst expression of efflux pumps was increased. Reduced OprD expression alone or the production of VIM-type MBLs showed similar contributions to a low to intermediate MIC(50) (minimum inhibitory concentration for 50% of the organisms) for carbapenems. Isolates with reduced OprD expression that simultaneously harboured bla(VIM) exhibited high levels of resistance to carbapenems, which implied that these two mechanisms had a synergistic effect on the MICs.