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Alterations of porin, pumps, and penicillin-binding proteins in carbapenem resistant clinical isolates of Pseudomonas aeruginosa.

Carbapenem-resistant clinical isolates of Pseudomonas aeruginosa from Sweden and Norway (n=27) were characterized regarding transcription of genes encoding OprD, efflux pumps MexAB-OprM and MexCD-OprJ, as well as penicillin-binding proteins (PBP) 2 and 3. Quantification of mRNA was performed with real-time RT-PCR. Levels of mRNA in clinical isolates were compared to ATCC 27853 and average transcription levels of four carbapenem-susceptible clinical isolates of P. aeruginosa. Sequencing of oprD, pbp 2, and pbp 3 was performed in selected isolates. An efflux inhibition assay was performed with Phe-Arg-beta-naphtylamide. Most carbapenem-resistant isolates had decreased levels of oprD mRNA. Among six isolates with normal amount of oprD mRNA, half of them had significant OprD sequence alterations. Increased transcription of mexB was observed in 16 of 23 meropenem-resistant isolates, and one isolate showed mexD hyperproduction. Decreased amount of pbp 2 and pbp 3 was found in two and three isolates, respectively. Sequencing of pbp 2 and 3 revealed no amino acid changes potentially leading to conformational changes. In conclusion, OprD changes were the predominant mechanism of high-level carbapenem resistance, and increased transcription of mexB was often present in meropenem-resistant isolates. Reduced transcription of pbp 2 and 3 may contribute to the carbapenem resistance.