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.

Evaluation of phenotypic tests for the detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa in a low prevalence country.

OBJECTIVES: To evaluate four phenotypic tests for the detection of metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa in a low MBL prevalence setting. METHODS: Sixty clinical isolates of P. aeruginosa resistant to imipenem and/or meropenem and seven MBL-positive control strains were examined by: (i) MBL Etest; (ii) combined imipenem discs supplemented with EDTA (IPM-EDTA); (iii) beta-lactam discs on dipicolinic acid plates (DF-DIPI); and (iv) the Cica-beta test. Spectrophotometric analysis of crude cell extracts for imipenem hydrolysis along with consensus PCRs for bla(VIM) and bla(IMP) was used as reference methods. RESULTS: Two clinical isolates (3%) were MBL-positive. The MBL Etest and IPM-EDTA test scored positive for all MBL-positive isolates, but showed specificities of 86% and 91%, and positive predictive values (PPVs) of only 20% and 29%, respectively. Adding resistance to ceftazidime (MIC >8 mg/L) as a criterion for MBL testing would reduce the number of isolates to be screened by 50% and increase the PPVs of the MBL Etest and IMP-EDTA test to 29% and 40%, respectively. The Cica-beta test correctly identified all MBL-negative isolates, but misidentified one MBL-positive clinical isolate as an extended-spectrum beta-lactamase (ESBL)-producer and one as inconclusive (producing multiple beta-lactamases). No reliable breakpoints could be defined for the DF-DIPI test due to overlapping inhibition zone diameters for MBL-positive and -negative isolates. CONCLUSIONS: None of the phenotypic tests were optimal due to low sensitivity or specificity, resulting in low PPVs. Including ceftazidime resistance to the MBL-screening criteria would significantly improve the performance of the MBL Etest and IPM-EDTA disc test.