Contributions of IMP-10 Metallo-β-Lactamase, the Outer Membrane Barrier and the MexAB-OprM Efflux System to High-Level Carbapenem Resistance in Pseudomonas aeruginosa

Abstract

Background: The emergence of carbapenem-hydrolyzing metallo-β-lactamases (MBLs) is a serious threat to clinical medication of carbapenems. We evaluated the contributions of IMP-10 MBL, the outer membrane barrier and the MexAB-OprM efflux system to high-level carbapenem resistance in Pseudomonas aeruginosa clinical isolates. Methods: MBL-producing strains were screened by ceftazidime and mercaptoacetic acid double-disk synergy testing. Genes were determined by PCR analysis and DNA sequencing. IMP-10 MBL activity was assayed using nitrocefin as a substrate. Results: The bla_IMP-10+ strains showed high-level resistance to carbapenems, with minimum inhibitory concentrations of 512 to >4,096 μg/ml. The minimum inhibitory concentrations were decreased by the inhibitors of MBLs, sodium mercaptoacetate (SMA) and ethylenediaminetetraacetic acid (EDTA), 16 and 64 fold, respectively. However, against the activity of the prepared IMP-10 MBL, the 50% inhibitory concentrations of SMA and EDTA were 5.5 and 211.6 μM, respectively, indicating that SMA was much more effective than EDTA in blocking the enzyme activity. The contradictory results may be explained by the additional effect of EDTA as an outer membrane permeabilizer because the susceptibility of the bla_IMP– strains to carbapenems was increased 2 fold by EDTA, but not by SMA. In the presence of carbonyl cyanide m-chlorophenylhydrazone, a proton motive force inhibitor, the susceptibility of the P. aeruginosa isolates to carbapenems was increased 1–4 fold. Conclusion: The acquired IMP-10 MBL is a crucial factor in the high-level resistance to carbapenems in P. aeruginosa clinical isolates, while the outer membrane barrier and the MexAB-OprM efflux system play a basic and minor role, respectively.