Contributions of IMP-10 metallo-beta-lactamase, the outer membrane barrier and the MexAB-OprM efflux system to high-level carbapenem resistance in Pseudomonas aeruginosa.
BACKGROUND
The emergence of carbapenem-hydrolyzing metallo-beta-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 microg/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 microM, 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.


CONCLUSIONS
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.
Zhao
Wei-Hua
WH
Department of Microbiology and Immunology, Showa University School of Medicine, Tokyo, Japan. whzhao@med.showa-u.ac.jp
Hu
Zhuting
Z
Chen
Gelin
G
Ito
Ribu
R
Hu
Zhi-Qing
ZQ
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D015780:Carbapenems / Q000494:pharmacology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial; D004492:Edetic Acid / Q000494:pharmacology; D005838:Genotype; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D010641:Phenotype; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013864:Thioglycolates / Q000494:pharmacology; D001618:beta-Lactamases / Q000096:biosynthesis*
D001425:Bacterial Outer Membrane Proteins; D015780:Carbapenems; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; C118597:MexB protein, Pseudomonas aeruginosa; C402263:OprM protein, Pseudomonas aeruginosa; D013864:Thioglycolates; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C017487:2-mercaptoacetate; D004492:Edetic Acid; C466680:metallo-beta-lactamase, IMP-10; D001618:beta-Lactamases