Background Carbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa(P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacter...Background Carbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa(P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacteria. The purpose of this study was to investigate the molecular epidemiology and carbapenem resistant mechanisms of P. aeruginosa isolated from a surgical intensive care unit(SICU) in China. Methods The molecular typing was analyzed by REP-PCR. Enzyme activity was measured with a 260 nm wavelength spectrophotometer. The levels of outer membrane proteins OprD and OprN were measured by Western blotting. The levels of mexA gene transcriptional expression were measured by quantitative real-time PCR. The metallo-beta-lactamase genes IMP, VIM, SPM, GES, and GIM were amplified by PCR. DNA fragments were sequenced by an automated ABI PRISM 3700.Results Forty-two strains resistant to carbapenems isolated from a SICU were analyzed. REP-PCR revealed 34 belonging to type A, a predominant strain in this SICU. But we did not find metallo-beta-lactamases IMP, VIM, SPM, GES, or GIM genes by PCR. With a three-dimensional extract test, we found 34 strains producing high levels of AmpC enzymes. We also observed the activity of beta-lactamases enzymes in the imipenem resistant group, which was statistically different from the sensitive group. Western blotting revealed that 23 strains showed loss of OprD, 18 strains had decreased OprD expression, and 14 strains expressed OprN. We discovered 27 strains that overexpressed mexA by quantitative real-time PCR, and the resistance rate to meropenem was statistically different between the overexpressing group and the lowexpressing group. Nucleotide sequences and deduced amino acid sequence analysis revealed that eight strains carried mutations in the mexR gene operon down regulating MexAB-OprM. The nucleotide sequences of mexR genes from PA36, PA41 and PA48 were submitted to the Genebank with accession numbers of AY899299, AY899300, and AY899301. Conclusions There was a predominant strain in the SICU of our hospital. Imipenem resistance is mainly mediated by OprD deficiency or loss, and high activity AmpC enzymes. Overexpression of MexAB-OprM is one of the mechanisms of meropenem resistance, which are partly upregulated by mutations in the mexR gene. The expression of MexEF-OprN also plays an important role in the carbapenem resistance.展开更多
基金This work was supported by grants from the National ＂863＂ High Technology Research and Development Project of China （No. 2002AA2Z341D）, National Natural Science Foundation of China （No. 30940080）, and Foundation of China-Japan Friendship Hospital, Ministry of Health （No. 2010-ms-54）.
文摘Background Carbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa(P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacteria. The purpose of this study was to investigate the molecular epidemiology and carbapenem resistant mechanisms of P. aeruginosa isolated from a surgical intensive care unit(SICU) in China. Methods The molecular typing was analyzed by REP-PCR. Enzyme activity was measured with a 260 nm wavelength spectrophotometer. The levels of outer membrane proteins OprD and OprN were measured by Western blotting. The levels of mexA gene transcriptional expression were measured by quantitative real-time PCR. The metallo-beta-lactamase genes IMP, VIM, SPM, GES, and GIM were amplified by PCR. DNA fragments were sequenced by an automated ABI PRISM 3700.Results Forty-two strains resistant to carbapenems isolated from a SICU were analyzed. REP-PCR revealed 34 belonging to type A, a predominant strain in this SICU. But we did not find metallo-beta-lactamases IMP, VIM, SPM, GES, or GIM genes by PCR. With a three-dimensional extract test, we found 34 strains producing high levels of AmpC enzymes. We also observed the activity of beta-lactamases enzymes in the imipenem resistant group, which was statistically different from the sensitive group. Western blotting revealed that 23 strains showed loss of OprD, 18 strains had decreased OprD expression, and 14 strains expressed OprN. We discovered 27 strains that overexpressed mexA by quantitative real-time PCR, and the resistance rate to meropenem was statistically different between the overexpressing group and the lowexpressing group. Nucleotide sequences and deduced amino acid sequence analysis revealed that eight strains carried mutations in the mexR gene operon down regulating MexAB-OprM. The nucleotide sequences of mexR genes from PA36, PA41 and PA48 were submitted to the Genebank with accession numbers of AY899299, AY899300, and AY899301. Conclusions There was a predominant strain in the SICU of our hospital. Imipenem resistance is mainly mediated by OprD deficiency or loss, and high activity AmpC enzymes. Overexpression of MexAB-OprM is one of the mechanisms of meropenem resistance, which are partly upregulated by mutations in the mexR gene. The expression of MexEF-OprN also plays an important role in the carbapenem resistance.
