Abstract:

Objective To investigate the resistance mechanism of Klebsiella pneumoniae towards carbapenem. Methods Carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from clinical samples between January, 2010 and December, 2013 were collected and identified by the automated microbial analysis system Vitek-2 Compact. Combined with disk diffusion method (K-B method), drug sensitivity test was conducted. The identification results were verified by MicroflexTM MALDI-TOF MS. Modified Hodge test was performed to screen the phenotype of carbapenemase. Carbapenemase genes such as blaKPC, blaGES, blaIMP, blaVIM, blaNDM-1, and blaOXA-48 and AmpC β-lactamases genes were detected by PCR and DNA sequencing was performed for positive amplified products. SDS-PAGE was adopted to detect the expressions of outer membrane proteins Ompk35 and Ompk36 and the plasmid conjugation experiment was conducted for strains carrying cabapenemase genes. Results For 75 CRKP strains, 71 of them were positive towards modified Hodge test. Among 71 strains, 69 of them carried blaKPC-2 gene with abnormal expression (68 strains) or no expression (1 strain) of Ompk36, 1 strain carried blaIMP-4 gene with abnormal expressions of Ompk35 and Ompk36, and 1 strain carried blaNDM-1 and blaCIT-1 genes with abnormal expressions of Ompk35 and Ompk36. For 4 strains that were negative towards modified Hodge test, 3 of them carried DHA-1 gene with abnormal expression (2 strains) or no expression (1 strain) of Ompk36, and 1 strain did not carry any drug resistant genes with no expression of Ompk36. The plasmid conjugation experiment of only 4 carbapenemase-producing strains was successfully. Conclusion The major resistance mechanism of Klebsiella pneumoniae isolated from clinical samples towards carbapenem is the production of KPC-2 carbapenemase and abnormal expressions of outer membrane proteins. Measures for preventing and controlling the nosocomial infection should be enhanced to avoid the dissemination of these strains.

Key words: Klebsiella pneumonia; , carbapenem, antibiotic resistance mechanism, carbapenemase, AmpC β-lactamases, outer membrane porin