Molecular study of carbapenemase genes in clinical isolates of Enterobacteriaceae resistant to carbapenems and determining their clonal relationship using pulsed-field gel electrophoresis.

Abstract

Enterobacteriaceae is a large family of Gram-negative bacteria that are considered as normal gut flora. They are the most common human pathogens. The main objective of this study was to investigate the carbapenemase genes in clinical isolates of Enterobacteriaceae resistant to carbapenem antibiotics and determine their clonal relationship using pulsed-field gel electrophoresis (PFGE). In the present study, bacteria were isolated and identified via conventional biochemical tests and API 20NE. Antibiotic susceptibility was evaluated by using the disc diffusion method and MIC was carried out using the E-test. Phenotypic determination of carbapenemases was performed by employing a modified Hodge test (MHT). Carbapenemase genes including IMP, VIM, KPC, NDM and OXA-48 were amplified by PCR. The relationships between their clonal types with l restriction enzyme were examined using PFGE. Out of 40 isolates that were resistant or moderately susceptible to carbapenem antibiotics, 29 (72.5 %) strains were positive for carbapenem enzymes phenotypically. Moreover, six isolates contained carbapenemase genes including IMP, VIM, NDM and OXA-48, but the KPC gene was not found in any of the isolates. PFGE results showed that E. coli strains in our area were clustered into eight pulsotypes (A-H), Klebsiella spp. isolates five pulsotypes (A-E) and Proteus spp. had two pulsotypes (A, B). The high resistance to antimicrobial agents in the A, B and F pulsotypes was attributed to E. coli clinical isolates. Our results could reflect some hospital multidrug-resistant strains in nosocomial infections. The widespread emergence of carbapenem-resistant isolates has caused increasing concern in recent years. Therefore, specific strategies should be designed and evaluated for the control of resistant strains.