CHARACTERIZATION OF CARBAPENEMASE-ENCODING GENES IN ENTEROBACTERIACEAE ISOLATES FROM THAI PATIENTS

Abstract

Carbapenem resistance in Enterobacteriaceae has been increasingly reported worldwide. Carbapenemase production is the main mechanism of carbapenem resistance. In this study, carbapenemase-encoding genes in Enterobacteriaceae isolates from Thai patients were characterized. A total of 3,854 Enterobacteriaceae isolates from King Chulalongkorn Memorial Hospital during August 2013 to January 2014 were screened for carbapenemase producers by using MacConkey agar containing ertapenem, cloxacillin and ZnSO4. A total of 104 isolates were positive on screening plates. MICs (minimum inhibitory concentrations) were performed by agar dilution method. Of the 104 isolates, 100 (96.2%) were resistant to ertapenem, 39 (37.5%) to imipenem, 48 (46.2%) to meropenem, 102 (98%) to cefotaxime, 102 (98%) to ceftazidime, 102 (98%) to ceftriaxone and 99 (95.2%) to cefoxitin. The prevalence of carbapenem resistace in Enterobacteriaceae was 2.7%. Carbapenemase genes, including blaIMP, blaVIM, blaGIM, blaSIM, blaSPM, blaAIM, blaDIM, blaBIC, blaKPC, blaOXA-48, blaNDM, were determined by multiplex PCR. Carbapenemase activity was determined by ertapenem modified Hodge test (MHT), boronic acid-based inhibition test and EDTA-meropenem combined-disk test. The detection of ESBL and AmpC phenotypes was performed by combination disk test and cefoxitin-MHT, respectively. There were 19 isolates carrying carbapenemase genes and had carbapenemase activity. The blaIMP was found in one isolate (5.3%) of E. cloacae. The blaOXA-48 was present in one isolate (5.3%) of E. cloacae. The blaNDM was found in two isolates (10.5%) of E. cloacae, two isolates of C. freundii (10.5%), three isolates (15.8%) of E. coli and ten isolates (52.6%) of K. pneumoniae. The majority of carbapenemase producers coproduced CTX-M-15 ESBL (63.1 %). None of carbapenemase producers coproduced AmpC beta-lactamases. DNA sequence analysis revealed that carbapenemase genes were blaNDM-1, blaNDM-4, blaIMP-14a, and blaOXA-48. The transfers of resistance genes by conjugation showed that the blaNDM-1 and blaOXA-48 genes from 15 Enterobactericeae isolates were successfully transferred to E. coli recipient strain. The PFGE patterns demonstrated that E. coli and K. pneumoniae isolates carrying blaNDM and blaCTX-M-15 genes were associated with clonal spread. Southern blot hybridization showed that the plasmid carrying the blaOXA-48 gene was approximately 51 kb in size and the plasmids carrying the blaNDM-1 gene were approximately 24 kb and 159 kb in size. The results showed that NDM-1 was the most common carbapenemase among Enterobacteriaceae isolates. This is the first report of NDM-4 in E. coli from Thailand.