Molecular epidemiology of carbapenem-resistant enterobacteriaceae

Dissemination of Carbapenem-Resistant Enterobacteriaceae (CRE) has raised a new challenge for health organizations all over the world. Acquisition of carbapenemase genes is the most worrisome among these CRE isolates. This study was constructed to investigate the dissemination of CRE isolates in Hong Kong and also to characterize plasmids harboring carbapenemase genes. CRE isolates were collected from public hospitals in Hong Kong from August 2006 to June 2012. Antimicrobial susceptibility of all CRE isolates was tested using disc diffusion method. Screening of carbapenemase genes (blaNDM , blaKPC, blaIMP, blaVIM and blaOXA-48) and ESBL genes (blaCTX-M and blaSHV) were also performed. Clonal relatedness was studied by multi-locus sequence typing. Characterization of plasmids was carried out by conjugation, S1-PFGE, hybridization and plasmid replicon typing. A total of 69 CRE isolates were collected including 50 K. pneumoniae, 15 E. coli, 2 E. cloacae, 1 E. aerogenes and 1 C. freundii. Eighteen carbapenemase producing Enterobacteriaceae were isolated from different patients with travel histories among these 69 isolates. Four K. pneumoniae were detected to carry blaKPC genes on different transferable plasmids as follows: 50 kb, IncX3 plasmid (ST258); 70 kb, un-typeable plasmid (ST258); 130 kb, un-typeable plasmid (ST11) and 140 kb, un-typeable plasmid (ST11). blaIMP genes were also detected in four CRE isolates to be harbored by different plasmids or located on chromosome: ST11 K. pneumoniae (50 kb, IncN), ST1 K. pneumoniae (150 kb, IncA/C), E. cloacae (130 kb, IncN-L/M) and ST899 K. pneumoniae (chromosomal located). NDM-1 (New Delhi Metallo enzyme) producing E. coli (n = 5), K. pneumoniae (n = 2), E. aerogenes (n = 1), E. cloacae (n = 1) and C. freundii (n =1) were also found in this study. Eight of them were isolated from patients travelled to different provinces of China blaNDM-1 was found to be carried by transferable plasmids in all ten isolates: IncX3 (n = 7, 50 kb), IncL/M (n = 1, 88 kb), IncA/C2 (n = 1, 140 kb) and FIIY- FIBS (n = 1, 110kb). Six of the seven IncX3 plasmids showed identical digestion profile while the other one only had two bands different from others using Restriction fragment length polymorphism (RFLP) analysis. An IncX3 plasmid pNDM-HN380 from a K. pneumoniae strain CRE380 was completely sequenced using Genome Sequencer FLX (Roche, USA). pNDM-HN380 was a 54,035 bp circular plasmid with 52 open reading frames (ORFs). The backbone of pNDM-HN380 was identical to those previous described IncX plasmids pIncX-SHV (accession number JN247852) and pEC14_35 (accession number JN935899). The blaNDM-1 gene was carried on an ISAba125 and IS26 flanked transposon-like element. And this element except IS26 and an interrupted ISAba125 was found to be identical to pNDM-BJ01 (accession number JQ001791). In conclusion, this is the first we describe a blaNDM-1 carrying IncX3 plasmid. This IncX3 plasmid was found to be predominant in the dissemination of blaNDM-1 in China. Future study of the nationwide dissemination of carbapenemase genes and also the novel IncX3 plasmids is needed.published_or_final_versionMicrobiologyMasterMaster of Philosoph