Analysis Of Genotype And Clonal Relatedness Of NDM-1-producing Enterobacteriaceae Stains In A Hospital Of Shandong Province,China

Objective: To explore the molecular epidemiology of the clinical carbapenem-resistant enterobacteriaceae strains isolating from patients in a hospital of Shandong Province and provide guidance for hospital infection control and prevention.Methods: Three Klebsiella pneumoniae stains and 16 Providencia rettgeri isolates were collected from a hospital in Shandong provience. The identification and antibiotic susceptibilities tests of carbapenem-resistant Klebsiella pneumoniae strains were done by using VITEK-2 compact system. The modified Hodge test（MHT） and MBL E-tests were done to detect putative carbapenemase producers, which were then further characterized by PCR and sequencing to detect resistance genes. Clonal relatedness was assessed by pulsed-field gel electrophoresis（PFGE） and multilocus sequence typing（MLST）. Conjugation experiments and Southern blot hybridization were performed to determine the transferability of Plasmids. The incompatibility groups of mobile plasmids were determined by the PCR-based replicon typing（PBRT） method.Results: Modified Hodge tests and MBL E-tests of 19 strains were all positive. Three Klebsiella pneumoniae isolates were highly resistant to most of tested antibiotics including carbapenems, and harbored bla_NDM-1,bla TEM-1,fos A3,oqx A and oqx B genes.Out of 16 Providencia rettgeri isolatess,seven harbored bla_NDM-1, bla TEM-116 and aac（6 ’）- I b genes,and others carried bla_NDM-1, bla TEM-116 genes. The three isolates of NDM-1 producing K.pneumoniae are categorized into one PFGE types, and all belong to ST22. Out of 16 Providencia rettgeri strains,11 are categorized into one type and others are categorized into being closely related strains. PCR-based replicon typing and Southern hybridization analysis revealed that plasmids that contained the NDM-1 gene in three K.pneumoniae stains and 7 Providencia rettgeri isolates were successfully transferred to E. coli J53 AziR by conjugation and that bla_NDM-1 genes were located on a 220 kb Inc A/C plasmid in K.pneumoniae stains and on a 130 kb Inc A/C plasmid in Providencia rettgeri isolates,respectively.Conclusion: The results of our study demonstrate that this is the first report of the coexistence of bla NDM-1,bla TEM-1 and fos A3 gens in a ST22 K.pneumoniae in worldwide and that there is no outbreak report of Providencia rettgeri carrying bla_NDM-1 and blaTEM-116 genes as well. Therefore there is an urgent need to strengthen the surveillance of carbapenem-resistant enterobacteriaceae.