Tandem Repeat of blaNDM-1 and Clonal Dissemination of a fosA3 and blaKPC-2 Co-Carrying lncR-F33: A-: B- Plasmid in Klebsiella pneumoniae Isolates Collected in a Southwest Hospital in China, 2010-2013

Introduction: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has been widespread in coastal cities of eastern China since 2009. However, how CRKP spreads and evolves in southwest China is unclear. Aim: We investigated the genetic characteristics and dissemination mechanisms of carbapenemase genes in forty-one non-repetitive CRKP isolates collected from a southwest hospital, Kunming, Yunnan, during 2010-2013. Methodology: Drug susceptibilities were analyzed by using VITEK 2 compact system. Genetic relationships were ascertained based on multilocus sequence typing (MLST) and Pulsed-field gel electrophoresis (PFGE) analysis. Genetic backgrounds of bla(KPC-2) and bla(NDM-1) were revealed by DNA walking and high-throughput sequencing. Results: All isolates were highly resistant to common antibiotics except for tigecycline. In total, 34 bla(KPC-2), 3 bla(NDM-1), 1 bla(IMP-4) and 3 bla(IMP-26) genes were identified and KP67 plasmid 1 co-harbored bla(NDM-1) and bla(IMP-26). Five sequence types, namely ST11, ST290, ST340, ST395 and ST437, were recognized by MLST. Surprisingly, bla(KPC-2) was only detected in ST11 strains. We described a clonal dissemination of fosA3-positive IncR-IncF33:A-:B- multireplicon plasmid carrying the gene cassettes IS 26-Delta Tn 3-ISKpn27-bla(KPC-2)-Delta ISKpn6-korC-klcA-Delta repB-Tn 1721 in all ST11 isolates. Three bla(NDM-1) positive isolates belonged to three different ST types and their bla(NDM-1) genetic backgrounds were also distinct. Interestingly, the flanking regions of bla(NDM-1) in KP67 and KP72 were duplicated into one to five copies in a form of tandem repeat by the transposition of IS 91 like element. The bla(NDM-1) of KP82 was carried on a common IncX3 plasmid. Conclusion: This study described the early epidemiological characteristics of bla(KPC-2)/bla(NDM-1)-carrying CRKP, and reported a new tandem repeat pattern of bla(NDM-1) cluster in Yunnan. These findings extend our knowledge on the carbapenemase gene evolutions.