Occurrence of two NDM-1-producing Raoultella ornithinolytica and Enterobacter cloacae in a single patient in China: probable a novel antimicrobial resistance plasmid transfer in vivo by conjugation

Objectives: To identify the general features of acquisition of drug-resistance genes in two multi-drug resistant Enterobacteriaceae strains isolated from a single patient in China. Methods: The whole-plasmid was sequenced by Illumina Hiseq 4000 and Pacbio RSII procedures. The plasmid conjugation transfer experiment were performed by the mating-out assay. Drug-resistance genes were amplified by PCR assay. Results: We identified two New Delhi metallo-β-lactamase type 1(NDM-1)-producing isolates, named Raoultella ornithinolytica B1645-1 and Enterobacter cloacae B1645-2, which shared the same sulfonamide-resistant dihydropteroate synthase sul2 gene and aminoglycoside O-phosphotransferase aph(3'')-Ib gene. A novel antimicrobial resistance plasmid pCYNDM01 was first discovered from the multi-drug resistant R. ornithinolytica B1645-1. Interestingly, plasmid pCYNDM01 carried a Gifsy-2 prophage gene. The blaNDM-1 gene was located on a novel complex class 1 integron with a structure of sul1-qacEΔ1-ΔISAba125-blaNDM-1-blaMBL-trpC-ISCR1-catb8-aacA4-IS1-IS6100-dfrA14-intI1. The carrying the blaNDM-1 gene plasmid pCYNDM01 was transferred to the E. cloacae B1645-2 recipient strain. This 149.44 kb plasmid pCYNDM01 belonged to the IncFII type. Conclusions: A novel antimicrobial resistance plasmid pCYNDM01 was first recovered from a multi-drug resistance R. ornithinolytica B1645-1 isolated from China. The novel complex sul1-type class 1 integron might play an essential role in the mobilization of the blaNDM-1 gene among different enterobacterial species. The occurrence of plasmid pCYNDM01 transfer from R. ornithinolytica to E. cloacae in vitro by conjugation showed that plasmid pCYNDM01 was a self-conjugative plasmid and might cause dissemination of drug-resistance genes within different enterobacterial species from a single patient in vivo by conjugation. The novel variant F-like T4SS of plasmid pCYNDM01 might be as a tool of R. ornithinolytica B1645-1 for resistance genes transfer. The emergence of the two NDM-1-producing Enterobacteriaceae strains should be attracted China attentions and required to prevent its future prevalence.