Comparative genomics of five different resistance plasmids coexisting in a clinical multi-drug resistant Citrobacter freundii isolate Jing Ouyang,1,2,* Fengjun Sun,1,* Dongsheng Zhou,3 Jiao Feng,3 Zhe Zhan,3 Zhijian Xiong,4 Bo Yang,1 Zhirui Liu,1 Tingbo Li,1 Yigang Tong,3 Peiyuan Xia1 1Department of Pharmacy, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China; 2Department of Pharmacy, Chongqing Public Health Medical Center, Chongqing 400038, China; 3State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 10007, China; 4Outpatient Department of August First Film Studio, Beijing 100161, China *These authors contributed equally to this work Background: Plasmid-mediated multi-drug resistance (MDR) has been widely found in Citrobacter freundii. C. freundii P10159 was isolated from a human case of postoperative urinary tract infection in a Chinese teaching hospital.Methods: The complete nucleotide sequences of five resistance plasmids pP10159-1, pP10159-2, pP10159-3, pP10159-4 and pP10159-5 from C. freundii P10159 were determined through high-throughput genome sequencing, and then compared with related plasmids sequences. Plasmid transfer, CarbaNP test of carbapenemase activity, and bacterial antimicrobial susceptibility test were performed to characterize resistance phenotypes mediated by these plasmids.Results: pP10159-1 carrying blaNDM-1 and pP10159-2 harboring blaIMP-4 plus qnrS1 were almost identical to IncX3 plasmid pNDM-HN380 and IncN1 plasmid pP378-IMP, respectively. The blaKPC-2-carrying plasmids pP10159-3, pHS062105-3 and pECN49-KPC were highly similar to each other, and constituted a novel group of plasmids belonging to an unknown incomparability group. The MDR plasmids pP10159-4 and pP10159-5 had the backbones highly similar to IncHI4 plasmid pNDM-CIT and type 2 IncC plasmid pR55, respectively, but their accessory resistance regions differed from pNDM-CIT and pR55, respectively. The five plasmids from the P10159 isolate contained a total of 24 different genes or gene loci, which contributed to resistance to 13 distinct antibiotic molecules or toxic compounds.Conclusion: This is the first report of co-occurrence of five different resistance plasmids, with determination of their complete sequences. Data presented here provide a deeper insight into co-selection and maintenance of multiple plasmids and an extremely large number of resistance genes in a single bacterial isolate. Keywords: Citrobacter freundii, multi-drug resistance, plasmids, mobile elements