Tracking A Hospital Outbreak Of Carbapenem-Resistant Klebsiella Pneumoniae With Whole Genome Sequencing

Objective:Carbapenem-resistant Klebsiella pneumoniae(CRKP)has emerged globally as an important pathogen of hospital infectionof which there are increasing isolate rate year by year,and the dissemination of carbapenemase gene and CRKP clone could lead to the hospital outbreak of resistant pathogens,causing a serious clinical threat.The aim of this study was to track and trace the outbreak of KPC-type carbapenemase-producing K.pneumoniae in a hospital by using the whole genome sequencing(WGS)based on its strong resolution and abundant information.Methods:Retrospective analysis of the K.pneumoniaestrainswere collected from several types of sterile samples from Quzhou People's Hospital,Chinaduring January 1,2012 to December31,2012.The minimum inhibitory concentrations(MICs)of all strains against common antimicrobial agents were determined by K-B and Etest methods;pulsed-field gel electrophoresis(PFGE)was performed for homology analysis;the modified Hodge test and polymerase chain reaction(PCR)were performed to confirm the producing of carbapenemases.S1-PFGE andSouthern hybridizations were used to indicateif the carbapenem resistance genes were located on plasmid or not.WGS was carried out by using the Illumina second-generation high-throughput sequencing platform.Relevant bioinformatics software was chosen to assemble and annotate the genome sequences and their phylogenetic structure was constructed by calculatingthe SNP differences between the outbreak strains,which help us to track the spread of these epidemic strains.Results:A total of 66K.pneumoniaestrains were collected from strile samples from Quzhou People's Hospitalin 2012.Based on the antimicrobial susceptibility testing results and PFGE molecular typing results,12 K.pneumoniaestrains were belonged to the same clone and exhibited high-level resistance against the carbapenems.The modified Hodge test and PCR amplification test of the 12 KP isolates were positive and the genotype was blaKPC-2 Southern hybridization confirmed that the blaKPC gene was located on two types of the different size plasmid(145 kb or 170 kb).SNP differences among 12 outbreak strains partitionedthem into three clades.The differences in the internal strains of different clade ranged from 700 to 2500 SNPs,however,thedifferent SNP numbers of the same clade were no more than 300.All 12 outbreak strains harbored one or more plasmids that encoded several acquired antimicrobial resistance genes such as blaKPC-2?blaCTX-M-65?blaTEM-1.Furthermore,the surrounding genetic structures of blaKPC-2gene in these plasmids are completely identical.Retrospective analysis of clinical information indicated these 12 K.pneumoniaestrains were isolated from 10 patients,some of which were showed the overlap of hospital stay unit or time,which may be the reason of pathogen transmission.Conclusion:Acquiring multidrug resistance from plasmid accelerates the outbreak and dissemination of resistant pathogen in hospital.Although 12 blaKPC-producing K.pneumoniae strains possessed similar PFGE pattern,the SNPs variations of whole genome divided these strains into three clades,suggesting that three independent transmission events led to hospital dissemination of the outbreak strains.