The Molecular Characteristics,Transmission Mechanism And Genetic Environment Of KPC-Producing Klebsiella Pneumoniae Isolates

BackgroundAs the broader application,abusing of carbapenem antibiotics is becoming increasingly serious problem,which followed by the emergence of carbapenem-resistant Enterobacteriaceae(CRE).Data show that CRE strains are widely distributed in humans,animals,and environment,and have been reported in many countries around the world.Further research shows that among CRE strains isolated in China,the United States and many European countries,carbapenem-resistant Klebsiella pneumoniae(CRKP)accounts for 60%-90%of all CRE strains,causing up to 40%-50%of clinical mortality.In 2016,the World Health Organization listed CRKP isolates as a priority concern.Carbapenemase-producing is the main resistance mechanism of Klebsiella pneumoniae to carbapenem antibiotics.Among them,KPC enzyme is the most common.KPC was first detected in the United States in 1996,after that KPC-producing Klebsiella pneumoniae(KPC-Kp)strains broke out in the United States.Currently,KPC-Kp strains have been found in many countries around the world,including Argentina,Brazil,Colombia,China,Greece,Israel,Italy,Poland,France and so on.There are multiple mutants of KPC enzyme,of which KPC-2 and KPC-3 are the most common.It is worth noting that KPC-Kp strains have multiple ST types,but the worldwide spread is limited to the CC258 clone complex,which include ST258,ST11,ST340 and ST512.Among them,ST258 is the most common type in North America,Latin America and Europe.In Asia especially in China,ST 11 type is the main clonal type,accounting for 60%of all CRKP strains.Previous studies showed that the pandemic of KPC enzyme was due to the clonal spread of the CC258 complex strains.Also,there were studies showed that blaKPC can be transferred horizontally between strains by means of plasmids,movable genetic elements.Moreover,blaKPC has been found in many different types of plasmid,including IncF,IncI2,IncX,IncA/C,IncR and ColE1.These complex modes of transmission make it difficult to control the spread of KPC-Kp strains.At present,the rapid development and application of second-and third-generation sequencing technologies and bioinformatics have allowed us to deeply study the drug resistance and molecular mechanisms of transmission.Combining epidemiological data and genomic data of strains,we can identify the strain's transmission route early and block the outbreak,of infections caused by KPC-Kp strains in time.Based on above,we designed this topic to explore the resistance mechanism of CRKP strains isolated in Hangzhou,and further study the molecular characteristics,genetic environment,and mode of transmission of KPC-Kp strains through sequencing technology and relative bioinformatics analysis,so as to provide laboratory basis for interrupting the spread of such bacteria.Method1.268 CRKP strains isolated from a tertiary hospital of Hangzhou and clinical data of the patients infected with CRKP strains were collected.Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF MS)analysis was used to re-identify the category of the strains,and polymerase chain reaction(PCR)was performed to analyze the common carbapenemase-resistant genes(blaKPC,blaNDM,blaIMP,blaVIM and blaOXA-48).After that,the PCR-positive products were sequenced.Finally,232 KPC-Kp strains were selected for the next experiments.2.The drug-resistant phenotype of the strains was determined by the agar dilution method and the microbroth dilution method.3.S1-PFGE was used to analyze the number and size of plasmids carried by the KPC-Kp strains.Southern blot hybridization were performed to locate the blaKPC,and the transferability of plasmids carrying blaKPC was determined by the conjugative experiments.4.Bacterial DNA was extracted for whole-genome sequencing(WGS),and the fasta files and gbk files of the bacterial genome were obtained through SPAdes and Prokka software,respectively.5.Plasmidfinder was used to search the plasmid replication type of KPC-Kp strains.Resfinder was used to search the drug-resistant genes carried by the strains.MLST database was used to analyze the ST type of the strains.Easyfig was used to construct the surrounding environment of blaKPC resistance gene.Result1.A total of 268 strains of CRKP strains were collected from November 1,2018 to June 30,2019,of which 232 strains carried blaKPC-2 and 8 strains carried blaNDM-1,two strains carry blaNDM-5.Among the 232 strains,one strain coexisted blaKPC-2 and blaNDM-1,the other coexisted blaKPC-2 and blaNDM-5.2.Most of the 232 strains were isolated from respiratory specimens,and the vast maj ority came from the intensive care unit.3.All 232 strains were multi-drug resistant,and all strains were mediated or resistant to piperacillin-tazobactam,cefotaxime,ceftazidime,and aztreonam.All but 3 strains were resistant to cefpirome,which were susceptible-dosedependent to cefpirome.The insensitivity rates of strains to imipenem and meropenem were 94.86%and 96.57%,respectively.The resistance rates of strains to amikacin,gentamicin,and ciprofloxacin were 82.86%,94.86%,and 97.97%,respectively.The resistance rates to trimethoprim and sulphame-thoxazole,fosfomycin,and chloramphenicol were 55.43%,56.00%,and 58.86%,respectively.The insensitivity rates to polymyxin and tigecycline were 8.62%and 5.17%,respectively.4.In addition to carbapenem resistance genes,232 strains carried a variety of other types of resistance genes,including extended-spectrum-?-lactamase resistance genes(ESBLs),cephalosporinase-related resistance genes,and quinolones,aminoglycosides,fosfomycin,tetracycline,chloramphenicol,macrolides,and sulphame associated drug resistance genes.5.ST11 was the main type of KPC-Kp strains.6.Strains carried 1-4 plasmids of different sizes and replication types,of which IncFII was the most common.All but one strains carried blaKPC on the plasmid.7.There were 21 kinds of genetic environment arounding blaKPC,among which ISKpn27-blaKPC was conserved sequence.Conclusion1.KPC-2-producing carbapenemase is the main drug resistance mechanism of CRKP strains in this region,and KPC-Kp strains can carry a variety of other drug-resistant genes,leading to failure of clinical treatment.2.The cloning and spreading of ST11 type KPC-Kp strains is the key factor to promote the dissemination of blaKPC gene in our hospital.3.The genetic environment of blaKPC is diverse,and ISKpn27-blaKPC-2 is its conserved sequence.