Whole-genome Sequencing Of A Multi-drug Resistant Klebsiella Pneumoniae ST37 Of Swine Origin And Comparative Genomic Analysis

Klebsiella pneumoniae is an opportunistic pathogen associated with acquired infections and is listed as a drug-resistant pathogen that urgently needs to develop new antibiotics by the World Health Organization(WHO).In veterinary clinics,K.pneumoniae which deserves attention can cause disease in swines and has become one of the pathogenic bacteria in large-scale swine farms.By now,researchers have mainly explored the drug resistance of K.pneumoniae from swines,but little is known about the evolutionary relationship,resistance and virulence genes of swine-derived ST37(Sequence type,ST)K.pneumoniae.The aim of this study was to understand the genetic evolutionary relationship of ST37 K.pneumoniae from swines,the distribution of drug resistance and virulence genes,and the structural characteristics of the plasmid carried,so as to guide the prevention and treatment of disease caused by K.pneumoniae in farms.In this study,we sequenced a multi-drug resistant K.pneumoniae strain KP200 isolated from the liver of a diarrhea pig in a farm of Guangdong province to obtain the genome characteristics of this strain,then compared this genome sequence with 160 ST37 K.pneumoniae in GenBank.In addition,comparative analysis with the existing plasmid sequences from Plasmid Database(PLSDB)were used to identify the structural characteristics of the two plasmids carried by KP200,and explore the formation of mobile elements at the molecular level.The results are as follows:1.The genomic feature of K.pneumoniae KP200 contained one circular chromosome of 5 257 665 bp with a 57.55%GC content,a total of 5 106 predicted open reading frames(ORFs).It also contained two circular plasmids with sizes of 213 254 bp and 75 320 bp,respectively.Plasmid 1 can encode 290 genes including aminoglycoside resistance aadA2,aadA5,armA,aph(3')-Ia and aphA1-1;sulfonamide resistance sul1 and dfrA1;quarternary ammonium compounds resistance qacE?1.Plasmid 2 can encode 107 genes including tetracycline resistance tetA;aminoglycoside resistance aadA16;florfenicol resistance floR;?-lactams resistance blaCTX-M-27;sulfonamide resistance sull and dfrA27;quinolone resistance qnrB2 and aac(6')-Ib-cr;quarternary ammonium compounds resistance qacE?1;rifampicin resistance arr-6.2.Multilocus sequence typing(MLST)have shown that the sequence type(ST)of KP200 was 37.Phylogenetic analysis based on single-copy genes of 161 K.pneumoniae ST37 genome sequences have shown that KP200 was more closely related to 5 human-derived K.pneumoniae strains.The distribution of virulence genes carried by these six strains were similar,indicating that the distribution of virulence genes was related to genetic evolution.Based on the the prediction of drug resistance and virulence genes of genome,161 strains carried the drug resistance genes(kpnE,lptD1,blaSHV)and virulence genes(type 1 fimbriae gene fimACFGI,type 3 fimbriae gene mrkDCHJ,enterobactin gene entABCEF and fepABC and capsule synthesis regulator rcsAB).KP200 and other 15 K.pneumoniae strains carried aerobactin transporter gene iutA.Furthermore,the nucleotide homology of sixteen iutA gene sequences was 73.49%?100%.Compared with the iutA nucleotide sequence carried by the hypervirulent K.pneumoniae NTUH-K2044,it was found that the iutA nucleotide carried by KP200 resulted in two amino acid substitutions due to two base mutations:Leu was replaced into Phe and Val was replaced into Leu.KP200 carried two type VI secretion system(T6SS)gene clusters,related regulators homologues TfoX and QstR,and natural competence proteins ComeA and ComeC by T6SS analysis.Bacteria carrying T6SS and natural competent proteins ComEA and ComEC have the ability to bacterial killing and absorb foreign genes.Based on integrative conjugative elements(ICE)analysis,ICEL1 carried by KP200 was 100%similar to novel ICEKpnHS 11286-2 carried by K.pneumoniae HS11286,which carried tfc type genes ability to horizontal transfer.3.Based on structure comparison analysis of the two plasmids pYhe2001 and pYhe2002 carried by KP200,two plasmids both contain small plasmid sequences and class 1 integrons.Class 1 integrons have ability to capture and spread drug-resistant genes.Plasmid pYhe2001 was a novel IncHI1B plasmid,which carried four types of heavy metal resistance genes(tellurium ter,mercury mer,silver sil and copper cop)and a new transposon Tn6897.Tn6897 carried several insertion sequences IS26,which mediate homologous recombination.The backbone of plasmid pYhe2002 was highly similar to the plasmid p19110124-2 carried by K.pneumoniae of swine origin from Henan,China by Basic local alignment search tool(BLAST).They both contained drug resistance genes tetA,floR,blaCTX-M-27,sull,qnrB2,qacE?1,aadA16,dfrA27,arr-6 and aac(6')-Ib-cr,suggesting that the drug resistance genes were spread in different K.pneumoniae strains by plasmids.4.The plasmids carried by KP200 transformed into Escherichia coli DH5? by electroporation and natural transformation at a frequency of 10-8 cells per recipient cell and 10-11 cells per recipient cell,respectively.The plasmids carried by KP200 transformed into Staphylococcus aureus RN4220 at a frequency of 10-12 cells per recipient cell by natural transformation.The transformants had increased MICs for chloramphenicol,oxacillin,ampicillin,ciprofloxacin,gentamicin,streptomycin,kanamycin and amikacin as compared with those of the parental strain.This finding indicated that plasmids contributed to chloramphenicol,?-lactams,quinolones and aminoglycoside antibiotics resistance in K.pneumoniae KP200.