Genome-wide Analysis Of 94 Strains Of Klebsiella Isolated From Sterile Body Fluids

Objectives To explore the molecular typing,drug resistance genes and virulence genes of Klebsiella pneumonia complex causing clinical infection by the high-throughput sequencing technology,and to explore characteristics of clinical infection and drug resistance of these strains,in order to describe popular features and provide the basis for clinical prevention and control.Methods 1 Between January 2018 and December 2020,94 strains isolated from sterile body fluids of non-duplicate strains were recovered from inpatients at three hospitals in three Chinese provinces（He Bei,Jiang Su and Sichuan Provinces）.The strains were isolated and cultured by clinical routine methods,and all strains were tested by MALDI-TOF-MS and VITEK2-Compact automatic microbial analyzer.2 Drug sensitivity test of drugs using broth VITEK2-Compact automatic microbial analyzer and Microdilution method.Mucoviscous phenotypes were determined through string test.3 The Next-generation sequencing technology（WGS）of Illumina was used to obtain the genome frame maps of94 strains.The thirdgeneration sequencing technology of Pac Bio Sequel was used to construct the complete maps of genome of representative strains.4 Multilocus sequence typing（MLST）method to explore the molecular typing and clonal epidemic characteristics of strains.BLAST method to screen strain resistance and virulence genes,and the characteristics of drug resistance plasmids and virulence plasmids carried by strains were analyzed by homology comparison.A phylogenetic tree was constructed based on the single nucleoitide polymorphism（SNP）of the strains to explore the relationship between the strains.Results 1 94 strains of Klebsiella pneumoniae complex from sterile body fluids were from Hebei Province（21 strains）,Jiangsu Province（57 strains）and Sichuan Province（16strains）,respectively.The distribution of departments was dominated by intensive care medicine（29.79%）.The main type of specimen was blood,accounting for 41.49%.2 Drug sensitivity:The drug susceptibility spectrum of strains presented diversity.There were carbapenem-resistant Klebsiella pneumoniae（CRKP）（24.47%）,CSKP（30.85%）and holo-sensitive strains（44.68%）.The resistance rates to cefotaxime and ciprofloxacin were relatively high（35.11%）.However,the resistance rates to ceftazidime（3.19%）,tigecycline（2.13%）and polymyxin（1.06%）were low.3 MLST and capsular serotypes:MLST presented clonal polymorphisms.ST23/K1 accounted for the highest proportion and five new STs were be found.4 Carrying status of drug resistance genes:The highest carrying rate of 94 strains was fosfomycin resistance gene fos A accounted for 98.94%,followed by the quinolone resistance gene oqx AB（94.68%）.The bla _KPC-2 was the main carbapenemase resistance gene（73.91%）.The detection of bla _OXA-232/ST15 and bla _IMP-4/ST268 of Klebsiella pneumoniae from sterile body fluids belongs to the earlier research reports in China.5 Carrier status of virulence genes:The 94 isolates carried the highest siderophore-related gene was iuc A（52.13%）.But wzy1 carrier rate was the lowest at 5.32%.The number of virulence genes carried by the same strain ranged from 0 to 6.The ST23clonotype showed the characteristics of concentrated virulence genes.And there were 37strains（39.36%）of the same strain carrying three or more virulence genes at the same time,and the corresponding positive rate of the string test was 100%（37/37）.6 Analysis of drug-resistant plasmids and virulence plasmids of key research strains:The drug-resistant plasmids include Inc FII/Inc R carrying bla _KPC-2,Inc X3 carrying bla _NDM-1,plasmid Col KP3carrying bla _OXA-232 and Inc N carrying bla _IMP-4.The virulence plasmids carried by the 8strains were the same as the internationally recognized the virulence plasmids carried by hypervirulent Klebsiella pneumoniae（hv KP）NTUH-K2044 have high homology.During the development and evolution of strain SC-YY49,plasmid SC-YY49-1 captured the three-part DNA sequence fragments of Xer D-IS102-IS1A,ISSpu2-iro BCDN-IS102 and153498bp-Pin R-IS102 from plasmid SC-YY49-2,making the plasmid SC-YY49-1acquired the iro BCDN-rmp A gene cluster.The phenomenon that SC-YY49 carried two virulence plasmids at the same time.7 SNP phylogenetic results:94 isolates were clearly distinguished into three species.They were K.pneumonia（86.17%）,K.quasipneumoniae（10.64%）and K.variicola（3.19%）,and all of which belonged to the Klebsiella pneumoniae complex.Conclusions 1 The Klebsiella pneumoniae complex that causes clinical infection was characterized by species diversity,drug susceptibility diversity and MLST clonal polymorphism.2 The strains of Klebsiella pneumoniae complex showed a polyclonal distribution characteristic of dominant clonal group ST23/ST11,supplemented by other clonal groups.The strains mainly carried the carbapenemase gene bla _KPC-2.3 In addition to the detection of 5 new ST types,The detection of OXA-232/ST15 and IMP-4/ST268 types in Klebsiella pneumoniae from sterile body fluids were relatively new study in China.4The Klebsiella pneumoniae complex that causes clinical infection carries virulent plasmids that were highly virulent and spread rapidly.The developmental evolution phenomenon of carrying two virulence plasmids appeared in individual strains.5 Highly virulent strains were no longer the only threat to invasive infection with sterile body fluids,and highly resistant strains（especially CRKP strains）also bring challenges and burdens to clinical treatment.6 Currently,only whole-genome sequencing technology can clearly distinguish different species of bacteria in the Klebsiella pneumoniae complex,and a safer and more convenient clinical identification method needs to be developed.Figure 12;Table 11;Reference 149...