Relationship Between CRISPR In Klebsiella Pneumoniae And Drug Resistance And Isolation Sites

Background Klebsiella pneumoniae(KP)is an important pathogen causing community and nosocomial infections.With the widespread use of antibiotics,drug-resistant KP is becoming more common,which brings great challenges to clinical anti-infective treatment.The CRISPR /Cas system consisting of clustered regularly interspaced short palindromic repeats(CRISPR)and their related sequences(CRISPR associated sequences,Cas)is an acquired new type widely found in bacteria and archaea.The microbial defense system can respond to the conjugation or transformation of plasmids and gene introduction caused by phage infection,and limit the horizontal genes transfer(HGT)such as drug resistance or virulence.At present,research on CRISPR mainly focuses on E.coli and Shigella,while KP research is rarely involved.Objective1.By analyzing the relationship between CRISPR distribution in KP and the drug-resistant phenotype and drug-resistant genes of the strain,the influence of CRISPR distribution in the strain on the drug-resistant phenotype and drug-resistant genes of the strain was explored.2.By analyzing the relationship between the drug resistance information of Spacer homologous plasmid or bacteriophage and the drug resistance information of Spacer host strain,the regulatory mechanism of CRISPR on drug resistance in Kp was explored.3.To explore the relationship between the location of KP separation and its CRISPR distribution and drug resistance phenotype.Method1.The CRISPR locus and drug-resistant genes of KP were amplified by Polymerase Chain Reaction(PCR)and sequenced.The drug-resistant phenotypes of strains were detected by modified Kirby Bauer K-B)method,and SPSS19.0 was used to analyze the correlation between CRISPR locus and drug-resistant phenotype and drug-resistant genes.2.Using CRISPR Target to find spacer homologous plasmids or bacteriophages and download their genomes in NCBI.Then uploading the genomes to the Center for Genomic Epidemiology website to obtain information about resistance genes.In combination with the resistance genes carried by the strains detected by PCR and analyzing whether there was a crossover between the two resistance genes.3.Collect information on the location of the isolates and use ClustalX 2.1 for a consistent analysis of the spacer.Combining the location information of isolates,the consistency analysis results and the drug-resistant phenotype to analyze the relationship between the location of isolates,the distribution characteristics of CRISPR and the drug-resistant phenotype.Result1.In terms of the positive rate of drug-resistant genes,CRISPR positive strains were less than CRISPR negative strains(except drug-resistant genes VIM,IMP and qurA),and there were statistically significant differences between CRISPR carrying and distribution of drug-resistant genes blaOXA,TEM,tetA,tetB and sul2(P<0.05).The drug resistance rate of strains carrying CRISPR to other single antibiotics was all lower than that of strains without CRISPR(except NOR and AMP),and there was a statistically significant difference between the distribution of resistance to C,TE and SXT antibiotics and the presence or absence of CRISPR sites(P<0.05).Antibiotic C,TE,and SXT,the proportion of the corresponding sensitive strain CRISPR1/2 containing multi-spaced sequences wasgreater than that of the resistant strain CRISPR1/2 containing multi-spaced sequences,and the distribution difference between the number of the CRISPR1 and CRISPR2 spacing sequences and the resistance of single antibiotic C,TE,and SXT was statistically significant(P<0.05).2.Drug resistance genes carried by the strain were found in all drug resistance genes detected by spacer homologous plasmid or phage,and the drug-resistant phenotype of the strain was highly consistent with the drug-resistant genes it carried.3.The strains isolated from the First Affiliated Hospital of Xinxiang Medical University had identical CRISPR distribution and nearly identical drug-resistant phenotypes.The drug-resistant phenotypes of strains isolated from the Center Hospital of Xinxiang.Conclusion1.The presence of CRISPR in KP strains greatly reduces the resistance of strains to antimicrobial drugs and the key role lies in the spacer.Meanwhile,CRISPR in KP strains can prevent the transmission of drug-resistant genes to a certain extent.2.KP regulated drug resistance by integrating foreign plasmid drug-resistant genes fragments into the bacterial genome.3.The drug-resistant phenotypes and CRISPR characteristics of strains isolated from the same site are highly similar,and the distribution characteristics of Spacer in CRISPR can be used to infer the past living environment of strains.