Resistance And MLST Classification Analysis Of Campylobacter Jejuni Isolated From Parts Of Sichuan

Campylobacter jejuni(C jejuni)is a zoonotic pathogen that has attracted researchers' widespread attention at home and abroad for more than a decade,and it is also one of the major pathogens of food-borne infections.Therefore,the prevention and control of the bacteria has important public health significance.In recent years,with the extensive use of antibiotics in animal husbandry and medical clinics,the resistance spectrum of this bacteria has been extensive,and the cause of drug resistance has become more complicated,which has increased the difficulty in preventing and controlling the diseases caused by this strain.In this study,Campylobacter jejuni was isolated from poultry cecal contents,feces and cow feces collected from many regions in Sichuan.The aim is to understand the correlation between the resistance phenotypes of isolates and the biological information of the CRISPR-Cas system contained in the isolates,and to explore the genetic evolutionary relationships among isolates based on molecular typing for the epidemiological detection and prevention of the bacteria.1.Isolation,identification and analysis of drug resistance of strainsA total of 828 caecal contents and faeces and 649 bovine manure samples were collected from many regions in Sichuan.Campylobacter jejuni were isolated and the isolates were identified by PCR.A total of 98 Campylobacter jejuni isolates were obtained from poultry,and there were 34 isolates of Campylobacter jejuni isolated from cattle,and the isolation rates were 11.7%(98/828)and 5.2%(34/649),respectively.The results of drug sensitivity showed that the isolates had higher resistance to penicillin,ciprofloxacin,nalidixic acid,and clindamycin were 84.5%(112/132),88.63%(117/132),and 78.64%(104/132),77.96%(103/132),tetracycline 65.9%(87/132),chloramphenicol 59.1%(78/132),gentamycin 40.9%(54/132),erythromycin 54.54%(72/132)and florfenicol 15.9%(21/132),respectively.The results of resistance gene of Campylobacter jejuni showed that the cat gene was 93.6%(73/78).The erythromycin-resistant isolates 94.4%(68/74)had a base A ? G substitution at position 2075 in the V region of 23 S rRNA.And randomly selected ciprofloxacin-resistant strains WJC2-6,JT-4,PX-3 Had the mutation of base C?T(Thr-86?Ile)occurred at position 257.The carrying rate of the resistance gene cluster aadE-sat4-aphA-3 in the aminoglycoside-resistant isolates was 31.5%(17/54).Most of the Class I gene cassettes contained the aadA2 aminoglycoside resistance gene cassette.The multiple drug resistance rate of the isolates was 89.4%(118/132).2.Bioinformatics analysis of CRISPR-Cas system of strains and its correlation with drug resistanceBioinformatics methods were used to predict the CRISPR-Cas system contained in the Campylobacter jejuni published in NCBI.Conservative alignment analysis and GC content analysis of repeat sequences,and RNA secondary structure prediction were performed based on Cas protein sequence and repeat sequence to constructs genetic phylogenetic trees.The results showed that the repeat sequences of the CRISPR-Cas system contained in Campylobacter jejuni were highly conserved,the GC content was lower than 28.57%,and the differences were mainly present in the free parts of the two ends.The CRISPR polymorphism due to large gaps in CRISPR space sequence.The homology of the Cas protein sequence was higher than 96.8%,which was consistent with the characteristics of gene level transfer.The primers were designed according to the conserved segment of each gene of Campylobacter jejuni CRISPR-Cas system for detection of the isolates and biological information was analyzed.The statistically analysis of the drug resistance positive rate and positive rate of each gene in isolated CRISPR-Cas system aimed to explore the correlation between CRISPR-Cas system and drug resistance by used the SPSS20.0 software.The results showed that 56.1%(74/132)strains had a complete CRISPR-Cas system,and some strains lacked the system or lacked part of the gene structure of the system;the conserved repeat sequences in cas and CRISPR sequences were comparable.The consistency of the repeat sequence was 88.9% and the consistency of the cas9 gene sequence was 98.47%.Repeat sequences of stem-loop-like RNA secondary structures depend on their inherent structure.There was no significant correlation between CRISPR sequences and drug resistance genes.There was no statistically significant difference between the positive rate of drug resistance and the positive rate of cas1 and cas2 genes in the strains.There was a significant correlation between the positive rate of erythromycin-resistant strains and the positive rate of cas9 gene.3.MLST typing of isolatesAccording to the MLST database,the Campylobacter jejuni MLST genotypes aspA,glnA,glyA,gltA,tkt,pgm and uncA were selected for amplification and sequenced,followed by cluster analysis to initially explore the genetic relationship between strains.The results showed that there were 38 different sequence types in 92 isolates,belonging to 11 different clonal lines,CC-21,CC-353 and CC-464 clonal lines were relatively more.The genetic phylogenetic trees showed that there are genetic diversity among isolates from different hosts and regions.