To determine the pathogenic bacteria of acute death pigs due to respiratory symptoms in a farm in Heilongjiang Province,and to explore its main biological characteristics,the visceral tissues of sick and dead pigs were collected,and the isolated bacteria were identified by bacterial isolation,Gram-staining,biochemical identification,PCR amplication of 16S rRNA gene and sequencing,and constructing phylogenetic tree of 16S rRNA gene.Antimicrobial susceptibility of this isolate to 8 types of antibiotics was detected using microbroth dilution method.Complete genome sequencing was performed using Illumina PE150,and resistance genes were detected using ResFinder software,and their correlation between resistance phenotypes and resistance genes was further analyzed.The detection and analysis of virulence genes were completed by VFanalyzer software and BLASTN comparison.The results showed that the strain formed a single colony with smooth surface and β-hemolytic ring after cultured on 10%defibrated sheep blood plate medium for 24 hours.Gram-staining results showed that it was blue-purple corynebacterium parvum.The biochemical identification results showed that the biochemical characteristics of the isolate were consistent with those of T.pyogenes.16S rRNA gene PCR results showed that the target sequence with a length of 1397bp was amplified,and the sequence alignment with the GenBank database showed that the strain shared 99%homology with the reported T.pyogenes.Phylogenetic tree of 16S rRNA gene analysis showed that it was in the same branch with TZQ 01,a bovine-derived T.pyogenes strain from Shenyang,Liaoning Province,and it was in a close branch with the isolate NIAH13531 of T.pyogen from Japanese pigs The above results showed that the isolate was T.pyogenes and named as FY-4-Z-1.The results of antimicrobial susceptibility testing showed that the strain was sensitive to ceftiofur of cephalosporin,penicillin and amoxicillin of penicillins,erythromycin of macrolide,tiamulin of pleuromutilin,and was resistant to tetracycline of tetracyclines and streptomycin of aminoglycosides.The bacterial genome sketch was successfully obtained by assembling the obtained whole genome sequencing data using the assembly software SPAdes,showing that the genome size was 2399.961kb.ResFinder software was used to detect and analyze the antimicrobial resistance genes of the obtained bacterial genome sequence,and it showed that the bacterial genome contained three antimicrobial resistance genes,namely,aminoglycoside resistance gene ant(6)-la,macrolide-lincosamide-streptogramin B resistance gene erm(X)and tetracycline resistance gene tet(W),among which ant(6)-la gene was detected for the first time in T.pyogenes.The results of antimicrobial susceptibility test of tetracycline and streptomycin were consistent with the results of the phenotype of the resistance genes,while the erythromycin sensitive phenotype may be related to the frameshift mutation of erm(X)gene.The virulence genes were detected using BLASTN and VFanalyzer softwares,and it showed that a total of 8 known virulence genes and 24 candidate virulence-related genes were detected.This study further enriched the relevant research on swine derived T.pyogenes,providing important basis and medication guidance for the prevention and control of the infection with T.pyogenes in large-scale pig farms.
NETL
NSTL
维普
万方数据
