Foodborne Hemolytic Streptococcus Isolation And Identification And CPS2J Virulence Gene Cloning And Sequence Analysis Of Guangxi Area

This paper intend to study the prevalence and the intramolecular biological characteristics of foodborne hemolytic streptococcus in some regions of Guangxi province to provide a scientific basis for the foodborne pathogens epidemiological and the estabilish of bacterial species and gene library of foodborne pathogens of Guangxi.Method:(1) using conventional bacterial isolation and culture, biochemical tests, animal virulence test and PCR technology to isolate and identify the hemolytic streptococcus, which got from the 1257 fresh meat simples, collected from different markets in some regions of Guangxi, during from September,2014 to March,2016.While carrying on susceptibility test by using K-B disk diffusion method. (2) on the reference GenBank published streptococcus CPS2J virulence gene sequences, which designed and synthesized primers, a length of 1152 bp, using the PCR method to amplificate, clon and sequence the isolated 60 hemolytic streptococcus by CPS2J virulence gene, obtained 45 purpose strains, and analysis and comparison of their nucleotide sequence and homology.Result:(1) using conventional method to isolate and indentify bacteria, got 60 hemolytic streptococcus from 1257 fresh meat simples, contend 12 strains from Liuzhou、27 strains from Guilin、9 strains from Laibin、7 strains from Shanglin、 5 strains from Nanning. Selected 11 strains of the strains inoculated mice, then all the test animals died after 8h to 24h. This result showed that the isolated 11 strains of hemolytic streptococci had strong pathogenicity on mice. (2) Susceptibility test results of Guilin、Laibin、Shanglin、Nanning、Liuzhou isolation strains were resistant to amoxicillin, resistance rate was 59%、33%、43%、40%、42%; Guilin、 Laibin isolation strains were resistant to clindamycin, resistance rate was 15%、 11%; Guilin、Laibin、Shanglin、Liuzhou isolation strains were resistant to penicillin, resistance rate was 41%、22%、29%、58%; Guilin、Shanglin、Nanning、Liuzhou isolation strains were resistant to vancomycin, resistance rate was 19%、29%、20%、 17%; Guilin isolation strains were resistant to gentamicin, resistance rate was 11%. (3) 45 of 60 strains isolated hemolytic streptococcus got CPS2J virulence genes by PCR, comparied with nucleotide sequence homology of domestic and international standards strain, the result showed that compared with domestic and international standard strains 98015、SC17、SC22、SC152 and ZYS8, the 8 isolated strains of Laibin (GLB-5、GLB-9、GLB-12、GLB-13、GLB-15、GLB-25、GLB-26、GLB-33) had 83.7%-96.3% nucleotide homology with CPS2J, the 9 isolated strains of Liuzhou (GLZ-1、GLZ-2-3、GLZ-3、GLZ-6、GLZ-8、GLZ-11、GLZ-14、GLZ-22、 GLZ-23) had 84.4%-96.7% nucleotide homology; the 4 isolated strains of Shanglin (GSL-1、GSL-2、GSL-7、GSL-13) had 84.2%-96.5% nucleotide homology; the 22 isolated strains of Guilin (GGL-2、GGL-2-2、GGL-2-4、GGL-2-10、GGL-2-16、 GGL-2-18、GGL-2-28、GGL-3-4、GGL-3-19、GGL-4、GGL-4-1、GGL-4-2、 GGL-4-5、GGL-7、GGL-10、GGL-12、GGL-13、GGL-18、GGL-24、GGL-26、 GGL-27、GGL-29) had 79.8%-96.5% nucleotide homology; the 2 isolated strains of Dashatian (GDST-2-2, GDST-5) had 81.2%-90.5% nucleotide homology. Compared with International standard strains (98015、SC17、SC22 and SC152), the nucleotide homology of the 8 strains from Laibin was 83.7%-96.3%; the nucleotide homology of the 9 strains from Liuzhou was 84.4%-96.7%; the nucleotide homology of the 4 strains from Shanglin was 84.2%-96.5%; the nucleotide homology of the 22 strains from Guilin was 79.8%-96.5%; the nucleotide homology of the 2 strains from Nanning was 81.2%-90.5%. Compared with domestic standard strain (ZYS8), the nucleotide homology of the 8 strains from Laibin was 84.8%-94.8%; the nucleotide homology of the 9 strains from Liuzhou was 85.8%-95.4%; the nucleotide homology of the 4 strains from Shanglin was 85.6%-96.3%; the nucleotide homology of the 22 strains from Guilin was 84.2%-93.6%; the nucleotide homology of the 2 strains from Nanning was 81.2%-90.5%. Compared with the 22 strains of Guilin, the CPS2J nucleotide homology of the 4 strains of Shanglin was 86.7%-99.9%, the CPS2J nucleotide homology of the 9 strains of Liuzhou was 77.8%-100.0%; Domestic and international standard strains (98015、SC17、SC22、SC152 and ZYS8) had closer genetic relationships. But the isolated strains of Guangxi (GGL-2-4、GGL-20-1、 GLZ-2), had distant relationship with domestic and international standard strains, it is more Independent. ZYS8 strain had closer genetic relationships with GGL-26 and GLB-25. SC152 strain had a closer genetic relationship with GGL-27.Conclusion:The 60 strains hemolytic streptococcus isolated from 1257 fresh samples collected from Guangxi had obvious resistance on drugs partly. There are 45 strains of the isolated strains containing CPS2J virulence genes, and have a strong pathogenicity on mice. Compare the 45 isolates CPS2J virulence genes with domestic and international standards strains, its homology exists some differences. This made the establishment of livestock and poultry products in Guangxi foodborne pathogens in risk assessment and early warning technology system provide theoretical basis.