| Objective: To investigate the virulence factors of non-typhoid Salmonella(NTS)in our region. Application PCR and DNA sequencing to identify virulence genes in non-typhoid Salmonella. The relationship between different serotypes, genotypes, resistance phenotypes of NTS and virulence genes have been analyzed. It can offer help for prevention and treat of non-typhoid Salmonella infection.Methods: 1, Detection of virulence gene: The strains isolated from stool specimens of patients with acute diarrhea in intestinal outpatient in Second Hospital of Tianjin Medical University, strains were collected from May to October period of 2012 and 2013 year. It including 12 serotypes, 23 kinds of PFGE graph types and 13 STs; The virulence genes(sit C, sii E, hil A, mgt C, pho P, sop B, sif A, sse L, prot6 E, pef A, spv B, cdt B) was performed by PCR amplification, then the strains of cdt B postive were screened for the cdt B, plt A, and plt B genes by a PCR. 2, DNA sequence analysis: The DNA sequencing was used for one strain of positive. The sequences obtained were compared to those registered in Gen Bank. The dendrograms of Cdt B, Plt A, and Plt B orthologs of the strains of cdt B postive using the BLAST and Clustal X software. 3, The relationship between resistance phenotypes and virulence genes: SPSS 20.0 software was used for statistical analysis. The Fisher’s exact test was used to study the association between virulence genes and resistance to antibiotics(P < 0.05).Results: 1, The results show that the carried rate of the virulence genes from the isolated strains indicated that sit C, sii E, hil A, mgt C, pho P, sop B was 100%. sif A, sse L was 97.78%. prot6 E was 31.11%. pef A, spv B was37.78%. cdt B was 2.22%. The homology of sit C were 98%, hil A were 98%, sii E were 99%, spv B were 100%, prot6 E were 100%, sif A were 99%, mgt C were 100%, sop B were 99%, sse L were 100%, pef A were99%, pho P were 100%, cdt B were 99%. 2, The virulence genes of SPI1~5: sit C, hil A, mgt C, sii E, sop B were observed in all isolates. The 406 were not detected sif A. The 25 were not detected sse L. 3, The virulence plasmid genes: prot6 E, spv B, pef A were present in 14 isolates of serovar Enteritidis. In 9 isolates of serovar Typhimurium, spv B and pef A were present in 3 isolates of ST19, spv B and pef A were not observed in other 6 isolates of ST34. prot6 E, spv B, pef A were not present in Other serotype. 4, cdt B genes: cdt B were present in 1 isolate of serovar Grampian. The size of cdt B, plt A, plt B was 819 bp, 729 bp, 414 bp, respectively. The length of Cdt B, Plt A, Plt B were 269 aa, 243 aa, 140 aa. The homology of cdt B, plt A, plt B were 93.1% ~ 99.5%, 81.5% ~ 99.6%, 64.3% ~ 99.0%, respectively. The homology of Cdt B, Plt A, Plt B were 92.6% ~ 99.3%, 78.2% ~ 100.0%, 69.1% ~ 100.0%. 5, The relationship between resistance phenotypes and virulence genes: We found significant associations between the presence of prot6 E, spv B, pef A and nalidixic acid(P<0.001), tetracycline(P=0.044, 0.017, 0.017, respectively). The significant associations between streptomycin and prot6 E were found(P=0.046).Conclusion: 1, SPI1~5 were distributed in all isolates. It is necessary to invase host in Non-typhoid Salmonella. 2, prot6 E gene located on the serovar Enteritidis specifically. specific genes. The virulence plasmids in serovar Typhimurium is closely related to MLST. 3, cdt B, plt A, plt B genes were observed in serovar Grampian first time, the DNA sequence and amino acid sequence were different from other NTS. We must High alert S. Typhimurium MLST19 become more and more resistance stronger in Tianjin. 4, The resistance and virulence genes in Salmonella can spread and persistence together. |
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