The Molecular Epidemiology Study Of Typhi Toxin Gene In Nontyphoid Salmonella

Objective:To investigate the molecular epidemiology of typhoid toxin?TT?coding genes?tt?in clinically isolated non-typhoid Salmonella?NTS?by detecting and analyzing the presence and characterization of cdtB,pltA and pltB genes in different serotypes and PFGE types of NTS isolates and comparing DNA sequences to corresponding genes in GenBank database.This research will help to understand and explore distribution,prevalence,and evolution of cdt/tt,and harm to human health of tt-positive NTS further.Method:1,PCR amplification of tt genes:108 non-repetitive strains of NTS were isolated from stool specimens of outpatients with acute diarrhea in the Second Hospital and General Hospital of Tianjin Medical University from May to October of 2014.cdtB,pltA and pltB genes were amplified by PCR with specific primers.2,PFGE analysis:the genomic DNA was extracted from the 108 NTS isolates and analyzed through pulsed field gel electrophoresis?PFGE?with the restriction endonuclease Xba?.3,Sequencing of tt genes:DNA sequencing of cdtB and pltA/B were performed in each PFGE type of tt-positive Salmonella isolates serotypes.DNA and amino acid sequences were analyzed by Blast and comapared to cdt/tt-positive strains in GenBank.4,Amino acid phylogenetic tree of tt genes:a phylogenetic tree was constructed by MEGA5 to compare CdtB,PltA and PltB sequences among tt-positive S.isolates from this study and GenBank.Results:1,cdtB and pltA/B were detected in 20?18.5%?of 108 NTS isolates,which 3 strains of S.Goldcoast,16 strains of S.Schwarzengrund and 1strain of S.Essen.None of cdtB,pltA/B was positive in else NTS isolates.3 S.Goldcoast and 16 S.Schwarzengrund had 3 different PFGE patterns,respectively.2,cdtB and pltA/B PCR products were sequenced in 3 S.Goldcoast,1 S.Essen and 3S.Schwarzengrund with different PFGE patterns.The deduced CdtB amino acid sequences of all 7 isolates contained the two conserved sites His-160?catalytic activity?and ASP-195(Mg²⁺binding)in CdtB of S.Typhi CT18.3,cdtB and pltA/B sequences of 3 S.Goldcoast were identical.Comparing with corresponding genes of S.Typhi CT18,the similarity of DNA was 99.51%,99.04%,97.34%,and the homology of amino acid was 99.26%,99.59%and 97.81%,respectively.4,cdtB and pltA/B sequences of 3 S.Schwarzengrund with different PFGE patterns,1S.Essen and S.Schwarzengrund strain CVM19633 isolated from US were identical.Comparing with the corresponding genes in S.Typhi CT18,the similarity of DNA sequences was 99.88%,98.63%and 96.86%,the homology of amino acid was 100%,98.76%and 95.62%,respectively.5,S.Paratyphi A ATCC 9150 was gathered into the same clusters with S.Goldcoast in PltA and PltB phylogenetic tree but separate in CdtB phylogenetic tree.S.Typhi CT18 and S.Javiana strain CFSAN001992 were in the separate clusters in PltA and PltB phylogenetic tree but gathered into one cluster in CdtB phylogenetic tree with S.Schwarzengrund,S.Essen isolates in this study and S.Schwarzengrund CVM19633from US.6,PltA/PltB amino acid sequences were identical but there were 3 different amino acids in CdtB between S.Paratyphi A ATCC 9150 and S.Goldcoast isolates.CdtB sequences were identical but 1 amino acid in PltA and 2 amino acids in PltB were different between S.Paratyphi A ATCC 9150 and S.Typhimurium Sal199.7,The CdtB homology between tt-positive NTS isolates in hits study and Escherichia coli Vir68 from GenBank was less than 50%,and the homology between PltA/B and CdtA/C was less than 20%.Conclusion:1,cdtB and pltA/B were detected in clinical isolates of S.Essen,S.Schwarzengrund and S.Goldcoast in Tianjin,of which S.Goldcoast carrying tt genes has not been reported in the literature.2,NTS clinical isolates in this study have a very high degree of similarity of tt genes with S.Typhi despite slight polymorphism.Deduced CdtB sequences in our NTS isolates showed the conserved sites in S.Typhi.3,Key subunit CdtB sequences show some degree of serotype-dependent.The identical CdtB sequences could be found among the same serotype isolates with distinct PFGE patterns or origins from different countries.4,The evolutions between CdtB and PltA/B subunits may be independent among the Salmonella isolates with different serotypes.