Antimicrobial Resistance And Salmonella Typhi Virulence Genes In Salmonella Goldcoast

Objective:To investigate antimicrobial resistance,resistance mechanisms to quinolones and β-lactams,and Salmonella Typhi-associated virulence genes of clinically isolated Salmonella Goldcoast.The aim of this study is to learn the trend of resistance and virulence of NTS for prevention and control of NTS infection and rational use of antimicrobial agents.Method:1.The clinical data and stool samples of patients with acute diarrhea from two teaching hospitals were collected from April to October of 2014 in Tianjin.Enrichment culture,biochemical identification and serotyping analysis were used to isolate and identify NTS strains.2.Susceptibility to 17 antimicrobial agents of S.Goldcoast was performed as Kirbty-Bauer and MIC determination.3.Molecular phenotypes: multiple locus sequence typing(MLST).4.Quinolone resistance mechanisms analysis: quinolone resistance determining region(gyr A,gyr B,par C,par E)and plasmid-mediated quinolone resistance genes of S.Goldcoast were amplified by PCR.Sequences of positive PCR products were analyzed by Blast and Clustal X.5.?-lactamase genes detection: ?-lactamase genes(TEM,SHV,CTX-M,OXA)of S.Goldcoast were amplified by PCR.Sequences of positive PCR products were analyzed by Blast and Clustal X.6.cdt B-islets and Salmonella Typhi-associated virulence genes detection analysis:coding and non-coding genes of cdt B-islet,hly E,tai A and tcf A were amplified by PCR.Sequences of positive PCR products were analyzed by Blast,Bio Edit and MEGA5.Result:1.A total of 3 strains of S.Goldcoast were isolated and identified from stool specimens of patients with acute diarrhea from two teaching hospitals in 2014 inTianjin,accounting for 0.03%(3/108).Two of the three patients with S.Goldcoast infection were diagnosed with acute gastroenteritis and the other one was diagnosed with acute bacterial dysentery.2.All 3 strains of S.Goldcoast were multidrug resistant.They were resistant to ampicillin,piperacillin/tazobactam,pefloxacin,chloramphenicol,and compound sulfamethoxazole,and intermediate to Amoxicillin/clavulanic acid,ciprofloxacin,and levofloxacin.2 isolates were intermediate or resistance to tetracycline,ampicillin/sulbactam,streptomycin,gentamicin,and etimicin and azithromycin.All were sensitive to nalidixic acid,ceftriaxone and ertapenem.3.Among 3 strains of S.Goldcoast,2 isolates are ST358 and 1 is ST2529.4.Comparing with the corresponding genes in FQs susceptible S.Typhimurium,the similarity of DNA and amino acid sequences of gyr A,gyr B and par E of 3 S.Goldcoast were 100%.The similarity of DNA sequence of par C was 99% and a single amino acid substitution(Thr57-Ser)occurred.5.All 3 strains of S.Goldcoast were positive for qnr S PCR.Sequence analysis showed that qnr S sequences among S.Goldcoast isolates were identical.Comparing with qnr S1 gene in S.Typhimurium,the DNA similarity was 99% and the homology of amino acid was 100%.PCR amplification of S.Goldcoast for qnr A,qnr B,qnr C,qnr D,aac(6’)-Ib-cr,qep A,and oqx AB were all negative.6.All 3 strains of S.Goldcoast were amplified with the expected fragment length of TEM gene.Comparing with TEM-1b gene of S.Typhimurium,the similarity of DNA sequence was 99% and amino acid was 100% in S.Goldcoast isolates.The detection of SHV,OXA and CTX-M genes were negative.7.All 3 strains of S.Goldcoast were amplified with the expected fragment length of sty1887,sty1889 and phage genes.Comparing with the corresponding genes in S.Typhi CT18,the similarity of DNA and amino acid sequence were 100% in S.Goldcoast isolates.Sequencing analysis showed that cdt B-islet of S.Goldcoast contained cdt B,sty1887,sty1889,plt A,plt B,and phage genes,of which the order and transcriptional direction were exactly the same as S.Typhi CT18.8.All 3 strains of S.Goldcoast were positive for hly E,tai A and tcf A genes PCR.Comparing with the corresponding genes in S.Typhi CT18,the similarity of DNAsequence was 99%,100% and 99% and amino acid was 100%,100%,99%,respectively.Conclusion:1.We identified 3 multidrug resistant isolates of S.Goldcoast from patients with 2ST358 and 1 ST2529.2.Qnr S1 and TEM-1b genes were positive in all 3 strains of S.Goldcoast,mediating intermediate or resistance to fluoroquinolones,?-lactamase/β-lactamase inhibitors,respectively.3.All 3 isolates of S.Goldcoast carried Typhi-related virulence genes cdt B-islet,hly E,tai A,and tcf A,which effects on pathogenicity of S.Goldcoast requires the further study.4.The emergence of clinically isolated multiple drug resistant S.Goldcoast harboring S.Typhi-associated virulence genes urges that we should pay more attention to and investigate continuously the tendency of resistance and virulence of NTS.