| Salmonella is the leading factor in food borne diseases and is considered to be the priority target for the control of food borne pathogenic bacteria in the world.Human and animals can be severely suffered from gastroenteritis after being infected by Salmonella and can be resulted in food poisoning even death.Salmonella is a major threat to human health and has also caused huge economic losses.Because the phenotypic characteristics of Salmonella are not stable,the related detail information between the strains cannot be obtained by phenotyping,and it is also impossible to diferenciate the strains of the same biochemical characteristics and serotypes.For the purposes of better investigation of the epidemiology,traceability and the rapid response to the food emergency events,the molecular typing technology with high throughput,high sensitivity,good repeatability,easy to apply is essential and necessary.In this study,two genotyping techniques were used to investigate the fresh meat original Salmonella of predominant serotypes in Guangxi.The purpose of this study was to find a molecular technique that could trace the source of food contaminated Salmonella.The quinolones resistance status of the fresh meat original Salmonella isolates was also studied.Totally 71 isolates of S.derby and 21 isolates of S.agona of the predominant serotypes of Salmonella from the fresh meats of chicken and duck,pork and beef sold in the wet markets and the supermarkets in Guangxi were used in the genotyping by a enterobacterial repetitive intergenic consensus based polymerase chain reaction(ERIC-PCR)and totally 13 representative isolates based on the years and regions of isolation and the ERIC-PCR genotypes were used in the genotyping by multilocus sequence typing(MLST).The results showed that the ERIC-PCR fingerprints of all the 92 Salmonella isolates had a genus ’s specific band at 250bp.The similarity coefficients of 71 S.derby isolates’fingerprints were 0.74～1.00 and the isolates were divided into 6 genotypes,of which type I and III were the dominant genotypes,accounting for 54.93%(39/71)and 19.72%(14/71),respectively.The similarity coefficients of 21 S.agona isolates’ fingerprints were 0.68～1.00 and the isolates were divided into 6 genotypes,of which genotype A(28.57%,6/21)and B(33.33%,7/21)were the dominant genotypes.Further investigations also found that different kinds of meats in the same market had the same Salmonella fingerprint,the same kind of meat had different Salmonella fingerprint and the same kind of meat with different sampling time had the same Salmonella fingerprint.The results of MLST genotyping showed that 7 S.derby isolates were belong to genotype ST40(19-20-3-20-5-22-22)and 6 S.agona isolates were belong to genotype ST13(3-3-7-4-3-3-7).Totally 71 isolates of S.derby were studied for the antibiotics resistance of five kinds of commonly used quinolones including natifloxacin,ciprofloxacin,ofloxacin,norfloxacin and gatifloxacin.The resistance phenotype of the strains was firstly detected by the conventional drug sensitive disk(K-B)method.Then the plasmid mediated quinolone resistance(PMQR)genes including qnrA,qnrB,qnrS,aac(6’)-lb-cr,oqxA,oqxB,and qepA were detected by PCR,and the quinolone resistance determinant genes gyrA and parC were sequenced and the amino acid site mutations were also analyzed.Finally,the relationship between the results of phenotyping and resistance-gene detection was also analyzed.The results showed that 71 S.derby isolates was strong resistance to the first generation of quinolone nalidixic acid(NA)by 19.72%,and the insensitivity rate was totally 43.66%.They were sensitive to the third generation of quinolones ciprofloxacin(71.83%),ofloxacin(74.65%)and norfloxacin(70.42%),but the intermediary rates of them were also high by 23.94%,25.35%,25.35%,respectively.The fourth generation of quinolone gatifloxacin was the most sensitive(84.51%)in the test,and the intermediary rate is 14.08%.PMQR genes was detected in 76.06%(54/71)of all the tested isolates,among them oqxA was the highest(53.52%),then followed by oqxB(49.30%),qnrS(29.58%)and aac(6’)-Ib-cr(25.35%),while those of qnrA(4.23%)and qnrB(5.63%)were low and qepA gene was not detected at all.32.39%(23/71)of the isolates carried 2 PMQR genes,and 12.68%(9/71)and 11.27%(8/71)of the isolates carried 3 and 4 PMQR genes,respectively.The coincidence rates of genes oqxA and oqxB positive and the NA resistant phenotype were all more than 70%,while those of aac(6’)-Ib-cr and qnrS were more than 40%.Totally 13 out of 14(92.86%)isolates that were resistant to NA,were found to have totally 21 amino acid mutation sites within the genes gyrA and parC.Among them,2 mutations D87N(35.71%,5/14)and S83I(21.43%,3/14)were found in gyrA and the major mutations of parC were T57S(42.86%,6/14)and T57V(21.43%,3/14).The results of the study demonstrated that the Salmonella genotypes of fresh meat sources were diverse in Guangxi,indicating that the Salmonella pollution had a wide range of sources.ERIC-PCR typing can be used to trace the source of Salmonella contaminated in the fresh meats.The resistance of Salmonella to quinolones was considerable serious,the site mutations of genes gyrA and parC were the main mechanisms of the resistance,while the carrying of PMQR gene/gens was also another important mechanism. |
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