羊源沙门氏菌的分离鉴定及致病性和耐药性分析

Isolation,Identification and Analysis of Pathogenicity and Drug Resistance of Salmonella from Sheep

【目的】分离张家口某地区羊源沙门氏菌并检测其血清型、毒力基因、耐药性及耐药基因,分析分离株表型和基因的相关性和差异性。【方法】将样品用选择培养基增菌并纯化培养,挑选疑似的沙门氏菌单菌落进行革兰氏染色、镜检和生化鉴定。根据沙门氏菌属特异性基因invA的核苷酸序列进行PCR和血清型鉴定。利用SPF昆明小鼠对分离株进行致病性试验并测定其半数致死量。PCR扩增毒力岛基因hilA、avrA、sseL、ssaQ、mgtC、siiD和sopB,肠毒素基因stn,质粒毒力基因spvR。采用Kirby-Bauer纸片扩散法进行药敏试验,PCR扩增β-内酰胺酶耐药基因bla_(TEM)、bla_(CMY)和bla_(OXA),氟喹诺酮耐药基因qnrS、oqxA和oqxB,磺胺类耐药基因sul1、sul2和sul3,四环素类耐药基因tetB及氨基糖苷类耐药基因aadA1。根据PCR检测结果,将扩增的毒力基因和耐药基因进行测序,并与NCBI中相应的参照基因进行BLAST比对分析。【结果】分离得到4株羊源沙门氏菌,血清型鉴定均为鼠伤寒沙门氏菌。分离株对小鼠具有致病性,4株分离株的半数致死量为5.67×10^(7)~6.45×10^(7) CFU。分离株可检出毒力岛基因hilA、avrA、sseL、ssaQ、mgtC、siiD和sopB,肠毒素基因stn及质粒毒力基因spvR,检出率均在50%以上。分离株对复方新诺明、利福平、林可霉素、青霉素、氨苄西林耐药,对庆大霉素、环丙沙星敏感。分离株可检出β-内酰胺类耐药基因bla_(TEM)、氟喹诺酮耐药基因qnrS、磺胺类耐药基因sul1和sul2。【结论】本研究分离的羊源沙门氏菌的毒力表型与染色体和质粒携带的毒力基因有关。分离菌株携带β-内酰胺类、磺胺类耐药基因,与耐药表型相符,临床上可将庆大霉素和环丙沙星作为首选药。

【Objective】The study was aimed to isolate and detect the serotype,virulence gene,drug resistance and drug resistance gene of Salmonella from sheep in Zhangjiakou,and analyze the correlation and difference between phenotype and gene.【Method】The collected samples were inoculated and purified with selective medium.Suspected single colonies were selected for Gram staining,microscopic examination and biochemical identification.PCR identification and serotype identification were carried out according to the nucleotide sequence of Salmonella specific gene invA.SPF Kunming mice were used to test the pathogenicity of the isolates and determine the median lethal dose.Then the virulence island genes hilA,avrA,sseL,ssaQ,mgtC,siiD and sopB,enterotoxin gene stn,plasmid virulence gene spvR were amplified.Kirby-Bauer disk diffusion method was used for drug sensitivity test to amplify drug resistanceβ-lactamase genes including bla_(TEM),bla_(CMY),bla_(OXA),fluoroquinolone genes qnrS,oqxA,oqxB,sulfonamide resistance genes sul1,sul2,sul3,tetracycline resistance gene tetB and aminoglycoside resistance gene aadA1.According to the PCR results,the target virulence gene and drug resistance gene were sent to sequence,and BLAST comparison analysis was carried out with the corresponding reference gene in NCBI.【Result】4 strains of Salmonella from sheep were isolated and identified as Salmonella Typhimurium.The isolates were pathogenic to mice,and the median lethal doses of 4 isolates were between 5.67×10^(7) and 6.45×10^(7) CFU.The isolated strains could detect virulence island genes hilA,avrA,sseL,ssaQ,mgtC,siiD,sopB,enterotoxin gene stn and plasmid virulence gene spvR,and the positive detection rates were all>50%.The isolates had strong resistance to compound sulfamethoxazole,rifampicin,lincomycin,penicillin and ampicillin,and were sensitive to gentamicin and ciprofloxacin.Drug resistance was detected in the isolates,β-lactam gene bla_(TEM),fluoroquinolone gene qnrS and sulfonamide resistance genes sul1 and sul2 were positive.【Conclusion】The virulence phenotype of Salmonella isolated from sheep was related to the virulence genes carried by chromosomes and plasmids.The isolated strains carriedβ-lactam and sulfonamide resistance genes,which were consistent with the drug resistance phenotype.Gentamicin and ciprofloxacin could be used as the first choice in clinic.