适于制备参考菌株用沙门氏菌的鉴定和特性研究

Identification and Characteristic of Salmonella that could be Potentially Used for Reference Strain

目的:对分离自陕西、广东、福建、上海、北京、河南和四川等7省(市)的118株沙门氏菌进行染色镜检、生化和16S rDNA鉴定,β-内酰胺酶编码基因遗传稳定性和药敏性测定,为制备适于携带β-内酰胺酶类编码基因食源性致病菌检测用参考菌株奠定材料基础。方法:采用革兰氏染色、VITEK生化鉴定、MALDI-TOF-MS鉴定、16S rDNA基因扩增和序列测定分析鉴定菌株;采用临床和实验室标准协会推荐的琼脂稀释法测定沙门氏菌的药敏性;采用传代培养、PCR扩增、基因序列测定和基因库在线比对方法,确定沙门氏菌携带的与β-内酰胺类抗生素耐药相关编码基因的遗传稳定性。结果:118株沙门氏菌的生化特性、16S rDNA、MALDI-TOF-MS鉴定结果均符合沙门氏菌属的典型特征。菌株对氨苄西林、磺胺异噁唑和头孢噻呋(100.00%)耐药最为普遍,对四环素和萘啶酮酸等7种抗生素的耐药率菌均在50%以上。76株(64.41%)菌株携带bla_(TEM-1),其次分别为bla_(CTX-M-55)(60,50.85%)、bla_(OXA-1)(19,16.10%)、bla_(CTX-M-3)(18,15.25%)、bla_(CTX-M-15)(2,1.69%)、bla_(TEM-116)(2,1.69%)、bla_(CTX-M-123)(1,0.85%)和bla_(CTX-M-64)(1,0.85%)。筛选出的12株沙门氏菌,其携带的相关编码基因在传代过程均未丢失或发生突变。结论:4种方法对118株沙门氏菌的鉴定结果均与沙门氏菌属的特性吻合,12株代表性沙门氏菌携带的bla_(TEM-1)、bla_(CTX-M)和bla_(OXA-1)基因可在15代传代过程稳定遗传,具有制备β-内酰胺类抗生素耐药性编码基因检测用参考菌株的潜能。

Objective:One hundred and eighteen foodborne Salmonella isolates that previously recovered from 7 provinces(cities),which including Shaanxi,Guangdong,Fujian,Shanghai,Beijing,Henan and Sichuan,were reidentified and characterized via Gram staining morphology,biochemical characterization,16S rDNA gene sequencing;antimicrobial susceptibility;the genetic stability ofβ-lactam antibiotic resistance encoding genes of 12 representative isolates;for reference strain preparation forβ-lactam antibiotic resistance encoding gene detection among foodborne pathogens.Methods:To reidentify the isolate,Gram stain,VITEK biochemical and MALDI-TOF MS identification,and 16S rDNA gene amplification and sequence analysis were used.The antibiotic susceptibility was tested via agar dilution method issued by the Clinical and Laboratory Standards Institute.The genetic stability ofβ-lactam antibiotic resistance encoding genes was determined using subculture,PCR,DNA sequencing and online BLAST software of the GenBank database.Results:The biochemical characteristics,sequence of 16S rDNA,flight mass spectrometry of proteins of 118 Salmonella were consistent with those of typical Salmonella.Among 118 Salmonella isolates,antibiotic resistance was commonly found to ampicillin(100.00%),sulfisoxazole(100.00%)and ceftiofur(100.00%);more than 50%of the isolates resisted to 7 kinds of antibiotics.blaTEM-1(64.41%)was most frequently detected among the isolates,the detection rates of blaCTX-M-55,blaOXA-1,blaCTX-M-3,blaCTX-M-15,blaTEM-116,blaCTX-M-123 and blaCTX-M-64 positive isolates were 50.85%,16.10%,15.25%,1.69%,1.69%,0.85%and 0.85%,respectively.No loss or mutation was detected among the base or gene fragment ofβ-lactam antibiotic resistance encoding genes of the 12 screened representative Salmonella isolates during subcultures.Conclusion:Results of 4 different identifications consisted to those of the typical Salmonella should to be;the antibiotic resistance encoding genes blaTEM-1,blaCTX-Ms and blaOXA-1 carried by the isolates could be stably inherited during subculture,the 12 representative Salmonella were verified to have the potentiality to produce reference strain forβ-lactam antibiotic resistance encoding gene detection.