适于β-内酰胺类抗生素耐药机制检测用参考菌株的研制

Preparation of Reference Strain forβ-Lactam Antibiotic Resistance Mechanism Detection

目的:为了满足系列食品中食源性致病菌携带的β-内酰胺类抗生素耐药机制快速检测需求,开展了携带此类抗生素耐药性编码基因沙门氏菌参考菌株定性标准样品的研制,以提供溯源性强、评价准确度高、易在各检测机构间开展耐药微生物检测的技术保障。方法:活化实验室-80℃保存的3株沙门氏菌,通过PCR、DNA测序和BLAST比对验证其所携带的β-内酰胺类抗生素耐药机制编码基因,同时检验目标基因的遗传稳定性。在真空冷冻干燥条件下制备活菌量为10^(6)~10^(7) CFU/瓶的菌株标准样品。按照GB/T 15000.3-2008要求对参考菌株标准样品进行均匀性检验,使用方差分析法(F检验)对结果进行统计分析、均匀性评价。分别于37,4℃和-80℃模拟运输和贮藏条件,检验参考菌株的稳定性。委托具有检验资质的7家实验室对参考菌株进行联合定值。结果:3株候选参考菌株中1株同时携带β-内酰胺类抗生素耐药机制编码基因blaCTX-M-55/blaTEM-1,1株同时携带blaTEM-1/blaOXA-1/blaCTX-M-l5,1株携带blaCTX-M-3,在15代内均可稳定遗传。制备的参考菌株均匀性良好。在37℃贮藏13 d,4℃贮藏90 d,-80℃贮藏360 d,活菌量基本稳定在×10^(4) CFU/瓶以上。7家实验室联合定值结果表明,制备的参考菌株特性值与目标一致,可作为标准样品使用。结论:遵循标准样品制备标准和规范,研制了3株适于β-内酰胺类抗生素耐药机制检测用参考菌株,可用于食源性细菌β-内酰胺类抗生素耐药机制的快速检测。

Objective:In order to quickly detect the mechanism ofβ-lactam antibiotic resistance carried by food-borne pathogens in a series of foods,the qualitative standard samples of Salmonella reference strain carryingβ-lactam antibiotic resistance coding genes were developed.It will provide the technical support of strong traceability,high evaluation accuracy and be conductive to widely carry out the detection of drug-resistant microorganisms among various testing institutions.Methods:Salmonella isolated previously and stored at-80℃were subcultured on LB agar plate.3 Salmonella representatives were selected and the encoding genes ofβ-lactam antibiotic resistance carried by the 3 strains were verified via PCR amplification,DNA sequencing and BLAST.The genetic stability of the target genes was simultaneously identified.The standard samples of the reference strain with live bacteria amount of 10^(6)~10^(7) CFU/vial were prepared by vacuum freeze-drying technique.On the basis of GB/T 15000.3-2008,the three reference strains were tested for homogeneity,the results were statistically analyzed via F test to evaluate their uniformity.The prepared reference strains were stored at 37℃,4℃and-80℃simulated,respectively,to test their transport stability and storage stability.Seven laboratories with checkout qualifications were commissioned to jointly evaluate the reference strains'characteristic values.Results:Theβ-lactam antibiotic resistance mechanism associated encoding genes of blaCTX-M-55/blaTEM-1 in one Salmonella,of blaTEM-1/blaOXA-1/blaCTX-M-l5 in one Salmonella,and of blaCTX-M-3 in one Salmonella can stably inherit in 15 generations.The homogeneity of the prepared reference strains meets the necessary requirements.When stored at 37℃for 13 d,4℃for 90 d and-80 for 360 d,the viable bacteria amount was basically stable and above×10^(4) CFU/vial.The characteristic values of the reference strains were consistent with 7 laboratories'and indicated that the prepared strains could be used as the standard samples.Conclusions:In accordance with the standard sample preparation standards and specifications,three reference strains carryingβ-lactam antibiotic resistance encoding genes were developed.They meet the requirements of standard samples and can be used for rapid detection ofβ-lactam antibiotic resistance mechanism of food-borne bacterial.