益生乳杆菌质粒抗生素抗性基因与其耐药性的相关性探讨

采用K-B药敏纸片法检测了5株具有潜在益生乳杆菌的耐药性,通过质粒消除,分析了菌株质粒与耐药性之间的联系,应用PCR确定了质粒决定的耐药基因。5株乳杆菌对万古霉素、多粘菌素B以及链霉素等7种抗生素普遍表现出抗性,但主要对四环素敏感。采用SDS与SDS-高温两种方法消除戊糖乳杆菌CH8的质粒后,菌株CH8表现出头孢噻吩和氯霉素敏感性。设计β-内酰胺类抗性基因blr、ECP-1569和nps-1以及氯霉素抗性基因cmlA、cat和cmlA1的引物进行PCR,与目的产物测序比对表明,戊糖乳杆菌CH8的质粒上含有β-内酰胺类抗性基因blr,该基因与其头孢噻吩抗性有关。该研究为探讨乳酸菌的抗药基因转移性提供了前期基础,有助于益生乳杆菌安全性评价体系的建立与完善。 

Possible connections between the antibiotic-related plasmids of potentially probiotic Lactobacillus strains and the antibiotic resistance

The tolerances of 5 potentially probiotic lactobacilli strains to antibiotics were investigated with K-B paper-diffusion method. The antibiotic-related resistance genes obtained from PCR were sequenced. Five lactobacilli strains showed their susceptibility to tetracycline, but tolerated to 7 antibiotics including bacitracin, polymyxin B, kanamycin, and nalidixic acid. All of five strains, Lactobacillus pentosus CH8 was used as case for further study. After both SDS and SDS-heating methods were chosen to remove the plasmids from strain CH8 cells, this strain showed its susceptibility to cephalothin and chloromycetin. Analysis of the PCR products, targeted from the primers of β-lactam resistance-related genes including blr, ECP-1569 and nps-1 as well as chloromycetin resistance-related genes including cmlA, cat and cmlA1, indicated that the plasmid of L. pentosus CH8 contained the blr gene that caused this bacteria to tolerate cephalothin. The elimination of plasmids by SDS or SDS -heating confirmed that there might be the potential relationship between the plasmid ’s presence of these strains and their antibiotic resistances. The detection of the plasmid’s presence should provide a novel way for the safety evaluation of potential probiotic lactobacilli bacteria, and thus be helpful for the further investigation of resistance gene transferability in human body at a molecular level.