生牛乳中耐药芽孢杆菌的分离鉴定及四环素抗性基因tetL定位研究

Antibiotic resistance of Bacillus spp.isolated from raw milk and the localization of tetL gene

为检测生牛乳中耐热芽孢杆菌的耐药性并定位耐药基因,本研究对34个生牛乳样品分离的芽孢杆菌抗生素耐药性进行研究。耐药实验结果表明:66株芽孢杆菌在固有抗生素耐药性中氨苄西林耐药率高达71.21%,青霉素耐药率高达75.75%。在获得性耐药中7株芽孢杆菌具有克林霉素抗性,1株沙福芽孢杆菌具有红霉素抗性,5株蜡样芽孢杆菌具有四环素抗性。获得性耐药的菌株中,四环素抗性的蜡样芽孢杆菌S7和S11携带有质粒。进一步通过芽孢杆菌四环素耐药基因的PCR筛查及测序分析,确定S7和S11中四环素抗性基因为tetL。通过反向PCR及DNA测序确定蜡样芽孢杆菌S7的tetL基因位于质粒pBCs7上。进一步的序列分析表明质粒pBCs7能够编码的移动蛋白Mob,而Mob蛋白一般能够介导质粒的接合转移。因此,推测质粒pBCs7可能具有转移性,蜡样芽孢杆菌S7的tetL基因存在水平转移风险。

In order to study the antibiotic resistance and the localization of antibiotic resistance gene,the Bacillus strains isolated from 34 raw milk samples were investigated for the antibiotic resistance.Antibiotic resistance tests showed that the antibiotic resistance rate of 66 Bacillus strains to ampicillin and penicillin reached up to 71.21%and 75.76%,respectively.Among the acquired antibiotic resistance,7 Bacillus strains were resistant to clindamycin.One Bacillus safensis was resistant to erythromycin.Five Bacillus cereus strains were resistant to tetracycline.Plasmid profiling indicated that Bacillus cereus S7 and S11 showed tetracycline resistance harbored plasmid.In order to determine the tetracycline resistance gene,specific PCR and DNA sequencing were performed.The tetracycline resistance gene was found to be tetL in S7 and S11.Moreover,the tetL gene in S7was determined in the plasmid pBCs7 by inverse PCR.Plasmid sequencing and analysis showed that the plasmid pBCs7 also harbored gene mob_1and mob_2 encoding plasmid mobilization protein,suggesting the probability of its conjugational transfer.Therefore,the tetracycline resistance gene tetL in Bacillus cereus S7 could be a risk of horizontal gene transfer.