牛源大肠杆菌四环素类抗生素耐药相关基因的筛选及鉴定

Screening and identification of tetracycline antibiotic resistant genes of bovine Escherichia coli

为筛选与鉴定牛源大肠杆菌耐四环素类药物的耐药相关基因,本研究从67份犊牛腹泻病料样品中分离细菌。采用生化和PCR方法鉴定分离菌,通过K-B法检测分离菌的药物敏感性,并选定一株多重耐药菌;通过杂交的方法,将供体菌携带的转座子插入到该分离菌(亲本菌)的染色体中,并通过转座子携带的卡那霉素抗性筛选转座子的插入突变株文库;分别利用含1/2 MIC四环素、米诺环素、强力霉素和卡那霉素的LB培养板对插入的突变株进行筛选,以获得对四环素类药物敏感的突变株;利用Taq I对四环素类药物敏感突变株基因组酶切后,以随机方式连接linker并作为模板经套式PCR扩增并测序,将测序结果与大肠杆菌O157株的染色体序列比对以确定转座子破坏的基因。结果显示,通过细菌的分离鉴定与药敏试验选定了一株耐18种抗生素的牛源大肠杆菌;经杂交方法及卡那霉素抗性筛选共获得3 000株转座子插入突变的大肠杆菌;且共筛选出10株对四环素类抗生素均敏感的突变株;通过套氏PCR扩增及序列比对分析结果显示,四环素类抗生素突变株中转座子破坏的基因为磷酸烯醇式丙酮酸蛋白磷酸转移酶I(ECs4877)和H-NS核蛋白(ECs1739)。本研究结果首次证实Ecs4877和Ecs1739基因是牛源大肠杆菌潜在的四环素类药物的耐药相关基因。本研究筛选出的耐药相关基因为深入了解大肠杆菌的耐药机制、寻找药物作用的新靶点具有重要意义。

To screen and identify tetracyclines antibiotic resistance genes of bovine Echerichia coli. In this study, bacteria isolated from 67 samples of diseased calf with diarrhea were identified by biochemical and PCR methods, and drug sensitivity tests were performed on clinically isolated E.coli strains for multidrug resistant strains. A mariner transposon was used to construct transposon mutant library of E.coli, and the transposons carried kanamycin resistance was used to screen the transposon mutant library. The mutants were screened by LB culture plates containing 1/2 MIC antibiotics to obtain the mutants sensitive to tetracycline. Taq I was used to digest the genomic DNA of tetracycline drug-sensitive mutants. Linker was connected in a random way and used as a template for nested PCR amplification and sequencing. The sequencing results were compared with the genome sequences of E.coli O157 to determine the genes disrupted by transposons. The results showed that a bovine E.coli strain resistant to 18 kinds of antibiotics was selected by isolation, identification and drug sensitivity test. A total of 3 000 transposon mutants of E.coli were obtained by hybridization and kanamycin resistance screening. The tetracyclines antibiotics were used to screen for the antibiotics sensitive mutants from the library, and ten mutants, which were sensitive to tetracyclines, were selected from the library.The transposon insertion sites were then determined by nested PCR and sequence alignment. Transposon insertion sites analysis results showed that the disrupted genes were ecs4877(PEP-protein phosphotransferase enzyme I) and ECs1739(global DNAbinding transcriptional dual regulator H-NS). These results confirmed for the first time that Ecs4877 and Ecs1739 genes were potential tetracycline resistance related genes in bovine Escherichia coli. The genes obtained were of great significance to understand the genetic basis of drug resistance of E. coli and searching for new drug targets.