猪源屎肠球菌生物学特性分析及电转化条件的优化

Identification of biological characteristics of pig-derived Enterococcus faecium and optimization of its electro-transformation conditions

为了获得屎肠球菌基因工程菌株,试验首先用选择性培养基对猪新鲜粪便中的屎肠球菌进行分离鉴定,然后对屎肠球菌分离株进行毒力基因检测、药敏试验、耐受试验及抑菌效果检验,针对该分离株进行电转化条件(甘氨酸添加量、培养时间、洗涤缓冲液、质粒大小、质粒质量浓度、电压、复苏时间、电转杯规格)的优化,并采用PCR方法对转化菌株进行验证,最后分析培养基中的氯霉素和葡萄糖对转化菌株生长的影响。结果表明:从猪新鲜粪便中分离到1株屎肠球菌,该分离株不含有毒力基因Asa1、cylA、efaA、esp、gelE和hyl,对β-内酰胺类抗生素(氨苄西林、头孢噻吩、青霉素G)、四环素类抗生素(米诺环素)和氯霉素类抗生素(氯霉素)敏感,对氨基糖苷类抗生素(阿米卡星、卡那霉素、庆大霉素)、大环内酯类抗生素(红霉素)和四环素类抗生素(四环素)耐药;在25℃生理盐水中静置培养12.5 h的存活率没有显著变化(P>0.05),50℃以下水温下静置15 min存活率接近100%,对强酸和胆盐具有一定的耐受能力,对大肠杆菌、金黄色葡萄球菌和沙门氏菌具有一定的抑制作用。将该分离株作为感受态细胞进行电转化,感受态细胞制备过程中培养基中甘氨酸添加量为2 g/mL、培养时间为16 h、用洗涤缓冲液Ⅴ(300 g PEG1000与700 mL超纯水的混合溶液)进行洗涤可获得较高的转化效率,电转化过程中质粒pAM4010k、质粒质量浓度为0.878 g/L、电压为1400 V、复苏时间为120 min、用1 mm电转杯可获得较高的转化效率。经PCR验证,该分离株可成功导入目的质粒。培养基中是否含有氯霉素对转化菌株生长没有明显影响,含葡萄糖对转化菌株的生长具有促进作用。说明本试验分离到的屎肠球菌是1株潜在的益生菌,具有开发为基因工程菌的潜力。

In order to obtain a genetically engineered strain of Enterococcus faecium,in this experiment,firstly,the Enterococcus faecium in fresh feces of pigs was isolated and identified by selective medium,and then virulence gene detection,drug susceptibility test,tolerance test and bacteriostatic effect of the isolated strain were tested.The electro-transformation conditions(glycine addition,culture time,washing buffer,plasmid size,plasmid mass concentration,voltage,recovery time,and transformation cup specifications)were optimized for the isolated strain,and the transformed strains were verified by PCR.Finally,the effect of chloramphenicol and glucose in the medium on the growth of transformed strains was analyzed.The results showed that one strain of Enterococcus faecium was isolated from fresh pig feces,which did not contain the virulence genes Asa1,cylA,efaA,esp,gelE and hyl,being sensitive toβ-lactam antibiotics(ampicillin,cefothiophene,penicillin G),tetracycline antibiotics(minocycline),and chloramphenicol antibiotics(chloramphenicol),and resistant to aminoglycoside antibiotics(amikacin,kanamycin,gentamicin)and macrolides(erythromycin)and tetracycline antibiotics(tetracycline).The survival rate of static culture in normal saline at 25℃for 12.5 h did not change significantly(P<0.05),and the survival rate was close to 100%while standing for 15 minutes at water temperature below 50℃,with a certain tolerance to strong acids and bile salts;it had a certain inhibitory effect on Escherichia coli,Staphylococcus aureus and Salmonella.The isolated strain was made as competent cells for electro-transformation;and the glycine addition amount of 2 g/mL in medium,the culture time of 16 h,and the washing buffer V(300 g PEG1000 mixed with 700 mL ultrapure water)for washing was used to obtain high transformation efficiency during the working process of competent cells.During the electro-transformation process,plasmid mass concentration of 0.878 g/L pAM4010K,voltage of 1400 V,recovery time of 120 min,and 1 mm transformation cup could obtain high conversion efficiency.PCR proved that the bacterium could successfully import the target plasmid.Whether chloramphenicol was contained in the medium had no significant effect on the growth of transformed strain,and glucose contained had a promoting effect on the growth of transformed strain.The results suggested that the Enterococcus faecium isolated in this test was a potential probiotic strain with the potential to develop into genetically engineered bacteria.