南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (12): 1809-1815.doi: 10.12122/j.issn.1673-4254.2021.12.08

• • 上一篇    下一篇

健康妇女生殖道内格氏乳杆菌的分离鉴定与安全性评价

苏炳森，操龙斌，郑介婷，张 琳，张子康，邱 峰   

  1. 广东省中山火炬开发区医院检验科，广东 中山 528437；南方医科大学第七附属医院医学检验科，广东 佛山 528244；南方医科大学中医药学院，广东 广州 510515
  • 出版日期:2021-12-20 发布日期:2022-01-05

Isolation, identification and safety evaluation of Lactobacillus gasseri strain LGV03 isolated from the vagina of healthy women

SU Bingsen, CAO Longbin, ZHENG Jieting, ZHANG Lin, ZHANG Zikang, QIU Feng   

  1. Department of Clinical Laboratory, Zhongshan Torch Development Zone Hospital, Zhongshan 528437, China; Department of Laboratory Medicine, Seventh Affiliated Hospital of Southern Medical University, Foshan 528244, China; School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
  • Online:2021-12-20 Published:2022-01-05

RichHTML

18

PDF

412

摘要： 目的 对健康妇女生殖道内分离得到的菌株LGV03进行鉴定及安全性评价。方法 通过稀释平板涂布法和划线分离法在MRS固体培养基上对女性生殖道分泌物中的菌株进行分离纯化和菌落形态观察；采用革兰氏染色鉴别分离菌株的形态与革兰氏阴阳性；采用VITEK ANC鉴定卡对菌株LGV03进行生理生化性质的鉴定；采用DNA提取、PCR扩增、16S rDNA和pheS产物测序和数据分析对菌株LGV03进行分子生物学鉴定和系统发育分析鉴定；采用血琼脂平板检测菌株LGV03的溶血性；采用E-test法检测菌株LGV03对青霉素、氨苄西林、美罗培南、万古霉素、红霉素、利奈唑胺、克林霉素的敏感性；菌株LGV03以体积分数5%接种于MRS培养基中，于37 ℃发酵后，运用生化分析仪检测其发酵液中的乳酸浓度；最后，通过小鼠急性毒性试评价菌株LGV03的安全性。结果 菌株LGV03经鉴定，其培养特性为：在MRS固体培养基上，37 ℃厌氧生长良好，菌落呈圆形、白色、表面湿润、不透明、边缘整齐。菌株LGV03革兰氏染色呈阳性，细胞呈杆状，菌体大小为0.4~0.5 μm×0.9~6.3 μm。结合生理生化试验结果可初步鉴定分离菌株LGV03为乳杆菌属。同时，经分子生物学鉴定和系统发育树显示，菌株LGV03被鉴定为格氏乳杆菌。溶血性试验结果显示格氏乳杆菌LGV03在血平板上未产生溶血环。药敏试验结果显示，格氏乳杆菌LGV03对青霉素、氨苄西林、美罗培南、万古霉素、红霉素、利奈唑胺敏感，最小抑菌浓度分别为0.048、0.19、0.38、0.75、0.064、0.75 μg/mL，而对克林霉素耐药最小抑菌浓度为6 μg/mL。菌株LGV03在MRS培养基中可产生乳酸，约18 h后达到饱和浓度，最终乳酸浓度达到1.72 mg/mL。小鼠毒性实验结果显示，与生理盐水组相比，格氏乳杆菌LGV03组小鼠的体质量变化无统计学意义（P> 0.05）。格氏乳杆菌LGV03组小鼠的器官质量和脏器指数与生理盐水组相比差异均无统计学意义（P>0.05）。结论 从女性生殖道中分离的菌株LGV03被鉴定为格氏乳杆菌，其具有潜在的益生效应和安全性。

关键词: 格氏乳杆菌；分离鉴定；安全性评价

Abstract: Objective To characterize the bacterial strain LGV03 isolated from the reproductive tract of healthy women and evaluate its safety. Methods The bacterial strains were isolated and purified using MRS agar by dilution plating and streak plate method. Gram staining was used to observe the morphology of the isolated strains and identify Gram-negative and -positive strains. The physiological and biochemical properties of the bacteria was assessed using VITEK ANC card. Strain LGV03 was identified by molecular biology and phylogenetic analysis and tested for hemolysis on blood agar plates. E-test method was performed to examine the sensitivity of strain LGV03. The concentration of lactic acid in the culture supernatant of LGV03 was measured using a biochemical analyzer. The safety of LGV03 was evaluated in mice using acute oral toxicity test. Results The isolated bacterial strain LGV03 showed good growth under anaerobic conditions at 37 ℃ on MRS medium and formed round, milky white, moist, and opaque colonies with neat margins. The strain LGV03 was positive for Gram staining, rod-shaped with a size range of (0.4-0.5) × (0.9-6.3) μm, and was identified as Lactobacillus gersonii. LGV03 was sensitive to PG, AM, MP, VA, EM and LZ. In MRS medium LGV03 could produce lactic acid, which reached its saturation concentration after 18 h with a final concentration of 1.72 mg/mL. The mice with acute oral exposure to LGV03 showed no significant changes of body weight or organ weight as compared with normal saline-treated mice. Conclusion The bacterial strain LGV03 isolated from the female reproductive tract was identified as Lactobacillus gersonii with potential probiotic effects and good safety profile.

Key words: Lactobacillus gersonii; separation and identification; safety evaluation