Application Of Culturomics In The Study Of Vaginal Microbiota And The Interactions Relationship Between Vaginal Microbiota

Objective: 1.High-throughput culture of female vaginal samples of healthy and bacterial vaginosis was conducted using culturomics to explore the significance of high-throughput culture methods for vaginal microbiota research.2.Based on the culture results of the culturomics,the interaction between Lactobacillus and other vaginal bacteria from different vaginal microbial types was investigated.Methods: The vaginal secretions of seven healthy women and eight women with bacterial vaginosis were collected.Seven different media were used to culture the vaginal secretions in high-throughput and extract the total bacterial DNA.The bacteria isolated and cultured were identified by Clin-ToF time-of-flight mass spectrometry in combination with 16 S rRNA sequencing.High-throughput sequencing was also performed using the 16 S rRNA V1V2 variable region.Based on the results of the culturomics,strains isolated from one healthy woman and two women with bacterial vaginosis were selected for bacterial interaction studies.After co-culture of Lactobacillus with seven vaginal-related bacteria for 0h,24 h,48h,and72 h,the real-time quantitative PCR(qPCR)method was used to analyze the interaction of vaginal bacteria.Results: 1.A total of 1328 vaginal bacteria were isolated from vaginal secretions in seven healthy women and eight bacterial vaginosis women,belonging to 33 genera and 92 species.The most commonly isolated genus is Lactobacillus,Gardnerella,and Streptococcus,and 535 strains,221 strains,and 146 strains were isolated,respectively.At the same time,16 genera were detected by high-throughput sequencing of 16 S rRNA V1V2 region in seven healthy women and eight bacterial vaginosis women.The detection overlap rate of the two is only24.24%.2.In the same body,Lactobacillus crispatus isolated from the healthy sample HV4 inhibited the growth of Staphylococcus epidermidis and Streptococcus anginosusLactobacillus jensenii isolated from bacterial vaginosis samples BV4 and BV7 also inhibited the growth of Staphylococcus epidermidis,Streptococcus anginosus,Streptococcus agalactiae,and Atopobium vaginae.But Lactobacillus iners did not inhibit the growth of Streptococcus anginosus and Streptococcus agalactiae.3?The ability of Lactobacillus to inhibit non-lactobacillus is different in different individuals.The Lactobacillus crispatus in the sample HV4 inhibited the non-lactobacillus in the sample BV4,but did not inhibit the non-lactobacillus in the sample BV7.The Lactobacillus jensenii in the sample BV4 had no inhibitory effect on Streptococcus anginosus in the sample HV4,but had an inhibitory effect on Staphylococcus epidermidis;it did not inhibit the non-lactobacillus in the sample BV7.The Lactobacillus jensenii and Lactobacillus iners in the sample BV7 had an inhibitory effect on the non-lactobacillus bacteria except the Streptococcus anginosus in the sample HV4.Conclusion: 1.The vaginal microbiota of the same sample was studied by the culturomics method and high-throughput sequencing method.The results were quite different,and the detection overlap rate of the two samples was only 24.24%.2.This study found that four strains of Lactobacillus from the vaginal source have some antagonistic effects on non-lactobacillus,but the inhibitory ability of different strains is significantly different,indicating that there may be differences in the inhibition of Lactobacillus strains.