A Study On The Relationship Between Vaginal Microbiota And The Occurrence Of Bacterial Vaginosis

BackgroundsBacterial vaginosis (BV) is a disruption or drift of normal microbiota of bacteria in the vagina where high concentration of aerobia and anaerobia take the place of lactobacillus and become the dominant bacteria. It's a concurrent condition with high incidence among pregnant women and is the main cause of severe complications such as low birth weight of foetus, premature birth, neonatal death and secondary infection after delivery and is also the most important risk factor for premature rupture of fetal membranes (PROM). At present, the recurrence rate of BV is more than 30% after treatment by Metronidazole and (or) Clindamycin due to the laboratory diagnosis of BV is not based on determination of pathogenic bacteria. Some of the BV related bacteria are hard to culture and some are difficult to detect by traditional methods, which hamper the exploration of the cause of BV. Nowadays, the condition remains devoid of effective prevation and cure mehods.With the development and application of culture-independent molecubiological methods in recent years, it is possible for researchers to explore the structure and dynamic shift of microbiota. In this study, women with and without BV was recruited as the research subject and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing techniques were used to analysis the genetic information of 16S rRNA genes(16S rDNA) of the bacteria derived from swabs of these woman participants.The sequence information of 16S rDNA of every detected bacterium was identified with gene bank data to affiliate each to the proper species. In addition, DGGE Gel Image System was used to calculate the abundant proportion of every bacterial species to the whole microbial population in one sample (vaginal swab). Eventually, appropriate statistical methods was used to analyze the relationship between BV and bacterial composition, as well as the relationship between BV and other risk factors.ObjectivesTo explore the BV-related bacteria; to investigate the relationship of BV with the relative abundance of normal and BV-related bacteria to help researchers bring out a clue for the study on etiology of BV, also a basis for the prevention of BV.Contents and Methods1.The methods for detecting vaginal microbiotaThe diagnosis of BV in this study was based on the method of Gram stain and Nugent score with the help of a commercial diagnostic kit. Based on the technique of DGGE, we redesigned and made the key component of the DGGE device, i.e. the 'sandwich' gel-making plate to modulate the gel bands resolution and to extend the flexible application fields of DGGE device. The genetic information of 16S rDNA for every detected bacterium was identified to determine its affiliation to specific species by gel band excision and reamplification of DNA template imbeded in the corresponding band and sequencing of the reamplified DNA product. The species of microorganism were identified by 97.5% homology compared to standards in NCBI gene bank. DGGE Gel Image System was used to calculate the abundant proportion of every bacterial species to the whole microbial population in each sample (vaginal swab). A combined qualitative and quantitative analysis was completed by incorporating DAN sequencing with DGGE Gel Image.2. Association of the composition of vaginal microbiota and other risk factors with BVA questionnaire, which contains the demographic characteristics, habits of hygiene, marriage and birth history and gynecologic diagnoses was designed and used to investigate the women participants. A correspondence analysis was used to identify the BV-related bacteria. The difference of detection ratios of every bacterium between the two groups was analyzed by chi-square statistics and the difference of relative abundance of every bacterium between the two groups analyzed by t-test. A stepwise Logistic regression analysis was used to study the relation intensity of latent influencing factors to BV condition. All statistics were analyzed by SPSS 11.5 and SAS 9.2. ResultsPart I:A study on methods for analysis of microbiota in vagina1. Totally 59 species of bacteria were detected among 196 participant women, of wich 14 species were found in 68 normal women and 55 species identified in 128 BV affected women.2. Averagelly 4.05 kinds of bacteria were detected in vagina microbial community of BV(+) women, varing from 1 to 9 kinds. Whereas only 1-5 species were detected in each woman of BV(-) group with an average of 2.59 species per woman, which was significently lower than that detected in BV(+) women(t=5.66, P<0.001).3. There are 5 species at the top of prevalence that ranked as Lactobacillus iners, Lactobacillus ultunensis, Bifidobacterium bifidum, Gardnerella vaginalis and Bifidobacterium breve) for the normal group whereas the corresponding list of the 5 species for the BV positive group is composed of Lactobacillus iners, Gardnerella vaginalis), Lactobacillus ultunensis, Atopobium vaginae and Burkholderia fungorum.4. A phylogenetic analysis divide the total 59 bacterial species into 2 groups, respectively titled as normal vaginal bacteria and BV-related bacteria. The group of normal is composed of Bifidobacterium bifidum, Peptostreptococcus massiliae, Leptotrichia amnionii, Lactobacillus iners, Lactobacillus ultunensis and Lactobacillus johnsonii and the other 53 species belong to BV-related bacteria.5.There are 2 species to be found having<97.5% similarities in their 16S rDNA sequences compared with standard species in gene bank database(i.e 96.835% vs Lactobacillus ultunensis and 86.184% vs Mycoplasma muris).Part II:A study on risk factors of bacterial vaginosisThe statistical study was carried out in accordance with the study of Part I to evaluate th relation of vagina bacterial composition and other risl factors to BV condition:1. Vaginal bacteria related to bormal and to BV group:There are Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus iners, Lactobacillus ultunensis and Staphylococcus epidermidis to be found being the innate inhabitant in normal vagina microbiota whereas Algoriphagus aquatilis, Atopobium vaginae, Burkholderia fungorum, Gardnerella vaginalis and Megasphaera genomosp are presumed to have relation to BV.2. The structural characteristic of normal and BV positive vaginal microbiota:(1) Lactobacillus sp is the most predominant species both in normal and BV positive group, and the prevalence of this species is consistent between the two groups(92.97% vs 95.59%; exact P=0.548), but the mean proportion of Lactobacillus sp to the whole microbial population colonized in vaginal tract of normal women was sharply higher than that calculated from BV(+) women(72.84% vs 51.27%; t=4.440, P<0.001).(2) Gardnerella vaginalis, commonly considered as the indicator bacterium for the diagnosis of BV, is present in both groups, but the prevalence rate and relative abundant concentration are both higher in BV positive group than in normal group.(3) Atopobium vaginae and Prevotella timonensis are BV-related bacteria in accordance with previous reports.(4) Three newly discovered species in this study, i.e, Burkholderia fungorum, Algoriphagus aquatilis, and Megasphaera micronuciformis are more likely to be considered as BV-related bacteria.Their real association with BV need verification due to the lack of relevant study.(5) The load of total bacteria in BV positive group is higher than in normal group.3. The discovery of other risk factors of BVLesser marriage age, higher vaginal pH value, lower vaginal lactobacillus content are risk factors associated with BV; women in pregnancy have 36 times risk to affect BV than those already given birth to more than one child (P=0.0002);women who have not experienced gestation and parity are more likely to be infected with BV by a 4.08 times likelihood than those that have completed children-delivery, but the difference is not statistically significant. Frequently vagina douching (wash with water or with disinfectant) is a risk factor compared with occasionally vagina douching. Women with an educational background of high school or above have 4.98 times risk for BV than those with an educational background of junior school or below.Conclusions1. There are Algoriphagus aquatilis, Burkholderia fungorum, Megasphaera genomosp to be found out in this study as the indicative bacteria in addition to Gardnerella vaginalis and Atopobium vaginae that are reported in previous studies.2. Either the number of categories or the total amount of bacteria is higher in BV positive women than in normal women, whereas the relative abundance of Lactobacillus is higher in normal women than in women of BV positive. The species in vaginal microbiota have a ground genetic distance between BV positive and normal women.3. The risk factors for BV discovered in previous studies are higher vaginal pH value, lower vaginal lactobacillus content and frequently vagina douching. In this study, we found that Lesser marriage age, being in pregnancy, frequently vagina douching and high education are positively associated with BV.