Analysis Of Vaginal Microbiome In Bacterial Vaginosis By L6SrDNA Gene

Bacterial vaginosis(BV) is the common vaginal infection in women of reproductive age, and is the most common etiology of vaginal symptoms which prompt women to seek medical care. BV is a serious threat to the majority of women’s health. Numerous health problems including pelvic inflammatory disease, preterm birth, preterm prelabor rupture of membranes (PPROM), preterm labor resulting in low birth weight, recurrent abortion, histological chorioamnionitis. Bacterial vaginosis (BV) is associated with an increased risk of upper genital tract and sexually transmitted infections and with the acquisition of the HIV. The recurrence rate pf BV is more than30%after treatment by Metronidazole and Clindamycin.The exploration of the cause of BV, particularly the pathogenie baeteria is still lack of deep understanding. BV is caused by imbalance of naturally occurring bacterial flora that vaginal microbiota shifting from the normal lactobacillus dominating community to a more diverse community of non-lactobacillus bacteria. In healthy women of reproductive age, the common vaginal microbiota normal bacterial flora is mainly Lactobacillus crispatus and Lactobacillus iners. Lactobacilli include some hydrogen peroxide-producing species that help to prevent other vaginal microorganisms from excessive growth. Previous reports showed that in vaginal of BV the normal Lactobacillus reduced, accompanied by higher proportions of anaerobic and facultative bacteria. Alone the absence of Lactobacillus does not define an unhealthy state. The genus Gardnerella vaginalis and Mobiluncus is the most common flora of BV by culture methods, but also found in BV-negative individuals. Complementarily, the presence of solely bacteria does not define an unhealthy state. Therefore we must conduct a comprehensive analysis and comparison of adult healthy women and BV patients of vaginal microbial populations, to understand the etiology and pathological mechanism of the BV.In the past, the composition and diversity of human vagina microbiota were detected by cultural methods which had many limitations for the really circumstance. The advent of PCR based techniques and pyrosequencing made it possible to further examine this complex microbial niche. The small ribosomal subunit or16S rDNA gene was often used as the most common target for molecular identification of bacteria.. The16S rRNA gene is useful because it is present in all bacteria, and has regions of conserved sequence that can be targeted by universal or specific primers, yet also has areas of heterogeneity that can be used to identify bacteria. We can PCR amplification16S rDNA heterogeneity fragments and construct the clone library to sequencing. The sequences obtained are aligned and compared to large databases of16S rDNA sequences, to characterize and identify the bacteria of origin, inferring phylogenetic relationships.16S rDNA fragments can PCR amplification and construct the clone library to sequencing. The sequences obtained are aligned and compared to large databases of16S rDNA sequences, to characterize and identify the bacteria of origin, inferring phylogenetic relationships.While conventional sequencing techniques have provided us with a framework, but due to the limination of amount of the clone libraries, the true extent of bacterial diversity in the vaginal niche is poorly understood. An alternate approach for large numbers of sequences is new generation sequencing technology(Roche454and Illumina Genome Analyzer). The new generation sequencing technology was applied on a small scale level to identify isolates by analyzing the signature sequences regions of the16S rDNA gene, which contains nine variable region of ten conserved regions and targets of V3area (about60bp) and V6area (about70bp) identify most bacteria. The extracted DNA from the vaginal sample can be amplified using broad range primers (modified with adaptor sequences) and high-throughput sequenced targeting the V3region orV6region. The sequences are analyzed for the information of species classification, population structure of bacterial flora and community comparison.The climate and situation of southern and northern area of China is different obviously. The population is difference in habits and individual immune constitution of, same as the environmental bacterial flora. Comparison of the species composition of vaginal bacterial communities of BV women in both areas, partly account for the knowledge of disparities in the susceptibility of BV women in these area groups.Part1:Comparative study of vaginal flora between bacterial vaginosis and non-bacterial vaginosis patientsIn this study, we used the full-length16S rDNA to determine the diversity of vaginal microbiota in10BV+and10BV-women from Haerbin in Northern China.16S rDNA PCR product was cloned into pMD19T vector for library construction and screened by X-gal white clones for Sanger sequencing. Sequence data was processed by filtering low-quality data and removing vector primer sequences, followed by blast with the database for analysis. The diversity and composition of sample vaginal flora at taxonomy levels was evaluated, and calculate the abundance of classification species. Multiple library community structure was compared by multivariate analysis. The species distribution was statistical comparised between positive and negative groups, to get the basic data for the bacterial imbalance when BV occurred.At a97%sequence similarity cutoff, the number of operational taxonomic units (OTUs) per10subjects with BV was nearly three times greater than10subjects without BV by29.4±9.3versus11.7±7.8(P<0.001). According to the Shannon, Simpson, Chaol, ACE, Good’s coverage and evenness index, the specie categories of BV positive group is more various, and the distribution is more evenness.The processed sequences was aligned to RDP databases for Seqmatch, and noted microbial strains identification. The vaginal bacterial communities detected in subjects with BV were much more taxon rich and diverse than those without BV. At phylum level, All sequences belonging to six bacterial phyla. Firmicutes was the most members in both group Aclinobacteria and Bacteroidetes was more abundance in BV+group. At genus level, the significant difference (P<0.001) was found between the subjects with BV and that without BV according to the mean number of taxa in each sample of Mean±SD with the11.8±4.44in BV+group and3.9±3.35in BV-group. The dominant microflora is Lactobacillus in subject of BV negative group, including L. iners, L. jensenii, L. crispatus, L. vaginalis and L. gasseri. Liners and L. crispatus prevalence account for83.73%and10.21%of all bacterial communities, significantly more than the BV positive group. In contrast, subjects with BV possess a diverse array of vaginal bacteria. Several bacteria have been found to be highly associated with BV, including Atopobium, Prevotella, Gemella, Sneathia, Peptoniphilus, Anaerococcus, Parvimonas, and Dialiste. Gardnerella vaginalis was found in the4subjects in BV+group and one subject in BV-group with no significant difference of abundance and presence. Based on result of classification, four possible novel phylotype microorganisms were found. Two uncultured bacterium classified into the Ruminococcaceae family were apparent to be associate with BV. One additional unidentified bacterium classified into Coriobacteriaceae bacterium and another classified into Clostridiales bacterium were also found in this study, although not associated with BV. The non-culture methods can find some bacteria which current culture methods are not able isolation and identification. Principal component analysis was performed at genus levels. Most BV+samples clustered together, only exception close to the cluster of BV-samples.Lactobacillus and Anaerococcus contributed to the formation most.Our data confirmed that a shift in the abundance of bacterial species present in the vaginal environment when compared with BV and non-BV groups. The dominant bacteria of BV negative samples is Lactobacillus, in which Liners and L.crispatus are protective bacteria. Different species found in women of different races type. The host genetic factors and local immune in vagina, deciding plays a very important role in the vaginal microbial community composition. Several bacteria such as Atopobium, Prevotella, Parvimonas, Peptoniphilus, Dialiste and Sneathia were found to be associated with BV. Flagyl drug resistance in various species of the genus, so as to high recurrence after treatment of BV. Gardnerella vaginalis was found in BV negative samples with low abundance, which can’t be the specific markers of disease. Diagnosis of BV with specific PCR targeted at G. vaginalis and Atopobium and Prevotella, will improve the sensitivity and targeted therapy. Comprehensive study of vaginal microbial ecosystem structure and richness is essential for understanding the vaginal disease etiology, prevention and treatment of disease. Part2:Diversity analysis of bacterial vaginal in southern and northern area of China with deep sequencingIn order to access the complete information of the flora in sequencing depth and library coverage,, we characterize the vaginal microbial of10BV women in southern and10in northern area of China using Illumina high-throughput sequencing the V6region of16S rDNA gene. Raw sequence was trimmed by removing barcode adapter and low-quality data, match to the reverse primer. The PE reads of clean data were overlapped to assemble the final V6Tag sequences. The tag sequences were used for species classification, OTU and diversity analysis, comparative of multiple samples, to understand the extremely weak changes of vaginal microbial variation under different environment conditions.A total of1,545,668tag sequences, approximately30%of the total raw sequencing reads passing the quality control and were included in our data analysis. The length of almost sequence tags is75bp. We get unique tags that are identical to the reference sequence. Operational Taxonomical Units (OTUs) then were assigned pre-cluster tags at the97%identity cutoff. All the sequences were assigned into829OTU, and two groups shared168OTUs accounting for20.26%. Southernn group sample classified into80.2±32.8OTU, no significant difference (P=0.775) with northern group sample of84.5±33.3OTU. The Alpha diversity calculated base on OTU classification, indicated that the microbial of most samples are abundance and evenness. Individual samples55and107in the southern subject have more abundant diversity, while samples19and135relative simple. OTU rarefaction curves were created to evaluate of the amount of sequencing and richness, and the majority samples curves reached saturation level. The high-throughput sequencing generates a large number of sequences for almost all species coverage.The unique tags were aligned to16S rDNA database using BLASTN and identified based on the best match. The sequences were assigned to a family level as the best species classification level. At phylum level, all library sequences belong to10bacterial phyla. Among the bacterial community of southern subjects, Actinobacteria is the highest abundance, followed by Firmicutes and Bacteroidetes. Northern subjects has different distribution as Firmicutes highest abundance, followed by Actinobacteria and Bacteroidetes. The distribution and abundance of Phylum, Class, Order, Family between two groups have no significant difference. Previously reported pathogenic species or BV associate bacteria were found with low abundance in each sample. Subjects with high abundance of Bifidobacteriaceae, are also accompanied by the presence of Prevotellaceae with high abundance. Within all subjects tag,99.96%sequences of Lactobacillaceae were identified as L.iners. We generated the heatmap of the abundence of family classification for20subjects, to compare the similarity.Comparison of the distribution and proportion of the vaginal microbial, Micrococcacea, Pseudomonadalese, Burkholderiaceae, Lactobacillaceae are the significant different factors. The beta diversity at the best classification level between two groups reflect the different distance in diversity among samples. Northern subject52and southern55differ least, while northern19and Southern122differ most. Bifidobacteriaceae and Lactobacillaceae contributed most to the difference of samples by PCA analysis, followed by Lachnospiraceae and Prevotellaceae. The distinguish result by cluster analysis verified that the main effect factor of cluster is the microscopic diagnosis information of clue cell and the bacteria of the vaginal flora. Multivariate analysis can reveal the potentially distinction of samples with Lactobacillaceae predominate and others.Using Illumina high throughput sequencing16SrDNA V6of the BV subjects in Southern and the Northern China, We processed the data through the screening of effective filtering analysis, and obtained richness sequences.We selected the "family" levels for the classification of species as the best level of OTU and Tags species notes, to ensure the balance between the level of species and the number of comments to the Tag. The dominant family in the two groups are similar with Bifidobacteriaceae,Prevotellaceae,Lachnospiracea,Lactobacillaceae,Leptotrichiace ae,Ruminococcaceae, Veillonellaceae, in which the first four contribute larger in the difference among samples. These strains accord to the BV-associated bacteria identificated by clone library method. The differences factors between groups are Micrococcacea, Pseudomonadalese, Burkholderiaceae, Lactobacillaceae, maybe affected by different external environment or individual immune reaction. The results of cluster analysis validated that microscopic examination of clue cells in clinical diagnostic are the main affecting factors of classification, while the environmental factors are relatively weak. Due to the complexity of individual immune reaction, a more comprehensive survey of the vaginal environment in similar samples is necessary.Using modern molecular methods (culture-independent) and bioinformatics, we study the composition and richness of the vaginal microbial ecosystem in BV and health state, in BV women of southern and northern areas.16SrDNA sequence analysis made it possible to detect the uncultured bacterium and indentify the diversity. The study will provide the depth insight in the etiology of BV and the basis for prevent and control of BV at the view of microbial ecosystem in women.