The Next Generation High Throughput Sequencing Reveals Diverse Vaginal Microbiomes Associated With Bacterial Vaginosis Or Vulvovaginal Candidiasis

BackgroundVaginal microbial community for the maintenance of the vaginal environment stability, prevention of reproductive tract and urinary tract infection is very important, therefore, to clear the vaginal microbial community composition and dynamics for our understanding of these diseases pathogenesis and treatment and preventive measures will undoubtedly help more.At present, we based on microscopy and traditional culture method to obtain the vaginal microbial community information. Basic recognized Lactobacillus vaginal dominant bacteria, the space occupying, nutrient competition, produce lactic acid, H2O2, biotin, stimulation of immunity mechanism to maintain the acidic environment of the vagina (3.8-4.2), inhibiting the growth of other bacteria in the vagina. However, because of many vaginal bacterias, some of which are very difficult to learn by method of traditional pure culture and isolation and identification. In recent years, with the rapid development of molecular genetics and molecular biology technology, some new methods, give us further clarify the vaginal microbial community composition may be brought. Since the polymerase chain reaction (PCR) was established, the development of this technology is very rapid, the application is also increasingly diversified.16S rRNA gene PCR amplification is currently the most commonly used detection of vaginal microbial non-culture based on classification and identification, making identification of microbe from phenotypic characteristics, deepen for gene identification. With the development of the second generation of high-throughput sequencing technologies, such as Roche454pyrosequencing platform and Illumina Solexa high-throughput sequencing platforms, the sequencing throughput and accuracy has been greatly improved, but due to the cost factor, there is only limited in several groups use. Zhou Hongwei in the second generation of high-throughput Solexa sequencing platform, through paired end sequencing method for the determination of16S fragments, and fusion of bar code (barcode) technology, established the Barcoded Paired-End Illumina Sequencing (BIPES) method, which is a new second generation of high-throughput sequencing method has a simple operation, low cost, good repeatability, simple, fast make clinical comprehensive, system, structured information of microbial community structure become possible.We know, vulvovaginal candidiasis (VVC) and bacterial vaginosis (BV) is a clinical common two kinds of vaginitis disease, its incidence is non microbial infections from exogenous, and the composition and the vagina microbial communities and their microenvironment change, but, for the onset and after treatment, the vagina microbial population will show what kind of change, no conclusion has been reached so far. This study to detect healthy women of childbearing age, VVC patients, VVC patients and BV patients were cured after vaginal flora using BIPES, in order to know more comprehensive and accurate on the vaginal flora in low cost conditions, provide the basis for further discussion of the causes of WC and BV and its prevention and treatment.Methods1. The experimental object and purpose: Experiment one:Comparison of different parts of the colony composition differences, randomly choose27cases of outpatients, aged21-47years old, the median age was34years old, menstrual regularity, after informed consent, using sterile cotton swab to vaginal fornix and low-vaginal1/3segments each take two tube secretions.Experiment two:Detection of WC before and after treatment of vaginal microbial population structure,①Group WC:there were44cases, aged20to45years old, the median age was32years old. In2011June to2011November in Zhujiang Hospital of Southern Medical Universit y obstetrics and gynecology clinic patients. Inclusion criteria:in accordance with the higher school textbook seventh edition of "obstetrics and Gynecology" diagnostic criteria.②The WC recently after the cure group:VVC group was treated with the regimen:fluconazole150mg, oral,1times a day, for4consecutive days; terbinafine hydrochloride vaginal effervescent tablets1times a day,1capsules each time, the medicine on the vagina, for7consecutive days; chushizhiyang lotion, bath,10to14days to weeks withdrawal a total of28patients with all the symptoms disappeared, and vaginal secretions microscope Candida negative.③Normal control group:39cases, for the same period in our hospital out-patient medical health of women of childbearing age, between the ages of21to49years old. Through the detailed history, vaginal cleanliness classification, vaginal secretion of Nugent score, the inclusion criteria in order to satisfy the following three items:①no clinical symptoms;②vaginal cleanliness class Ⅰ-Ⅱ; the Nugent score in0～3.Experiment three:Detection of vaginal microbial populations in patients with BV.①group BV:a total of37patients, aged22to48years old, the median age was34years old, for the201201to201203months in patients of Obstetrics and gynecology clinic of Zhujiang Hospital of Southern Medical University. Inclusion criteria:the contents of4clinical diagnostic criteria for Amsel, which in order to find the clue cells for any necessary items and the other3in the2positive, while Nugent score^7points for the diagnosis of BV.②Women of childbearing age group of health:a total of37cases. In accordance with the inclusion criteria in experiment two normal controls.The above groups meet the following conditions:no systemic application of antibiotics and topical vaginal in patients with a history Within one month,3days of a sexual life history, non pregnancy, lactation or menstrual period, without diabetes mellitus, not also suffer from trichomonad sex vaginitis, or others.Experiment two or three groups of experimental subjects after informed consent, with sterile vaginal swabs in vaginal fornix take two tube secretions.2.Tthe forensic DNA Extraction Kit (Shenzhen Bioeasy) extracted total DNA, specific steps reference manual.3.Amplification of16s rRNA V6gene fragment and Illumina sequence:the sample total DNA as template, using the primers967F and1046R amplification of the16S rRNA V6gene. PCR products with equal concentrations of mixed evenly, double end100bp sequencing by Illumina HiSeq2000platform (BGI, Shenzhen).4.The measured sequence through the barcoded Illumina paired-end (PE) sequencing (BIPES) process for preliminary treatment. The use of TSC for clustering, extracted from each operational taxonomic units (OTU) represents the sequence after the use of GAST species, which belong to the lactic acid bacteria of the genus OTU using Megablast is further divided into different levels, the subsequent analysis including cluster analysis, analysis of Alpha diversity and beta diversity. Based on the Unifrac distance, uses QIIME to carry on the analysis of principal component of vaginal bacteria. Using linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) online tool for the analysis of patients with special vaginal flora.ResultsThe result of experiment one: 1. The vaginal microbial community of vaginal fornix and low-vaginal1/3segments tends to be the same.The results of experiment two:1. Structure of vaginal flora of patients with VVC can be divided into four types::(l)Tthe vaginal flora of VVC_Ⅰ type with Lactobacillus dominated (richness range from95.4%to99.7%, average98.5%), similar to healthy women of childbearing age of vaginal flora, there are two Lactobacillus strains of Lactobacillus iners or Lactobacillus crispatus;(2) In VVC_Ⅱ type still with Lactobacillus dominated, but visually abundance levels were lower in the VVC_Ⅰ type. Lactobacillus species richness ranged from73.2%to93.8%, average84.2%, no statistically significant difference between the Lactobacillus sp. between VVC_Ⅰ type and VVC_Ⅱ type. All of the VVC_type Ⅱ samples by Lactobacillus iners dominated, some specimens are small proportion of Lactobacillus crispatus, Lactobacillus jensenii or lactobacillus gasserii, VVC_Ⅱ type than in VVC_Ⅰ type significantly increased Alpha and beta diversity;(3) VVC_Ⅲ type samples show flora information than before the two set of more complex, lactobacillus is still relatively common, but significantly different from WC_Ⅰ type, richness decreased significantly, accounting for18.9%～48.8%, with an average of33.9%. In this group lactobacillus is still Lactobacillus iners species, Gardnerella become a dominant bacteria, accounting for24.3%～43.5%, with an average of33.9%;(4) WC_Ⅳtype vaginal flora Alpha diversity and beta diversity increased significantly, Lactobacillus decreased to3.2%, or even disappear, much to Gardnerella is dominant, Granulicatella, Prevotella, Anaerococcus, Parvimonas, Dialister, Sneathia, Mobiluncus, Sphingomonas species richness increased significantly.3.Clinical symptoms disappeared after antifungal therapy, pathogenic diagnosis of negative, but the majority of vaginal flora are not as expected to return to normal, individual differences. VVC_Ⅰ type before treatment is almost normal vaginal flora,4patients were that Gardnerella is increased compared with those before treatment, the remaining6vaginal flora changed little. There are2patients from the patient with VVC_Ⅱ type, their vaginal flora is that streptococcus is dominant, while the remaining4patients VVC_Ⅱ type referral patients, after treatment with Lactobacillus crispatus richness increased. After the treatment of WC_Ⅲ type Gardnerella richness decreased, Lactobacillus iners richness increased, but did not return to normal, and only2cases recovered to normal.2VVC_IVtype patients were, Granulicatella significantly less, but the vaginal flora is dominated by streptococcus. The other2VVC_type Ⅳ patients were, Gardnerella richness decreased, Sneathia, Prevotella spp. disappeared, also appeared a few streptococci.The results of experiment three:1.Through the dilution curve, Observed OTU and Shannon diversity index showed significantly increased BV vaginal flora diversity of Alpha than in healthy women of childbearing age;2.Through the PCoA Unifrac distance and primer clustering showed significantly increased BV vaginal flora diversity of beta than in healthy women of childbearing age;3.At the genus level, healthy women of childbearing age the highest relative abundance of Lactobacillus, accounting for about95%or even higher, in Lactobacillus species level, most people with Lactobacillus iners or Lactobacillus crispatus, fairly or both ratio, few people have a small amount of Lactobacillus jensenii or Lactobacillus gasserii, part of vaginal secretions within a small amount of Gardnerella^Granulicatella、Streptococcus、Prevotella、Escherichia and other genus;4.Patients with BV, Lactobacilli were significantly decreased, the relative ratio from45%to1%, with lactobacillus iners, some patients even detected, but there were some patients still L.gasser、L.acidophilus advantage, relative ratio from75%to50%, in addition to Lactobacillus, Gardnerella、Prevotella、Granulicatella、 Anaerococcus、Parvimonas、Peptoniphilus.harei、Peptostreptococcus、Dialister, increase.Conclusions1. The vaginal microbial community of vaginal fornix and low-vaginal1/3segments tends to be the same;2. WC patients with vaginal flora has four kinds of phenotype, characterized by diversity;3.Differences in healthy women of childbearing age and in patients with BV vaginal flora significantly. Healthy women of childbearing age vaginal bacteria were Lactobacillus dominated, mainly Lactobacillus iners or Lactobacillus crispatus, or both, and with a variety of bacteria exists;4. WC patients after antifungal therapy, the cure after vaginal flora change was not immediately returned to normal, and individual differences;5. The majority of patients with BV vaginal flora showed Lactobacillus significantly reduced or absent, small part of BV patients with rare Lactobacillus acidophilus or Lactobacillus gasseri dominant bacteria.