The Next Generation High Throughput Sequencing Reveals The Structure Of Vaginal Microbiomes Associated With Vulvovaginal Candidiasis Or Recurrent Vulvovaginal Candidiasis

BackgroundThe female genital tract is a complex ecological system of the fourth ecosystem of human body, composed by the vaginal anatomy, endocrine regulation and the microecological bacteria. The existence of more normal healthy women’s reproductive tract microbial communities, with the host, provide a mutual constraint, mutual coordination between environment, the relationship between the dynamic balance, is the host important microbial barrier against pathogenic bacteria or conditional pathogenic bacteria, and closely related to human health and disease. But in response to environmental changes, drugs stimulation, levels of the hormone and immune state of the host, the influence of such factors as the female reproductive tract microecological bacteria balance have been broken and imbalance, resulting in the occurrence of diseases. Therefore, a comprehensive understanding of vaginal flora composition and dynamic changes of microbial ecology, helps us to understand the reproductive tract infection disease occurrence, development process, further guide clinical take timely intervention measures, using the appropriate medication, avoid the abuse of drugs.Due to vaginal flora are mainly anaerobic bacterium, cultivate nutrition conditions demanding, the use of traditional culture and separated identification method is difficult to fully recognize the vagina flora composition, limited the vaginal flora of the microbial community diversity to further study and reveal the relationship with the disease. In recent years, with the rapid development of molecular biology techniques, the train dependency technology invention, people awareness of vaginal microbial flora has achieved breakthrough progress. The classification of microbial identification become from the phenotypic traits of deepening for identification of genetic traits.16s rRNA genes PCR (polymerase chain reaction, PCR) amplification is the most commonly used detection of vaginal microbial culture method by far. We can rapidly, comprehensively understand the vaginal microbial community structure information by Illumina high-throughput Sequencing platform of Solexa Sequencing, junction Sequencing platform based on fluorescence labels of double End Sequencing (Barcoded Paired-End Illumina Sequencing, BIPES).Vulvovaginal candidiasis (VVC) is a common vulva vagina inflammatory disease caused by Candida, about 5% developed to recurrent vulvovaginal candidiasis(RVVC). It become a clinical research difficulty because of its easy to break out repeatedly over a long period and the symptoms such as itching influence of women’s work and life. The onset of VVC and RVVC is associated with a variety of factors, and pathogenesis remains uncertain, the present study hints the disease associated with changes of the vagina structure itself and the microenvironment of microbial flora. But whether candida infection after vaginal flora changes, how to change, there is still no conclusion. Through Illumina high-throughput sequencing technologies and BIPES series research methods,the study compare the microbial community structure within healthy women of reproductive age, VVC and RVVC patients, track the dynamic changes of vaginal flora in the healthy women of reproductive age and WC patients during the menstrual cycle, in order to further explore the etiology, provide theory basis to treatment and prevention of VVC and RVVC.Research purposes1. Through study the vaginal microbial flora structure of healthy women of reproductive age, VVC and RVVC patients by Illumina high-throughput sequencing technologies and BIPES series research methods,we analyse the vaginal flora diversity change, find out the difference of vaginal flora in attack period.2. We track the change vaginal flora structure information in the menstrual cycle of VVC patients and healthy women of reproductive age, investigate the dynamic changes of the flora structure model and the relationship with the menstrual cycle.Methodsl.The experimental object:Experiment 1:We choose 12 cases of outpatients,average age (28.83±6.10)years, in the gynaecology outpatient department of the first people’s hospital of Shunde in Foshan city during April 2013 to October 2013. WC group have 5 cases, inclusion criteria: ① Symptoms:pruritus vulvae, burning pain, accompanied by urinary symptoms such as pain and pain during sex; Vaginal secretions increase. ② Signs:vulva flush, edema, visible scratches, and even cracked, white membrane attach to the inside of nympha and vaginal mucosa, vagina has more white secretions, curd shaped or shoddy. ③ Laboratory examination:find out candida spore or hypha in vaginal discharge by microscopic examination, or found white candida, nearly smooth candida, such as tropical candida yeast growth by cultivating. RVVC group have 5 cases, inclusion criteria:① The WC attacks more than 1 year 4 times. ② Laboratory examination: find out candida spore or hypha in Vaginal discharge by microscopic examination, or found white candida, nearly smooth candida, such as tropical candida yeast growth by cultivating. Healthy group have 2 cases, inclusion criteria:① No clinical symptoms. ② Vaginal cleanness is Ⅰ～Ⅱ level.③ Laboratory examination:no found candida spores or mycelium in Vaginal discharge by microscopic examination.Experiment 2:We choose 2 cases of healthy women of reproductive age,average age (27.00±3.00)years, in the gynaecology outpatient department of the first people’s hospital of Shunde in Foshan city during June 2013 to September 2013, incorporated in the Healthy group. Inclusion criteria: ①±③same as the experiment 1.④ Normal menstruation. WC group have 3 cases, average age (29.00±4.16)years. Inclusion criteria:①～③ same as the experiment 1.④ Normal menstruation.Groups above are the han nationality in Chinese, exclusion criteria:the objects of study within a month without systemic antibiotics and vaginal local medication history, within three days of asexual life history and vaginal lavage; Rule out during pregnancy and lactation; Eliminate systemic diseases such as diabetes, hormone therapy disease (such as acute or chronic nephritis); Eliminate trichomonas vaginitis,bacterial vaginal disease,or aerobic vaginitis, etc. Experiment 2 also need to satisfy the following conditions:now there is only one sexual partner, use condoms for contraception or natural contraception, no oral contraceptives.2. Sampling:in experiment 1 and experiment2, after made all the research objects informed consent, take vaginal discharge specimens in the vaginal wall period with sterile cotton swabs, then sent to check.In experiment 2, the sampling time of research objects is in the menstrual cycle of 2,4,7,14,16,21,27 days.3. Use DNA extraction kit (shenzhen Yi Rui) to extracte the total DNA of all samples, specific steps reference manual.4. Amplification 16s rRNA gene fragment and Illumina sequence:the sample total DNA as template,using the primers 514F and 805R amplification of the 16s rRNA V4 gene.PCR products with equal concertrations of mixed evenly,double end 100p sequencing by the Illumina HiSeq2000 platform (BGI, shenzhen).5. The measured sequence through the barcoded Illumina paired-end sequencing(BIPES)process for preliminary treatment. Using TSC for clustering, to extract the classification of each operating unit (the Operational taxonomy unit, OTU) on behalf of the sequence, using GAST to classification of species, which belong to lactic acid bacteria of the genus OTU using megablast assigned to the kind of level, further follow-up analysis including clustering analysis, Alpha and Beta diversity analysis. Based on Unifrac distance, uses QIIME to carry on the analysis of principal component of vaginal flora. Using linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) online tools to analyze patients with special vaginal flora.6. Statistical processing:SPSS 13.0 software was used to statistical analysis, with the proportion or rate counting data. Use analysis of variance to compare the Measurement data set within or between groups, use Wilcoxon rank and inspection to compare diversity index and the relative abundance of bacteria genus, P< 0.05 for the difference is statistically significant.ResultsExperiment 1:1. Sequencing BIPES flow used in the data analysis, adopting UCHIME remove chimeric sequences, there are 12 samples to sequence, eventually received 852992 sequences, each sample an average of 71083.2. According to the QIIME analysis process for beta diversity analysis, show the similarity within the three groups of flora. The highest Beta diversity is VVC group with widely distributed. The Beta diversity of RVVC group is compact distribution, so the flora structure similarity is high. The Beta diversity of healthy group is between RVVC group and VVC group.3. Through the PD whole tree to compare the Alpha diversity of three group samples, according to the vaginal flora Alpha diversity of VVC group is highest, that reveal its flora diversity increased significantly in the RVVC and healthy group (T-test, P< 0.05).4. To compare the bacteria of the genus level, Lactobacillus crispatus is the mainly predominant vaginal spcies in healthy group,accounted more than 95%. In VVC group,the constitute of Vaginal flora diversified change, lactobacillus genus richness decrease significantly with from 68% fallen to 0.2%, Gardnerella,prevotella, bifidobacterium richness increase significantly. RVVC group of Advantage bacterium is Lactobacillus iners, accounts for about 85%.5. Through LEfSe statistics analyse the species differences in three groups,we found that the abundance richness of Actinobacteria, Bifidobacteriaceae,Bifidobact-eriales,Chryseobacterium in VVC group increase than in RVVC group and healthy group. And Acidovorax is significantly higher in healthy group.Experiment 2:1. Sequencing BIPES flow used in the data analysis, adopting UCHIME remove chimeric sequences, there are 12 samples to sequence, eventually received 105796 clean sequences, each sample an average of 92610.2. With Chaol, Observed species, PD whole tree and Shannon four indicators show the alpha diversity changes in the vaginal flora of five research subjects in the menstrual cycle. The alpha diversity index in healthy group H2 and WC group V2 is lowest,so that the vaginal bacteria genera constitute of these two subjects is relatively single.3. through calculating Euclidean distance to display the volatility of the vaginal flora in 5 research objects in the menstrual cycle. Menstruation 7th day of vaginal flora structure make obviously distance with period 2nd day. It prompt that the vaginal flora of menstruation 7th day change obviously, the advantage bacterium in vagina rapidly grow, and the diversity of bacteria by complicate tend to simplification.4. Compared to the species level,with menstrual cycle dynamically, Lactobacillus crispatus is dominant in healthy group. WC group structure is relatively complex, and the advantage bacterium is Lactobacillus iners (2/3)or be used for Gardnerella (1/3). The vaginal flora is in alternating dynamic change with menstrual cycle. Vaginal flora diversity increased, the flora fluctuation in menstrual cycle is obvious.5. Pick out the largest number of 15 species, the vagina flora of 5 research objects in the menstrual cycle shows changes by the line chart. There are two models in the vaginal advantage bacterium group changes.The one model is alternating changes:after menstruation the vagina advantage bacterium rapidly grow to reach peak before ovulation, began to decline in oviposit period (menstruation 14th day),and down to the lowest point in menses, while other vaginal flora accordingly increased. Another model is to keep the absolute advantage. After the menstrual period, the vaginal advantage bacterium thrive, and always keep a higher abundance, then decline before menses.Conclusion1. The structure of vaginal microbiomes in Healthy women of reproductive age and RWC patients is simple, but the Flora diversity increased in WC patients.2. The advantage bacterium group in healthy women is Lactobacillus crispatus, and in RWC patients is Lactobacillus iners. the proportion of Lactobacillus.spp in the vaginal microbiota of the VVC Group is decreased,with increased the richness of Gardnerella, Prevotella, Bifidobacterium and other bacterial species.3. The vaginal flora is along with the change of the menstrual cycle to change,expecially before and after menstruation is the largest. The dynamic change of vaginal advantage flora is alternating changes or keeping the absolute advantage.4. The advantage flora in healthy women of reproductive age in menstrual cycle is still Lactobacillus, and in VVC group is Lactobacillus iners or other advantage flora, with increasing flora diversity changes.5. By 16 s rRNA genes and Illumina sequencing we found that there have been existing Lactobacillus crispatus and Lactobacillus iners in the vagina of healthy women of reproductive age and VVC patients in the menstrual cycle, with coexistence of varied microorganisms.