阴道细菌群落多样性及外阴阴道念珠菌病相关白念珠菌基因多态性研究
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摘要
第一部分454焦磷酸测序研究健康女性阴道细菌群落多样性
目的了解正常女性阴道微环境中各种细菌的种类和丰度。方法采集20例健康志愿者的阴道分泌物标本,直接提取基因组DNA,使用细菌16S rRNA通用引物扩增V1-V3区,产物进行焦磷酸测序。对测序数据进行有效数据统计,评估样本中的菌群的丰度和多样性,对菌群进行门、纲、目、科、属、种六个水平的分类学比较。结果20个样本中共检测到有效序列115329条,平均每个样本5766条,序列平均长度477bp。经聚类比对,共生成2363个操作分类单元。在门的水平共检测到了6个门,其中以硬壁菌门为最多。在60个属中,乳杆菌属占绝大多数,平均含量在90%以上。而种的水平上,L.crispatus是最常见的菌种,其他种的乳杆菌少见。结论健康女性阴道菌群以乳杆菌为主,且L.crisputs是最常见的组成部分。
第二部分南京地区妇女外阴阴道念珠菌病的分子流行病学研究
目的了解南京地区引起外阴阴道念珠菌病的白念珠菌的优势基因型,比较不同年龄、不同生理状态下白念珠菌基因型分布是否存在差异。方法收集2011年12月至2012年04月在南京市妇幼保健医院就诊的外阴阴道念珠菌病病例,获取患者的临床资料和阴道分泌物标本。分泌物均经镜检、培养纯化及分子生物学鉴定。对鉴定为白念珠菌的菌株基因组DNA使用CA Ⅰ微卫星标记进行扩增,产物经毛细管电泳后用ABI3700测序仪扫板后分析。结果共收集外阴阴道念珠菌病例246例,微卫星标记成功扩增了208株白念珠菌DNA,共检测到了51种基因型。其中占前三位的基因型分别是30：45,21：21和32：46,分别占所检测样本的29.3%、13.0%和12.0%。妊娠人群中主要的基因型是46：46、32：46和21：21,妊娠人群白念珠菌基因型与非妊娠人群分布存在差异。各年龄组间白念珠菌基因型分布未见明显差异。结论南京地区外阴阴道念珠菌病相关的白念珠菌基因型分布有着独特的分布特征。妊娠人群的基因型分布与非妊娠人群相比具有显著性差异。而年龄与基因型分布无关。
第三部分妊娠期外阴阴道念珠菌病相关的白念珠菌基因多态性的研究
目的了解引起妊娠妇女外阴阴道念珠菌病的白念珠菌基因型分布情况。方法收集中后期妊娠外阴阴道念珠菌病患者的阴道分泌物样本,经培养鉴定为白念珠菌后提取基因组DNA。对所提取的DNA使用7个管家基因进行扩增后测序。使用Mega5.1对获得的序列构建进化树,用eBURST进行进化分析。结果共收集了妊娠VVC病例80例,共分离出念珠菌83株,其中白念珠菌77株,光滑念珠菌7株。其中77株白念珠菌经ATTla、ACC1、ADP1、MPIb、SYA1、VPS13以及ZWF1b7个管家基因扩增后共形成60个序列型,这60个序列型在数据库中均未见收录。使用UPGMA法构建系统进化树可将所有菌株分成5类,其中最多的一类占所有菌株比例的58.4%。搜索数据库与国内报道的VVC菌株共同建立进化树发现都在同一类中。eBURST分析发现60个DST中最大的克隆复合体包含了23个DST。其中编号57号菌株对应的为分子进化的始祖DST。结论南京地区妊娠VVC相关的念珠菌由未报道过的序列型组成,具有独特的DST和特有的进化始祖,并呈现出基因的高度多态性。基因多态性的DST之间的遗传距离小。
Chapter1:Study on the vaginal microbiome in healthy women by using454pyrosequencing
Objective To understand the composition of bacterial communities in healthy Chinese women. Methods Genome DNA were extracted directly from vaginal fluids of20healthy women. the primers target V1-V3region of bacterial16S rRNA were used to amplify the DNA. The purified PCR products were subjected to pyrosequencing. Valid sequences were calculated and richness and diversity of bacterial communities among the20subjects were also assessed. Taxonomical analysis was done on six levels including phylum, class, family, order, genus and species. Results A total of115329sequences were finally analyzed. The average number of sequences is5766, with an average length of477bp. After clustering and alignment,2363operational taxonomy unites were recognized. A total of6phylum were detected predominated by Firmacutes. was the most prevalent genus in vaginal tract, composing over90%of the total bacterial communities. Conclusions Lactobacillus was the largest composition of normal micorbiome. L.crispatus was the most common species detected.
Chapter2Characterization of Candida albians associated with vulvovaginal candidiasis using microsatellite length polymorphisms
Objective:To characterize the genotype distribution pattern of Candida albicans associated with vulvovaginal candidiasis in Nanjing, China. Methods:A molecular epidemiological study was conducted to explore the genotypes of C.albicans associated with VVC in Nanjing. Generally, microscopical examination of a wet smear combined with culture was used for the diagnosis of vulvovaginal candidiasis (VVC). A questionnaire about information associated with VVC was obtained from each participant. The genotypes of clinical isolates were determined by the analysis of PCR products amplified by microsatellite primers of CAI. Chi-square test was used to compare the genotype distribution of strains isolated from pregnant women with non-pregnant ones. Comparison among different age groups was also performed by using Chi-square test. Difference of genotype distribution of VVC with different clinical conditions was calculated by Chi-square test. Results:PCR fragments containing30:45,21:21and32:46alleles were the three major genotypes among the51genotypes detected, accounting for29.3%,13.0%and12.0%of206clinical isolates respectively. The21:21genotype was more prevalent in younger groups compared with other groups. The46:46,32:46and 21:21were important genotypes causing VVC in pregnant women. The30:45was rare in pregnant women, only one strain demonstrated this genotype. Patients with different clinical conditions had similar genotype distribution.Conclusions:A distinctive genotype distribution of C. albicans was detected in Nanjing, China, compared to previous reports, with21:21being the major genotype besides previously reported30:45and32:46. A different distribution pattern was also observed in pregnant women compared with non-conceived ones. No difference distribution of C.albicans was found among different age groups.
Chapter3Genetic diversity of Candida albicans associated with vulvovaginal candidiasis in pregnant women
Objective To study the genetic diversity of C.albicans associated VVC in pregnant women in Nanjing. Methods Mid-term pregnant women diagnosed as VVC were collected for fungal detection and clinical information. Strains isolated from the discharge were subjected to morphologic and molecular identification on the genus level. Strains confirmed as Candida albicans were then conducted to perform multi locus sequence typing using the seven house-keeping genes. The sequence type of each strain was determined by comparison with database. Phylogenic analysis was done with the Mega5.1software. EBURST V3.0software was used to check the clonal complex of the sequence types. Results A total of84Candida strains were obtained from81pregnant women diagnosed as VVC. Among the84strains,77strains were finally identified as Canida albicans while the rest7belong the species Candida glabrata. Seven genes including TTla, ACC1, ADP1, MPIb, SYA1, VPS13and ZWFlb were successfully amplied. All the strains were nominated to60independent sequence types and all of them had not beed recognized before. Phylogenic analysis using the UPMGMA algorithm divived the sequence types into5groups and most of them were include in clade one.The largest clonal complex was consisted with23unique sequence types and the ancenstor was the sequence type drived from strain57. Conclusions The Candida albcians associated with VVC during pregnancy showed highly discrimative diversity by using MLST. However, the phylogenic analysis exhibited genetic similarity among the strains studied.
目的了解正常女性阴道微环境中各种细菌的种类和丰度。方法采集20例健康志愿者的阴道分泌物标本,直接提取基因组DNA,使用细菌16S rRNA通用引物扩增V1-V3区,产物进行焦磷酸测序。对测序数据进行有效数据统计,评估样本中的菌群的丰度和多样性,对菌群进行门、纲、目、科、属、种六个水平的分类学比较。结果20个样本中共检测到有效序列115329条,平均每个样本5766条,序列平均长度477bp。经聚类比对,共生成2363个操作分类单元。在门的水平共检测到了6个门,其中以硬壁菌门为最多。在60个属中,乳杆菌属占绝大多数,平均含量在90%以上。而种的水平上,L.crispatus是最常见的菌种,其他种的乳杆菌少见。结论健康女性阴道菌群以乳杆菌为主,且L.crisputs是最常见的组成部分。
第二部分南京地区妇女外阴阴道念珠菌病的分子流行病学研究
目的了解南京地区引起外阴阴道念珠菌病的白念珠菌的优势基因型,比较不同年龄、不同生理状态下白念珠菌基因型分布是否存在差异。方法收集2011年12月至2012年04月在南京市妇幼保健医院就诊的外阴阴道念珠菌病病例,获取患者的临床资料和阴道分泌物标本。分泌物均经镜检、培养纯化及分子生物学鉴定。对鉴定为白念珠菌的菌株基因组DNA使用CA Ⅰ微卫星标记进行扩增,产物经毛细管电泳后用ABI3700测序仪扫板后分析。结果共收集外阴阴道念珠菌病例246例,微卫星标记成功扩增了208株白念珠菌DNA,共检测到了51种基因型。其中占前三位的基因型分别是30：45,21：21和32：46,分别占所检测样本的29.3%、13.0%和12.0%。妊娠人群中主要的基因型是46：46、32：46和21：21,妊娠人群白念珠菌基因型与非妊娠人群分布存在差异。各年龄组间白念珠菌基因型分布未见明显差异。结论南京地区外阴阴道念珠菌病相关的白念珠菌基因型分布有着独特的分布特征。妊娠人群的基因型分布与非妊娠人群相比具有显著性差异。而年龄与基因型分布无关。
第三部分妊娠期外阴阴道念珠菌病相关的白念珠菌基因多态性的研究
目的了解引起妊娠妇女外阴阴道念珠菌病的白念珠菌基因型分布情况。方法收集中后期妊娠外阴阴道念珠菌病患者的阴道分泌物样本,经培养鉴定为白念珠菌后提取基因组DNA。对所提取的DNA使用7个管家基因进行扩增后测序。使用Mega5.1对获得的序列构建进化树,用eBURST进行进化分析。结果共收集了妊娠VVC病例80例,共分离出念珠菌83株,其中白念珠菌77株,光滑念珠菌7株。其中77株白念珠菌经ATTla、ACC1、ADP1、MPIb、SYA1、VPS13以及ZWF1b7个管家基因扩增后共形成60个序列型,这60个序列型在数据库中均未见收录。使用UPGMA法构建系统进化树可将所有菌株分成5类,其中最多的一类占所有菌株比例的58.4%。搜索数据库与国内报道的VVC菌株共同建立进化树发现都在同一类中。eBURST分析发现60个DST中最大的克隆复合体包含了23个DST。其中编号57号菌株对应的为分子进化的始祖DST。结论南京地区妊娠VVC相关的念珠菌由未报道过的序列型组成,具有独特的DST和特有的进化始祖,并呈现出基因的高度多态性。基因多态性的DST之间的遗传距离小。
Chapter1:Study on the vaginal microbiome in healthy women by using454pyrosequencing
Objective To understand the composition of bacterial communities in healthy Chinese women. Methods Genome DNA were extracted directly from vaginal fluids of20healthy women. the primers target V1-V3region of bacterial16S rRNA were used to amplify the DNA. The purified PCR products were subjected to pyrosequencing. Valid sequences were calculated and richness and diversity of bacterial communities among the20subjects were also assessed. Taxonomical analysis was done on six levels including phylum, class, family, order, genus and species. Results A total of115329sequences were finally analyzed. The average number of sequences is5766, with an average length of477bp. After clustering and alignment,2363operational taxonomy unites were recognized. A total of6phylum were detected predominated by Firmacutes. was the most prevalent genus in vaginal tract, composing over90%of the total bacterial communities. Conclusions Lactobacillus was the largest composition of normal micorbiome. L.crispatus was the most common species detected.
Chapter2Characterization of Candida albians associated with vulvovaginal candidiasis using microsatellite length polymorphisms
Objective:To characterize the genotype distribution pattern of Candida albicans associated with vulvovaginal candidiasis in Nanjing, China. Methods:A molecular epidemiological study was conducted to explore the genotypes of C.albicans associated with VVC in Nanjing. Generally, microscopical examination of a wet smear combined with culture was used for the diagnosis of vulvovaginal candidiasis (VVC). A questionnaire about information associated with VVC was obtained from each participant. The genotypes of clinical isolates were determined by the analysis of PCR products amplified by microsatellite primers of CAI. Chi-square test was used to compare the genotype distribution of strains isolated from pregnant women with non-pregnant ones. Comparison among different age groups was also performed by using Chi-square test. Difference of genotype distribution of VVC with different clinical conditions was calculated by Chi-square test. Results:PCR fragments containing30:45,21:21and32:46alleles were the three major genotypes among the51genotypes detected, accounting for29.3%,13.0%and12.0%of206clinical isolates respectively. The21:21genotype was more prevalent in younger groups compared with other groups. The46:46,32:46and 21:21were important genotypes causing VVC in pregnant women. The30:45was rare in pregnant women, only one strain demonstrated this genotype. Patients with different clinical conditions had similar genotype distribution.Conclusions:A distinctive genotype distribution of C. albicans was detected in Nanjing, China, compared to previous reports, with21:21being the major genotype besides previously reported30:45and32:46. A different distribution pattern was also observed in pregnant women compared with non-conceived ones. No difference distribution of C.albicans was found among different age groups.
Chapter3Genetic diversity of Candida albicans associated with vulvovaginal candidiasis in pregnant women
Objective To study the genetic diversity of C.albicans associated VVC in pregnant women in Nanjing. Methods Mid-term pregnant women diagnosed as VVC were collected for fungal detection and clinical information. Strains isolated from the discharge were subjected to morphologic and molecular identification on the genus level. Strains confirmed as Candida albicans were then conducted to perform multi locus sequence typing using the seven house-keeping genes. The sequence type of each strain was determined by comparison with database. Phylogenic analysis was done with the Mega5.1software. EBURST V3.0software was used to check the clonal complex of the sequence types. Results A total of84Candida strains were obtained from81pregnant women diagnosed as VVC. Among the84strains,77strains were finally identified as Canida albicans while the rest7belong the species Candida glabrata. Seven genes including TTla, ACC1, ADP1, MPIb, SYA1, VPS13and ZWFlb were successfully amplied. All the strains were nominated to60independent sequence types and all of them had not beed recognized before. Phylogenic analysis using the UPMGMA algorithm divived the sequence types into5groups and most of them were include in clade one.The largest clonal complex was consisted with23unique sequence types and the ancenstor was the sequence type drived from strain57. Conclusions The Candida albcians associated with VVC during pregnancy showed highly discrimative diversity by using MLST. However, the phylogenic analysis exhibited genetic similarity among the strains studied.
引文
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