فایل ورد کامل شناسایی تنوع باکتریهای واژینال و رابطه آن با واژینوز باکتریال تشخیص داده شده بالینی با دناتورینگ الکتروفورز ژل گرادیانی و تحلیل تطابق

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تعداد صفحات این فایل: ۲۱ صفحه

بخشی از مقاله انگلیسیعنوان انگلیسی:Identification of vaginal bacteria diversity and it’s association with clinically diagnosed bacterial vaginosis by denaturing gradient gel electrophoresis and correspondence analysis~~en~~

Abstract

Bacterial vaginosis (BV) is a common complex associated with numerous adverse health outcomes, affecting women of different ages throughout the world. The etiology of BV remains poorly understood due to the difficulty of establishing a molecular genetic criterion to recognize the vaginal microbiota of BV-positive women from that of the normal women. We used techniques of broad-range PCR-DGGE and gel imaging analysis system cooperated with 16S rRNA gene sequencing and statistical analysis to investigate the community structure of the healthy and BV-affected vaginal microbial ecosystems. The community of vaginal bacteria detected in subjects with BV was far more luxuriant and diverse than in subjects without BV. The mean number of microbial species in 128 BV-positive women was nearly two times greater than in 68 subjects without BV(4.05 ± ۱۹۶ versus 2.59 ± ۱۱۴). Our sequencing efforts yielded many novel phylotypes (198 of our sequences represented 59 species), including several novel BV-associated bacteria (BVAB) and many belonging to opportunistic infections, which remain inexplicable for their roles in determining the health condition of vaginal microflora. This study identifies Algoriphagus aquatilis, Atopobium vaginae, Burkholderia fungorum, Megasphaera genomosp species as indicators to BV and subjects with BV harbor particularly taxon-rich and diverse bacterial communities. Maybe Bifidobacterium, Staphylococcus or even more alien species are commensal creatures in normal vaginal microbiota.

۱ Introduction

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the normal vaginal bacterial microbiota to a heterogeneous state containing a complex population of anaerobic and microaerophilic organisms. BV is associated with sexually transmitted infections and adverse pregnancy outcomes such as low birth weight of foetus, premature birth, neonatal death and secondary infection after delivery and is also the risk factor for premature rupture of fetal membranes (PROM) (Goldenberg et al., 2008). Antibiotics treatment of the condition used to failure due to the uncertain variation of BV-associated bacteria (BVAB) (Beigi et al., 2004; Ferris et al., 2004). Many BV associated bacteria are resistant to culture and (or) difficult to detect by traditional methods, which thwart the exploration of the pathogenesis of BV. So far, there is a lack of effective prevention and treatment measures.

According to Nugent’s classification (Nugent et al., 1991), a score ranged from 7 to 10 is considered as BV, a clinical condition dominated by the morphological identification of Gram-negative and-positive bacteria, without evidence of Lactobacillus morphotypes. In contrast, the score from 0 to 3 is considered an undisturbed vaginal microflora dominated by the Lactobacillus genus, identified as the principal Gram-positive rods bacteria.

In general, race (nationality) (Ness et al., 2003; Uscher-Pines et al., 2009), behavioral habits (Bradshaw et al., 2013), personal hygiene status (Brotman et al., 2008), and so on have been considered as causes of BV. Although there is agreement in the literature that no single agent likely causes BV, there is no consensus about what constitutes a pathogenic bacterial community in this syndrome. Comprehensive cultivation-independent comparisons of the vaginal bacterial communities between subjects with and without clinically defined BV have been rare.

With the development and application of culture-independent nucleic acid amplification test (NAAT) in recent years, it is possible to explore the structure and dynamic shift of microbiota (Larin et al., 2007; Burton and Reid, 2002; Zozaya-Hinchliffe et al., 2010; Ravel et al., 2011). Molecular methods have identified in the vagina of healthy, nonpregnant women the Lactobacillus genus living with a spectrum of bacteria including Gardnerella, Atopobium, Eggerthella, Megasphaera, Leptotrichia, Prevotella, Enterococcus, Bifidobacterium, Staphylococcus, Corynebacterium, Streptococcus, Bacterioides, Mycoplasma, Escherichia, Peptostreptococcus, Ureaplasma, Veillonela, and Candida species (Zhou et al., 2004; Ling et al., 2013; Hyman et al., 2005; Vitali et al., 2007; Nam et al., 2007). However, at present the spectrum of bacterial species that are correspondent to clinical diagnosis, i.e. the Nugent’s score is not well defined.

In this study, we investigated the composition of BVAB in countryside women of reproductive age. Women with and without BV was diagnosed by Nugent’s criteria and 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 vaginal bacteria. The sequence information of 16S rDNA of every detected bacterium was identified with gene bank data. In addition, DGGE Gel Image System was used to calculate the relative abundance of each bacterial species to the whole microbial population of one sample (vaginal swab). Eventually, appropriate statistical method was used to analyze the relationship between BV and bacterial composition, as well as the relationship between BV and other risk factors.

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