فایل ورد کامل شناسایی و مشخصات فیلوژنتیکی واژینولیزین Gardnerella vaginalis در نمونه های برگرفته از زنان بلغاری مبتلا به واژینوز باکتریال

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تعداد صفحات این فایل: ۱۴ صفحه

بخشی از مقاله انگلیسیعنوان انگلیسی:Detection And Phylogenetic Characterization Of Gardnerella Vaginalis Vaginolysin In Samples From Bulgarian Women With Bacterial Vaginosis~~en~~

Abstract

Recently, the intensive use of more advanced methods, including molecular genetic analysis, has aided the studies on the etiological role of Gardnerella vaginalis as the major pathogen of bacterial vaginosis (BV). The objective of our study was to propose a polymerase chain reaction (PCR)-based method detecting the G. vaginalis vaginolysin gene (vly) and to perform a phylogenetic analysis in an attempt to find possible correlations between clinical manifestations and gene mutations. Vaginal samples were collected from 1145 women with symptoms for vaginitis and 378 asymptomatic ones of reproductive age. After PCR detection of G. vaginalis 289 DNA samples were tested for vly using primers targeted at amplification of the whole gene. The presence of vly was detected in all 289 samples with positive PCR for G. vaginalis. Sixteen vly-positive PCR products were sequenced. The phylogenetic analysis based on the reference nucleic acid sequences deposited in the NCBI GenBank, covering the whole G. vaginalis vly gene, placed the studied isolates into three main groups. In conclusion, our experiments confirmed the major G. vaginalis virulence factor to be vaginolysin. The multiplex PCR results are promising and further studies should be carried out to determine the sensitivity range of the method and its applicability to diagnostics. Our phylogenetic analysis results, where some samples were grouped together, might imply a possible correlation between the clinical signs, the tendency to chronification, the cytotoxicity of vaginolysin and the vly gene mutations.

۱ Introduction

Gardnerella vaginalis is a small, non-motile, Gram-variable coccobacillus that can grow in microaerophilic conditions. In the years following the discovery taxonomic status of this microbe remained uncertain due to some specifics in its cell wall structure: the cell wall stains Gram-positively in the exponential growth phase but, as the culture ages, the peptidoglycan layer becomes thinner and gives Gram-negative staining [1 ]. Thus, this Gram-variable microorganism was initially classified in the Corynebacterium and Haemophilus genera, but was later moved to a separate genus under the name G. vaginalis [ 1 ]. This bacterial species was proved to be a causative agent of genital tract infections in studies with volunteers [2 ] and in animal models [3 ]. It was also detected in exfoliated vaginal epithelium, in the absence of an inflammatory infiltrate. In recent years, the intensive use of more advanced methods, including molecular genetic analysis, has aided the studies on the etiological role of G. vaginalis, identifying it to be the major pathogen (in terms of frequency and quantity) in women with bacterial vaginosis (BV) [4–۶]. Several members of the genus Lactobacillus inhibit pathogenic bacteria in healthy women‘s vaginal ecosystem but some substances produced by G. vaginalis could provide a synergistic effect for the growth of the other pathogens as well as an inhibitory effect against lactobacilli [3 ]. BV is the most frequent vaginal infection among women in the reproductive age group, and surveys on different continents alarm that it is especially frequent in younger women, under 30 years of age [5,7].

Several attempts have been made to classify G. vaginalis strains based on the phenotype; however, this proved to be clinically irrelevant [8 ]. The major G. vaginalis virulence factor is an exotoxin with cytotoxic activity (including hemolytic activity) against human cells. The hemolysin triggers a local immune response and the synthesis of secretary immunoglobulin A (sIgA) can therefore be used as a diagnostic marker for BV. This toxic product is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins [9 ]. The G. vaginalis hemolysin, termed vaginolysin (VLY), shows selective toxicity and specifically targets host cells that carry complement regulatory molecule CD59. The cytotoxicity mechanism is based on VLY-mediated pore formation, with the proline residue in the undecapeptide of domain 4 of the toxin molecule playing the main role. Mutations in this region generate a VLY toxoid and could possibly aid in the development of a vaccine [9 ].

The aim of the present study was to propose a polymerase chain reaction (PCR)-based method for detection of the G. vaginalis vly gene and to perform a phylogenetic analysis in an attempt to find possible correlations between some clinical manifestations and certain gene mutations.

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