The Pathogenesis Of Bacterial Vaginosis And Its Sequelae With Respect To The Gardnerella Vaginalis Biofilm

In recent years, the prevalence of secondary infertility has been on the rise as the frequency of induced abortion and recurrent abortion increases. Studies have shown the history of pelvic inflammatory disease, complications arise from postpartum or after abortion and previous episodes of genital tract infections are potential risk factors that contribute to secondary infertility in women. The rate of infertility is approximately5.2%in Beijing and Shanghai according to statistical analysis and secondary infertility comprises50%, of which60-80%has a history of induced abortion. A follow-up survey shows that incidence of secondary infertility stands at3.3%. Lower genital infection includes recurrent episodes of vulvitis, vaginitis and chronic cervicitis, causes lower genital tract inflammation, salpingitis, tubal obstruction and endometrial inflammation that leads to subsequent infertility via ascending infection. The pathogens that implicated tubal inflammation usually occur after abortion or postpartum infection. However most of the tubal inflammation cases can have insignificant manifestation and pelvic examination is unremarkable therefore tends to be misdiagnosed and caused delay in prompt treatment, resulting in occurrence of chronic inflammation and obstruction of fallopian tube.Bacterial vaginosis (BV) is a common and complicated gynaecological disease, characterized by a disruption in the proportion of commensal vaginal microflora. Whether symptomatic or asymptomatic, BV is an independent risk factor for severe reproductive tract and obstetric sequelae such as premature rupture of fetal membrane and intrauterine infection inflicting preterm delivery and spontaneous abortion, salpingitis predisposing to infertility and ectopic pregnancy, ascending infection developing into pelvic inflammatory diseases, cervicitis, urogenital and post-surgery infection, which seriously hampers women’s health. BV is incriminated to be the primary cause of secondary infertile patients. Thus, considering its resulting dire sequelae, the importance of diagnosis and treatment of BV is highlighted.A retrospective clinical study on the vaginal cultures of secondary infertile patients and BV patients was performed to evaluate the dominant pathogens prevalent and its corresponding antimicrobial resistance in normal situation, with comparison to the lower genital infection in clinically-diagnosed BV category. Furthermore, Gardnerella vaginalis strains were isolated from BV patients and analysed the virulence property of biofilm forming capability, with respect to the resistance of antimicrobial, and the association of BV recurrence. Hence, this will provide evidence for preventing BV and its complications.The study is divided into three aspects:The first part is to evaluate the lower genital pathogenic infection and antimicrobial of secondary infertile patients admitted to Obstetrics and Gynaecology Hospital of Fudan University from January2010to December2012. The second part is to identify the BV-associated bacteria and antibiotic susceptibility test in a sample of887clinically diagnosed BV patients from January2012till February2013. The third part is to determine the biofilm capacity of58Gardnerella vaginalis strains isolated from the vagina of BV patients and the growth condition under variable pH environment. The ability to form biofilm by Gardnerella vaginalis may be associated with the recurrence of BV after treatment.Part I. The association of lower genital infection to Secondary Infertility and antimicrobial susceptibility analysis of microfloraVarious causes that contribute to infertility have become a pressing problem and highlight the need to address the problem. Infertility is categorized into primary and secondary. Secondary infertility refers to failure to conceive following a previous pregnancy for one year or more after coitus, without contraception. Female infertility is not an isolated disease; it entails various factors, encompassing the complication or end stage from numerous gynecological diseases. Pathogenic infection that arises to lower genital infection is one of the paramount factors that causes secondary infertility. Interventions to treat lower genital infection focus on the use of antibiotics, but resistant bacteria strains surface continuously, causing the effectiveness of such treatments to be disappointingly low. Thus to evaluate and differentiate the interindividual variation commensal microbiota profile and antimicrobial resistance of pathogenic organisms is especially crucial.A total of606pathogens isolated from vaginal discharge of secondary infertile female inpatients at Fudan Obstetrics and Gynaecology Hospital from January2010to December2012. A retrospective analysis was conducted to determine the distribution of causative organisms from the vagina of these patients and their antimicrobial resistance pattern. The genital microflora infection present in secondary infertile female patients and the antimicrobial susceptibility testing of pathogens isolated assists in rational use of medicine. It also aims to appraise whether the high prevalence of pathogens from this population is associated with reproductive sequelae.The most prevalent pathogens isolated from secondary infertile patients are namely Escherichia coli (E coli), Enterococcus faecalis and Candidia albicans. Mycoplasma infection consists of48.8%. Enterobacteriaceae such as E coli and Klebsiella pneumoniae have high antibiotic resistance to ampicillin, cefotaxime, ceftazidime, sulfamethoxazole, however a lower resistance is seen for imipeniem, menorpenum, amixicillin. Gram positive cocci has a higher sensitivity towards vancomycin, nitrofurantoin, linezolid. E coli has a higher sensitivity to ampicillin, hence should be recommended as first-line treatment. None of the Staphylococcus aureus strains are vancomycin-resistant. Ureaplasma urealyticum is highly sensitive to doxycycline and minocycline and can be used for mycoplasma infection.The causative pathogenic infection is analysed with respect to the antimicrobial susceptibility testing of vaginal microbes in secondary infertile patients. The main pathogens which caused female genital infection is identified and the corresponding antimicrobial susceptibility of these anaerobes, which would provide guidance for treating clinical antimicrobial infection more effectively. Our results indicate that is important to culture the vaginal discharge for the prevalence of some microorganisms (Gardnerella vaginalis, Enterobacteriaceae and Enterococci, Streptococcus agalactiae) in the population of infertile women need to be better analyzed, especially after the recent studies correlating idiopathic infertility with the presence of cervical cytokines in women with an abnormal vaginal flora.Part II. The vagina microflora distribution and antimicrobial susceptibility analysis in bacterial vaginosis patientsUnder normal conditions, the vagina harbours a diverse mutualistic microflora that primarily consists of lactobacilli which confer antimicrobial protection through producing lactic acid, hydrogen peroxide and bacteriocin which inhibit the growth and colonization of pathogens. Thus a balanced state of vaginal microbiota is maintained. Prolonged use of antibiotics, decreased immunity, vaginal douching, sexual intercourse and hormone fluctuation cause disturbances in the vaginal ecosystem, displacing the resident lactobacilli by other proliferating anaerobic organisms like Gardnerella vaginalis and Atopobium vaginae. The loss or depletion of hydrogen peroxide-producing lactobacilli results in bacterial vaginosis (BV). BV is associated with several gynaecological diseases, can arise to salpinginitis and pelvic inflammatory, increase adverse pregnancy outcomes and acquisition of sexually transmitted infections. Clinical approach to treating BV is currently prescribing metronidazole and clindamycin, however more than50%women will experience recurrence after initial treatment. One of the main reasons is a lack of profound understanding in the etiology of BV, especially the pathogenesis aspect, which contributes to ineffective prophylaxis and treatment. Therefore highlights the need to evaluate the pathogenesis and antimicrobial resistance research into BV, which aids in prevention, diagnosis and therapy intervention at an earlier stage, thus diminishing the complications caused by BV.This study evaluates the microflora distribution in the genital tract of887clinically diagnosed BV patients. Microbial culture of vaginal swab and antimicrobial susceptibility test were performed among these patients. Every pathogen isolated from BV patients were analysed according to its proportional rate of colonization and to evaluate the dominant causative agents which arises from imbalance of vaginal microbiota. This facilitates early prognosis, diagnosis and treatment. In the population of BV patients,48species of microorganisms were isolated, namely Candida species (25.14%), Streptococcus species (18.72%), Gemella species (15.57%), Gardnerella vaginalis (12.16%), Enterococci species (9.02%), Escherichia species (6.69%), Staphylococcus species (3.69%), both Klebsiella species and Listera species(2.19%each). The most frequent microorganisms cultured are as followed Candida species, Gemella mobillorum, Gardnerella vaginalis, Streptococcus agalactiae and Enterococcus faecalis.Part Ⅲ. Analysis of predisposing factors causing recurrent bacterial vaginosis induced by Gardnerella VaginalisThe disturbance of the vaginal ecosystem, loss of the hydrogen peroxide producing lactobacilli accompanied by massive anaerobic overgrowth and BV-associated bacteria, such as Gardnerella vaginalis in particular is observed in patients with bacterial vaginosis. After standard treatment regimen of metronidazole. although temporarily relief of symptom is achieved, Gardnerella vaginalis still adhered to the vaginal epithelium cohesively. Bacterial biofilm has not been eradicated and its persistence, after treated for BV, causes recalcitrant infection and recurring symptoms. Research has validated that Gardnerella vaginalis among other bacteria vaginosis associated pathogens to have a stronger capacity to form biofilm and possesses greater virulence. During treatment, the metabolism of Gardnerella vaginalis and BVAB ceases, but once treatment stops, their activities reactivate and cause disruption of vaginal microbiota, thus contributing to the recurrence of BV.The present study evaluates the association of Gardnerella vaginalis with bacterial vaginosis on its pathogenesis and therapeutic efficacy. It also assesses the antimicrobial resistance of clinical isolates of Gardnerella vaginalis towards the current commonly-used antimicrobial drugs, metronidazole and clindamycin. Under various modifications of in-vitro environmental factors, the growth and colonization of Gardnerella vaginalis can be appraised. These corresponding factors will predispose to bacterial vaginosis and the aim to show the association with its recurrence.Vaginal swabs were collected from women attending the gynaecology outpatient department and clinically diagnosed as bacterial vaginosis (BV) over a period of seven months from September2012to February2013. A total of58Gardnerella vaginalis isolates were identified and evaluated its growth alteration under various pH fluctuations. Antibiotic resistance assay of clinical isolates was determined utilising the disk diffusion and broth microdilution methods, and employment of semi-quantification microtiter plate to assess biofilm-forming capacity.Growth of Gardnerella vaginalis was restricted in acidic vaginal environment whereas alkaline condition was found be more conducive for its growth. High antimicrobial susceptibility testing of clinical isolates to clindamycin, erythromycin, ceftriaxone, penicillin and vancomycin was observed, whereas result of antibiotic resistance to levoflovaxin and metronidazole indicated67.2%and63.8%, respectively.48.3%of the strains exhibited strong biofilm-forming ability.A healthy female vagina usually confers an acidic environment which inhibits the overgrowth of pathogens. However, when pH elevates in the vagina of BV patients, Gardnerella vaginalis is more prone to colonize. This correlates its capacity to form biofilm with the propensity to cause recurrence of BV after treatment.