Mobiluncus species--their culture, identification and role in bacterial vaginosis

The authors present a brief account of the history of the genesis of a new genus of bacteria Mobiluncus. They describe their own experience with the cultivation and identification of this microorganism. From 300 specimens of vaginal smears it proved possible to cultivate Mobiluncus sp. in 15 instances whereby all positive cultivations correlated with the microscopic vaginal picture IIb. The majority of patients where Mobiluncus was proved by cultivation had clinical symptoms of the syndrome of bacterial vaginosis which suggests the participation of this organism in the aetiology of the disease.     

Immuno-microbiological aspects of chronic bacterial prostatitis pathogenesis

The paper deals with a role of the factors of the body's antiinfective resistance system in the pathogenesis of chronic bacterial prostatitis and with the identification of an etiological factor of the disease. Long-term studies have revealed that chronic bacterial prostatitis runs in the presence of impairments in the antiinfective resistance system, in failures of T lymphocytes, phagocytic activity, and opsonization in particular. Gram-positive cocci, predominantly Staphylococcus and Enterococcus alone or as part of 2-component associations have been found to be the leading agents in the etiology of chronic bacterial prostatitis.     

Nitrocellulose filter blots for species identification of Mobiluncus curtisii and Mobiluncus mulieris

Seventy strains of Mobiluncus, motile curved anaerobic bacteria associated with bacterial vaginosis, were correctly identified to species level by using bacteria fixed to nitrocellulose and hybridized with 32P-labeled DNA.     

Anaerobes in bacterial vaginosis

Four hundred high vaginal swabs were taken from patients attending gynaecology and obstetrics department of Govt. medical college, Amritsar. The patients were divided into four groups i.e. women in pregnancy (Group I), in labour/post partum (Group II), with abnormal vaginal discharge or bacterial vaginosis (Group III) and asymptomatic women as control (Group IV). Anaerobic culture of vaginal swabs revealed that out of 400 cases, 212(53%) were culture positive. Maximum isolation of anaerobes was in group III (84%) followed by group II (56%), group I (36%) and control group (15%). Gram positive anaerobes (69.2%) out numbered gram negatives (30.8%). Among various isolates Peptostreptococcus spp. and Bacteroides spp. were predominant.     

Pathogenicity islands and phage conversion: evolutionary aspects of bacterial pathogenesis

Horizontal gene transfer plays a key role in the generation of novel bacterial pathogens. Besides plasmids and bacteriophages, large genomic regions termed pathogenicity islands (PAIs) can be transferred horizontally. All three mechanisms for DNA exchange or transfer may be important for the evolution of bacterial pathogens.     

Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis

A predominance of Lactobacillus species in the vaginal flora is considered normal. In women with bacterial vaginosis, the prevalence and concentrations of intravaginal Gardnerella vaginalis and anaerobes are increased, whereas the prevalence of intravaginal Lactobacillus species is decreased. Because some lactobacilli are known to produce hydrogen peroxide (H2O2), which can be toxic to organisms that produce little or no H2O2-scavenging enzymes (e.g., catalase), we postulated that an absence of H2O2-producing Lactobacillus species could allow an overgrowth of catalase-negative organisms, such as those found among women with bacterial vaginosis. In this study, H2O2-producing facultative Lactobacillus species were found in the vaginas of 27 (96%) of 28 normal women and 4 (6%) of 67 women with bacterial vaginosis (P less than 0.001). Anaerobic Lactobacillus species (which do not produce hydrogen peroxide) were isolated from 24 (36%) of 67 women with bacterial vaginosis and 1 (4%) of 28 normal women (P less than 0.001). The production of H2O2 by Lactobacillus species may represent a nonspecific antimicrobial defense mechanism of the normal vaginal ecosystem.     

Mobiluncus species and other anaerobic bacteria in non-puerperal breast abscesses

Samples of 109 breast masses were cultured aerobically and anaerobically forMobiluncus spp. using selective and non-selective media. Two of the 103 evaluable samples were sterile and 57 (55%) yielded facultative bacteria only. The remaining 44 (43%) contained anaerobic growth alone or mixed infections. Seven (16%) of the 44 samples with anaerobes, six from women and one from a man, yieldedMobiluncus spp. Six were identified asMobiluncus curtisii and one asMobiluncus mulieris. There were two types of breast abscesses: one withStaphylococcus aureus predominating and one with anaerobes predominating. Most of the anaerobes belonged to the normal vaginal flora, but some were members of the normal oral flora. These results support the hypothesis that breast infections with these anaerobes result from sexual activities.     

Prevalence of bacterial vaginosis in antenatal women

Bacterial vaginosis is an established risk factor in pregnant women for premature rupture of membranes and preterm delivery. This study was carried out to find out the prevalence of Bacterial Vaginosis (BV) in antenatal women with vaginal discharge and the effect of treatment with Metronidazole gel on pregnancy outcome. One hundred and fifty symptomatic and fifty asymptomatic women in second trimester of pregnancy in the age group of 20-30 years were included in the study. Gram stained smears of vaginal discharge were examined for evidence of BV with a scoring system by Nugent et al and was found to be positive in 38.5% in symptomatic antenatal women. Intravaginal metronidazole gel application was found to be an effective therapeutic option. Incidence of preterm labour was more in untreated cases.     

Normal flora and bacterial vaginosis in pregnancy: an overview

Abstract

The female genital tract is an intricate, yet balanced ecosystem that hosts a variety of different residential microflora. The physiological changes that occur during pregnancy may disrupt this balanced ecosystem and predispose women to a potentially pathogenic microbiota. Bacteria that are associated with bacterial vaginosis (BV) are opportunistic pathogens that frequently form part of this microbiota. The overgrowth of and infections with these bacteria are linked to poor obstetric outcomes and increased transmission of other reproductive tract infections (RTIs). These infections increase women's susceptibility of acquiring HIV, the rates of HIV shedding and the development of acquired immune deficiency syndrome (AIDS) in HIV-infected patients. It is unknown how the plethora of bacterial species associated with BV contributes to the dynamics of this condition. The use of high-throughput methods have led to the in-depth investigation of different BV-related bacterial species and the functional capabilities of these species. However, the pathogenesis of BV is still poorly defined and the role of individual BV-related bacterial species in specific pregnancy complications is unclear and controversial. The majority of BV infections are asymptomatic and successful diagnosis is complicated by the lack of reliable and standardized diagnostic tests.     

Fibronectin binding of Lactobacillus species isolated from women with and without bacterial vaginosis.

Lactobacilli isolated from the vaginas of healthy women (39 strains) and from the vaginal discharge of women with bacterial vaginosis (15 strains) were investigated for their binding to 125I-fibronectin. Nine of the 54 strains bound fibronectin at pH 7.2. The binding capacity of these nine strains was about the same as that observed with Staphylococcus aureas Cowan 1. The binding was specific; an excess of unlabelled fibronectin or its amino-terminal 29-kDa fragment effectively competed for binding, whereas bovine serum albumin, human IgG and orosomucoid did not. Incubation of lactobacilli with fibronectin for different periods revealed a time-dependent increase in binding. Lowering the pH to 4.0 increased the binding capacity of all of the lactobacilli tested; binding occurred with strains that had previously failed to bind at pH 7.2. The increased binding of lactobacilli to fibronectin at a low pH may play a role in the maintenance of the ecological balance of the vagina.     

Antigenic distinctiveness of Mobiluncus curtisii and Mobiluncus mulieris

A total of 26 Mobiluncus strains (17 M. curtisii and 9 M. mulieris strains) were compared serologically by double immunodiffusion and immunoblotting against antisera prepared against representative isolates of each species. All strains from the same species were strongly reactive with homologous antisera but generally weakly reactive with antisera to the heterologous Mobiluncus spp. The antisera did not react with strains of the unrelated genera Campylobacter, Succinivibrio, Wolinella, Actinomyces, Anaerobiospirillum, and Anaerovibrio.     

Reversible deficiency of antimicrobial polypeptides in bacterial vaginosis

Bacterial vaginosis is a common condition associated with increased risk of sexually transmitted diseases, including human immunodeficiency virus infections. In contrast, vulvovaginal candidiasis has a much weaker association with sexually transmitted diseases. We found that vaginal lavage fluid from women with bacterial vaginosis is deficient in antimicrobial polypeptides and antimicrobial activity compared to fluid from healthy women or women with vulvovaginal candidiasis. Effective treatment normalized the concentrations of antimicrobial polypeptides in both bacterial vaginosis and in vulvovaginal candidiasis, suggesting that the abnormalities were a result of the diseases. Unlike in vulvovaginal candidiasis, the neutrophil attractant chemokine interleukin-8 (IL-8) was not increased in bacterial vaginosis, accounting for low concentrations of neutrophil-derived defensins in vaginal fluid. In organotypic cultures of human vaginal epithelium containing dendritic cells, treatment with Lactobacillus jensenii, a typical vaginal resident, induced the synthesis of IL-8 mRNA and the epithelial human beta-defensin-2 mRNA, but a typical bacterial vaginosis pathogen, Gardnerella vaginalis, had no effect. When the two bacteria were combined, Gardnerella vaginalis did not interfere with the immunostimulatory effect of Lactobacillus jensenii. The loss of normal immunostimulatory flora in bacterial vaginosis is thus associated with a local deficiency of multiple innate immune factors, and this deficiency could predispose individuals to sexually transmitted diseases.     

Production and characterization of monoclonal antibodies to Mobiluncus species.

Members of the genus Mobiluncus are anaerobic motile curved rods which are associated with bacterial vaginosis (BV). Murine monoclonal antibodies (MAbs) to the ATCC type strains of M. curtisii subsp. curtisii, M. curtisii subsp. holmesii, and M. mulieris were produced and characterized by enzyme-linked immunosorbent assay and indirect immunofluorescence assay. Four MAbs were subspecies specific and reacted with M. curtisii subsp. curtisii but not with M. curtisii subsp. holmesii; four were specific for M. mulieris. The remaining antibodies demonstrated some cross-reactivity: three were species specific and reacted with both subspecies of M. curtisii, and one defined an epitope shared by M. curtisii subsp. holmesii and M. mulieris but not by M. curtisii subsp. curtisii. None of the MAbs reacted with a panel of other bacteria commonly present in the vaginas of normal women or women with BV. Examination of the molecular specificities of the antibodies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed four antibodies which were specific for an 82,000-dalton molecule of M. curtisii subsp. curtisii and five antibodies which bound a major band of M. mulieris at 93,000 daltons. Selected MAbs reacted in the indirect immunofluorescence assay with 24 of 25 Mobiluncus spp. clinical isolates from local women with BV and could be used for direct detection of Mobiluncus spp. in vaginal fluid from a patient with BV.     

BVBlue test for diagnosis of bacterial vaginosis

Bacterial vaginosis (BV) is a disorder of the vaginal ecosystem characterized by a shift in the vaginal flora from the normally predominant Lactobacillus to one dominated by sialidase enzyme-producing mixed flora. It is the most common cause of abnormal vaginal discharge in adult women. The BVBlue system (Gryphus Diagnostics, L.L.C.) is a chromogenic diagnostic test based on the presence of elevated sialidase enzyme in vaginal fluid samples. BVBlue was compared to the standard method for diagnosing BV (Amsel criteria and Nugent score). Fifty-seven nonmenstruating women of > or =16 years of age who presented for a pelvic examination were recruited. Demographic features were collected via a self-administered questionnaire. The Amsel criteria were assessed based on three of four of the following characteristics of vaginal discharge: consistency, odor, pH, and presence of clue cells on Gram stain. BVBlue was compared to the Gram stain and Amsel criteria. The sensitivity, specificity, positive predictive value, and negative predictive value for BVBlue versus the Gram stain and Amsel criteria were 91.7, 97.8, 91.7, and 97.8% and 50.0, 100, 100, and 88.2%, respectively. A significantly greater proportion of patients with a vaginal pH of >4.5, a positive amine test, or with clue cells on vaginal Gram smear were found to have a positive BVBlue test (P < 0.001). Women previously treated for BV were 2.98 times more likely to have another episode of BV. BVBlue is a useful point-of-care diagnostic tool to provide a presumptive diagnosis of BV, especially in situations where microscopic capabilities are unavailable.     

Evaluation of liquid culture media to support growth of Mobiluncus species

Mobiluncus curtisii and M. mulieris are anaerobic, gram-negative, motile curved rods isolated commonly from the vagina of women with bacterial vaginosis. Hitherto, there has been difficulty in isolating and growing these bacteria and little attention has been paid to growth in liquid media. Reasons for establishing the means of attaining optimal growth in such media include production of antigens for diagnostic and immunological studies and production of the soluble cytotoxin. In this study the efficacy of 12 liquid culture media in supporting growth was examined. M. mulieris (strain A198) multiplied > or =10-fold in only five media - Schaedler broth, Columbia blood broth (CBB), peptone-starch-dextrose (PSD) broth, brain-heart infusion plus arginine and spent tissue-culture medium. Similarly, M. curtisii (strain A98) multiplied > or =10-fold in only three media -Schaedler broth, CBB and PSD. Some strains of both bacterial species grew very poorly or not at all, in all the media tested. With an inoculum of > or =10(5)/ml, CBB, or PSD plus 10% horse serum, supported the growth of some strains of both bacterial species to 10(9) organisms/ml within 48 h, and viable bacteria persisted longer in some media (e.g., CBB) than in others. While variation in growth of Mobiluncus spp. may occur between one laboratory and another, these observations provide the basis for optimisation of a universal liquid culture medium that should facilitate production of antigens and cytotoxin.     

Demonstration of heterogeneity among the antigenic proteins of Mobiluncus species.

The protein and antigenic profiles of the American Type Culture Collection type strains of Mobiluncus species and those of 114 clinical isolates were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and immunoblotting with homologous polyvalent antisera. The majority of isolates (82%) possessed characteristic protein profiles and could be identified to the species level by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The major protein bands were also antigenic, and some antigenic cross-reactivity was noted between the two Mobiluncus species. All of the isolates were examined for reactivity with a panel of 12 monoclonal antibodies previously prepared against the type strains. While 56 of 60 clinical isolates of Mobiluncus curtisii (93%) reacted with one or more of the monoclonal antibodies, only 23 of 54 clinical isolates which were identified as Mobiluncus mulieris by biochemical methods (48%) reacted with one or more of the monoclonal antibodies. One of the 4 M. curtisii isolates (25%) and 11 of the 31 M. mulieris isolates (35%) which did not react with the monoclonal antibodies also had atypical protein profiles. These results demonstrate a high degree of heterogeneity in the protein and antigenic profiles of Mobiluncus isolates and suggest that further taxonomic division may be appropriate.     

Molecular diagnosis of bacterial vaginosis: impact on IVF outcome

Bacterial vaginosis can increase obstetrical complications such as miscarriage, premature rupture of membranes and preterm delivery. The aim of our study was first to assess BV prevalence for infertile patients treated by in vitro fertilisation (IVF) using both the Nugent score and polymerase chain reaction (PCR), and then to assess the impact of BV on the pregnancy rate after IVF. Vaginal samples were obtained from women followed for IVF in our Assisted Reproduction Technology (ART) Unit between August 2010 and April 2011. For each patient, two techniques were performed to diagnose BV: Gram staining to assess the Nugent score and a quantitative molecular analysis using a specific real-time PCR assay. Two groups were studied: normal flora (BV?) and BV (BV+). The primary outcome measure was the implantation rate. The secondary outcomes were clinical pregnancy rate, early and late miscarriage, premature rupture of membranes, preterm delivery, mode of delivery and birthweight. A total of 307 patients were included. PCR revealed a prevalence of BV of 9.45 %. Among women who performed vaginal douching, 22.2 % were BV+, whereas 7.9 % of patients who did not douche were BV+ (p?=?0.028). The embryo implantation rate was decreased between the BV? and BV+ groups (36.3 % vs. 27.6 %, p?=?0.418), but it was not significant. Obstetrical outcomes did not present significant statistical differences among the groups. Vaginal douching significantly enhanced BV in women treated with IVF. We also observed a non-significant decrease of embryo implantation rate and clinical pregnancy rate for women treated by IVF.     

Sialidases (neuraminidases) in bacterial vaginosis and bacterial vaginosis-associated microflora.

Bacterial vaginosis, Prevotella species, and Bacteroides species have been associated with prematurity and upper genital tract infection. Prevotella (Bacteroides) species and Bacteroides fragilis have also been associated with preterm birth. However, the mechanism by which lower genital tract infection causes upper genital tract disease remains poorly understood. Sialidases (neuraminidases) are enzymes which enhance the ability of microorganisms to invade and destroy tissue. Elevated levels of sialidase activity were detected in 42 (84%) of 50 vaginal fluid specimens from women with bacterial vaginosis and none of 19 vaginal fluids from women without bacterial vaginosis (P less than 0.001). Vaginal fluid from women with bacterial vaginosis had a median specific activity of 9.8 U compared to 2.5 U of sialidase in women without bacterial vaginosis (P less than 0.001). In order to determine the probable source of sialidases in vaginal fluid, the microorganisms recovered from women with bacterial vaginosis before and after treatment were assayed. Of 28 specimens from women with bacterial vaginosis, 27 (96%) yielded sialidase-positive bacteria, at a median concentration of 10(6.5) CFU/ml of vaginal fluid. Prevotella and Bacteroides species accounted for the sialidase activity in 26 of the vaginal fluids, and Gardnerella vaginalis accounted for the sialidase activity in the remaining fluid. After treatment, sialidase was detected in the vaginal fluid of 1 (5%) of 22 women who responded to therapy and in all of 6 women for whom therapy failed. These data suggest that vaginal fluid sialidase is highly correlated with bacterial vaginosis and that the probable sources for this enzyme activity are the Bacteroides and Prevotella species present in the vagina.