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Objective To undertake the first comprehensive evaluation of the urinary microbiota associated with Hunner lesion (HL) interstitial cystitis/bladder pain syndrome (IC/BPS). Despite no previous identification of a distinct IC/BPS microbial urotype, HL IC/BPS, an inflammatory subtype of IC/BPS, was hypothesized most likely to be associated with a specific bacterial species or microbial pattern. Participants and methods The bacterial microbiota of midstream urine specimens from HL IC/BPS and age- and gender-matched IC/BPS patients without HL (non-HL IC/BPS) were examined using the pan-bacterial domain clinical-level molecular diagnostic Pacific Biosciences full-length 16S gene sequencing protocol, informatics pipeline and database. We characterized the differential presence, abundances, and diversity of species, as well as gender-specific differences between and among HL and non-HL IC/BPS patients. Results A total of 59 patients with IC/BPS were enrolled (29 HL, 30 non-HL; 43 women, 16 men) from a single centre and the microbiota in midstream urine specimens was available for comparison. The species abundance differentiation between the HL and non-HL groups (12 species) was not significantly different after Bonferroni adjustments for multiple comparisons. Similarly, the nine differentiating species noted between female HL and non-HL patients were not significantly different after similar statistical correction. However, four species abundances (out of the 10 species differences identified prior to correction) remained significantly different between male HL and non-HL subjects: Negativicoccus succinivorans, Porphyromonas somerae, Mobiluncus curtisii and Corynebacterium renale. Shannon diversity metrics showed significantly higher diversity among HL male patients than HL female patients (P = 0.045), but no significant diversity differences between HL and non-HL patients overall. Conclusions We were not able to identify a unique pathogenic urinary microbiota that differentiates all HL from all non-HL IC/BPS. It is likely that the male-specific differences resulted from colonization/contamination remote from the bladder. We were not able to show that bacteria play an important role in patients with HL IC/BPS.

Background Bacterial vaginosis (BV) is one of the most common vaginal infections worldwide. It is associated with the presence of a dense polymicrobial biofilm on the vaginal epithelium, formed mainly by Gardnerella species. The biofilm also contains other anaerobic species, but little is known about their role in BV development. Aim To evaluate the influence of different culture media on the planktonic and biofilm growth of six cultivable anaerobes frequently associated with BV, namely Gardnerella sp., Atopobium vaginae, Lactobacillus iners, Mobiluncus curtisii, Peptostreptococcus anaerobius and Prevotella bivia. Methods A total of nine different culture media compositions, including commercially available and chemically defined media simulating genital tract secretions, were tested in this study. Planktonic cultures and biofilms were grown under anaerobic conditions (10% carbon dioxide, 10% helium and 80% nitrogen). Planktonic growth was assessed by optical density measurements, and biofilm formation was quantified by crystal violet staining. Results Significant planktonic growth was observed for Gardnerella sp., A. vaginae and L. iners in New York City III broth, with or without ascorbic acid supplementation. Biofilm quantification showed high in vitro biofilm growth for Gardnerella sp., P. anaerobius and P. bivia in almost all culture media excluding Brucella broth. Contrary, only New York City III broth was able to promote biofilm formation for A. vaginae, L. iners and M. curtisii. Conclusions Our data demonstrate that New York City III broth relative to the other tested media is the most conducive for future studies addressing polymicrobial biofilms development as this culture medium allowed the formation of significant levels of single-species biofilms., This work was supported by the research project [PTDC/BIA-MIC/28271/2017] under the scope of COMPETE 2020 [POCI-01-0145-FEDER-028271], supported by the Portuguese Foundation for Science and Technology (FCT), and by the strategic funding of unit [UIDB/04469/2020]. Aliona S. Rosca received financial support from individual Grant [PD/BD/128037/2016]. Nuno Cerca received support from the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1, granted to Christina A. Muzny, MD, MSPH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., info:eu-repo/semantics/publishedVersion

Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species., Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species. The host response to flagellin mediated through Toll-like receptor 5 (TLR5) has not been explored in BV. Using independent discovery and validation cohorts, we examined the hypothesis that TLR5 deficiency—defined by a dominant negative stop codon polymorphism, rs5744168—is associated with an increased risk for BV and increased colonization with flagellated bacteria associated with BV (BVAB1, Mobiluncus curtisii, and Mobiluncus mulieris). TLR5 deficiency was not associated with BV status, and TLR5-deficient women had decreased colonization with BVAB1 in both cohorts. We stimulated HEK-hTLR5-overexpressing NF-κB reporter cells with whole, heat-killed M. mulieris or M. curtisii and with partially purified flagellin from these species; as BVAB1 is uncultivated, we used cervicovaginal lavage (CVL) fluid supernatant from women colonized with BVAB1 for stimulation. While heat-killed M. mulieris and CVL fluid from women colonized with BVAB1 stimulate a TLR5-mediated response, heat-killed M. curtisii did not. In contrast, partially purified flagellin from both Mobiluncus species stimulated a TLR5-mediated response in vitro. We observed no correlation between vaginal interleukin 8 (IL-8) and flagellated BVAB concentrations among TLR5-sufficient women. Interspecies variation in accessibility of flagellin recognition domains may be responsible for these observations, as reflected in the potentially novel flagellin products encoded by Mobiluncus species versus those encoded by BVAB1.

Transport systems are used to collect and maintain the viability of microorganisms. Two Amies media based transport systems, BD CultureSwab™ MaxV(+) Amies Medium without Charcoal (MaxV(+)) and Fisherfinest® with Amies gel Transport Medium without charcoal (Fisherfinest®) were compared to a Cary-Blair media based transport system, Starswab® Anaerobic Transport System (Starswab®), for their capacity to maintain the viability of 17 clinical microorganisms commonly isolated from the vagina (Lactobacillus crispatus, L. jensenii, L. iners, group B streptococci, Candida albicans, Escherichia coli, Enterococcus faecalis, Atopobium vaginae, Peptoniphilus harei, Mycoplasma hominis, Gardnerella vaginalis, Dialister microaerophilus, Mobiluncus curtisii, Prevotella amnii, P. timonensis, P. bivia, and Porphyromonas uenonis). Single swabs containing mixtures of up to five different species were inoculated in triplicate and held at 4°C and room temperature for 24, 48, 72, and 96 hours (h). At each time point, swabs were eluted into a sterile salt solution, serially diluted, inoculated onto selected media, and incubated. Each colony type was quantified and identified. A change in sample stability was reported as a ≥ 1 log increase or decrease in microorganism density from baseline. Overall, the viability of fastidious anaerobes was maintained better at 4°C than room temperature. At 4°C all three transport systems ma intained the viability and prevented replication of C. albicans, E. faecalis, GBS, and E. coli. Microorganisms having a ≥ 1 log decrease in less than 24 h at 4°C included A. vaginae, G. vaginalis, and P. uenonis in Starswab®, L. iners, A. vaginae, and P. amnii in MaxV(+), and A. vaginae, G. vaginalis, P. bivia, and P. amnii in Fisherfinest®. At 48 h at 4°C, a ≥ 1 log decrease in concentration density was observed for P. harei and P. amnii in Starswab®, G. vaginalis, P. bivia and P. uenonis in MaxV(+), and L. iners, P. harei, P. timonensis, and P. uenonis in Fisherfinest®. Overall, at 4°C the viability an d stability of vaginal microorganisms was maintained better in the Cary-Blair based transport system (Starswab®) than in the two Amies based transport systems.

Background Bacterial vaginosis (BV) is a poorly detected public health problem that is associated with preterm delivery and for which no reliable diagnostic tool exists. Methods Molecular analysis of 231 vaginal samples, classified by Gram stain-based Nugent score, was used to propose molecular criteria for BV; these criteria were prospectively applied to 56 new samples. A quantitative molecular tool targeting 8 BV-related microorganisms and a human gene was developed using a specific real-time polymerase chain reaction assay and serial dilutions of a plasmid suspension. The targeted microorganisms were Gardnerella vaginalis, Lactobacillus species, Mobiluncus curtisii, Mobiluncus mulieris, and Candida albicans (which can be identified by Gram staining), as well as Atopobium vaginae, Mycoplasma hominis, and Ureaplasma urealyticum (which cannot be detected by Gram staining). Results With use of the Nugent score, 167 samples were classified as normal, 20 were classified as BV, and 44 were classified as intermediate. Except for U. urealyticum, M. mulieris, and Lactobacillus species, DNA of the tested bacteria was detected more frequently in samples demonstrating BV, but the predictive value of such detection was low. The molecular quantification of A. vaginae (DNA level, > or = 10(8) copies/mL) and G. vaginalis (DNA level, > or = 10(9) copies/mL) had the highest predictive value for the diagnosis of BV, with excellent sensitivity (95%), specificity (99%), and positive (95%) and negative (99%) predictive values; 25 (57%) of the samples demonstrating intermediate flora had a BV profile. When applied prospectively, our molecular criteria had total positive and negative predictive values of 96% and 99%, respectively. Conclusions We report a highly reproducible, quantitative tool to objectively analyze vaginal flora that uses cutoff values for the concentrations of A. vaginae and G. vaginalis to establish the molecular diagnosis of BV.

Background: Bacterial vaginosis (BV) and vaginitis in cervical cancer patients might becaused by mixed aerobic, anaerobic, and atypical bacteria. Since genital tract infections can be complicated, early and accurate identification of causal pathogens is vital. Objectives:The purpose of this study was i) to determinate if currently used aerobic culture methods are sufficiently sensitive to identify pathogens that can appear in the cervix of women after cancer treatment; ii) to investigate if molecular methods can improve the diagnostic process of BV and vaginitis, as well as broaden the range of detectable pathogens that would otherwise be difficult to cultivate. Methods: A one-year hospital-based study was conducted in 2011/2012. Cervical swabs from 130 patients were examined by microbiological culture and multiplex PCR. Results: Swab samples were positive for 107 and 93 women by microbiological culture and multiplex PCR, respectively. The most common bacteria isolated from culture were: Escherichia coli, Enterococcus faecalis, Streptococcus agalactiae, and Staphylococcus aureus, and using the molecular technique were: Gardnerella vaginalis, Bacteroides fragilis, Ureoplasma ureoliticum/parvum, Mobiluncus curtisii and Atopobium vaginae. Conclusions: Multiplex PCR might contribute to the diagnosis of genital tract infections and it broadens the number of detectable microorganisms responsible for BV. Combination of these two methods may become the basis for standardized diagnosis of BV and vaginitis.

The purpose of this study was to determine whether microbial DNA hybridization is a useful method to study bacterial vaginosis in asymptomatic pregnant women.Vaginal specimens were collected at26 weeks' gestation from 230 women, and analyzed for bacterial vaginosis by both Gram stain using Nugent criteria and DNA hybridization for Gardnerella vaginalis, Prevotella bivia, Bacteroides ureolyticus, and Mobiluncus curtisii. Results were analyzed using McNemar's paired test and chi-square test for trend, with significance set at P.05.By Gram stain, 60 (26.1%) of 230 were positive for bacterial vaginosis, and 134 (58.3%) were negative. By DNA hybridization, 99 (43%) were positive for at least 1 pathogen, and DNA results were significantly associated with Gram-stain results (P.01). As the Nugent score progressed from normal to abnormal flora, the proportion with1 pathogen detected by DNA hybridization increased significantly (P.001).DNA hybridization may be a useful method to study shifts in vaginal flora during pregnancy.

It has long been thought that the genera Mobiluncus and Falcivibrio contain the same organisms. Using a polyphasic approach, it was found that Mobiluncus curtisii and Mobiluncus mulieris were the same as Falcivibrio vaginalis and Falcivibrio grandis, respectively. As the genus name Mobiluncus takes precedence, it is proposed that F. vaginalis and F. grandis be transferred to the genus Mobiluncus. In agreement with previous studies, results from phenotypic tests did not support the separation of M. curtisii strains into its two subspecies, M. curtisii subsp. curtisii and M. curtisii subsp. holmesii. Phenotypic complexity within M. curtisii dictates that the species should be treated as a complex until more in-depth analyses of the species have been performed. Phylogenetic analyses, based on 16S rRNA gene sequences, demonstrated that the genus Mobiluncus was associated with Varibaculum cambriense and the two subspecies of Actinomyces neuii, and that A. neuii is only distantly related to Actinomyces sensu stricto.

We tested the activity of Novispirin G-10, a novel antimicrobial alpha-helical octadecapeptide structurally related to cathelicidins and other innate immunity peptides, against Chlamydia trachomatis serovars L2, D, and E and three organisms associated with bacterial vaginosis (BV). The peptide's activity against C. trachomatis was measured in 48-h shell vial assays with McCoy cell targets. Exposure to 100 micro g/ml of Novispirin G-10 reduced the infectivity of serovars D and E by 99.4-100% and serovar L2 by 91.7-99.1%. At the same concentration of 100 micro g/ml, Novispirin G-10 rapidly killed >99% of Mobiluncus curtisii, Gardnerella vaginalis, and Prevotella bivia, in standard colony-forming unit (CFU) assays. Given its simple structure and relative lack of cytotoxic and hemolytic activity, Novispirin G-10 may be a useful component of microbicide preparations designed to prevent chlamydial infection and/or remediate the abnormal vaginal flora associated with BV.

Bacterial vaginosis (BV) is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR) assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs) related to symptomatic BV:Gardnerella vaginalis,Mobiluncus curtisii,Mobiluncus mulieris,Bacteroides fragilis,Mycoplasma hominis,Atopobium vaginae,Ureaplasma urealyticum,Megasphaeratype I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs) 1, 2, and 3,Sneathia sanguinegens, andMycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR) were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%), and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future.

On the basis of partial 16S rRNA gene sequences and the results of Southern blot analyses, we confirmed the division of the genus Mobiluncus into the species Mobiluncus curtisii and Mobiluncus mulieris. Division of M. curtisii into M. curtisii subsp. curtisii and M. curtisii subsp. holmesii was not supported by our data.

Background and objectives A decreased concentration or total disappearance of Lactobacillus acidophilus in the vagina constitutes a frequent observation in bacterial vaginosis. Goal of the study Cetyltrimethylammonium naproxenate has been evaluated in vitro to detect antiadhesive properties at subinhibitory concentrations for Gardnerella vaginalis and Mobiluncus curtisii to vaginal epithelial cells (VEC). Study design Bacterial strains 14C- and or 3H-labeled were tested for adherence and competition to binding sites in VECs before and after treatment at sub-MIC with cetyltrimethylammonium naproxenate. Results In control tests of adherence, G. vaginalis and M. curtisii had their maximal adhesion at pH 5.4, L. acidophilus at pH 4.4. Preincubation of G. vaginalis and M. curtisii with cetyltrimethylammonium naproxenate 2.5 mg/mL (subinhibitory concentration) at pH 5.4 reduced adherence to VECs respectively by 48.3% and 34.1%. The same treatment of L. acidophilus showed no statistically significant difference. Treatment of VECs alone did not modify adherence. Competition tests between L. acidophilus and G. vaginalis and between L. acidophilus and M. curtisii showed that, in small quantities, L. acidophilus could compete with G. vaginalis and M. curtisii for binding sites in VECs at pH 4.4, when pretreated with cetyltrimethylammonium naproxenate. At a higher pH (4.8 and 5.4), L. acidophilus in higher quantities did not compete for binding sites occupied by G. vaginalis and M. curtisii. Conclusions Cetyltrimethylammonium naproxenate at subinhibitory concentrations modifies the adhesiveness of G. vaginalis and M. curtisii to VECs, reducing it by 48.3% and 34.1%, respectively. Adhesion of L. acidophilus to VECs is not impaired by pretreatment with cetyltrimethylammonium naproxenate at pH 4.4, even if they are in low number and compete for binding sites against pathogens. At higher pH levels, L. acidophilus did not compete for binding sites occupied by G. vaginalis and M. curtisii.

The analysis of electrophoretic protein profiles of 21 Mobiluncus curtisii, 11 Mobiluncus mulieris and 3 reference strains (Mobiluncus curtisii subsp. curtisii ATCC 35241, Mobiluncus curtisii subsp. holmesii ATCC 35242 and Mobiluncus mulieris BV 64-5) demonstrated species-related patterns. A highly variable region appeared at 70-85 kDa for Mobiluncus curtisii and at 75-95 kDa for Mobiluncus mulieris, which was likely to correspond to cell surface located proteins. When performed under standardized conditions, PAGE-protein analysis allowed to define intraspecies clusters, from which some strains appeared identical. Thus, the method seemed to provide a useful additive to identify a strain at the species level and might be of epidemiological interest.

The most common bacterial species isolated from the vaginas of 37 healthy rhesus macaques were Streptococcus viridans, coagulase negative Staphylococcus, Mobiluncus curtisii ss. curtisii, Corynebacterium renale-like organisms, Peptostreptococcus anaerobius, Gardnerella-like organisms, and other Corynebacterium species. The vaginal flora of the rhesus macaque differs from that previously reported for five other primate species. A two-year retrospective review of clinical cases of vaginitis and metritis found Escherichia coli and coagulase positive Staphylococci to be the most common pathogens isolated.

Transport media should preserve the viability and stability of microorganisms in clinical specimens. In this study, the Port-A-Cul transport system and the Copan transport system without charcoal, both designed to preserve anaerobes, were evaluated. Dacron swabs were inoculated with two combinations of facultative and anaerobic organisms typically found in vaginal swab samples. Combination I contained Candida albicans , Escherichia coli , Enterococcus spp., group B streptococci, Lactobacillus crispatus , and Staphylococcus aureus . Combination II contained Lactobacillus iners , Peptoniphilus asaccharolyticus , Mycoplasma hominis , Prevotella bivia , Prevotella corporis , Porphyromonas asaccharolytica , Mobiluncus curtisii , Peptostreptococcus anaerobius , and Gardnerella vaginalis . Duplicate swabs were placed into the two transporters and held for 24, 48, 72, and 96 h at 4 and 24°C. Both transporters maintained the viability of organisms better at 4°C than at 24°C. Prevotella bivia and Prevotella corporis had a loss of viability in both transporters at both temperatures. However, at 24°C, there was a significantly greater loss of viability for Mycoplasma hominis , Prevotella bivia , Prevotella corporis , and Peptoniphilus asaccharolyticus when the organisms were stored in Copan transport medium than when they were stored in Port-A-Cul transport medium for 96 h ( P < 0.002). Some organisms proliferated in the transport media, but when transporters were held at 24°C for 96 h, a significantly greater increase in the concentrations of group B streptococci and Candida albicans , Escherichia coli , and Enterococcus spp. organisms in Copan medium than in Port-A-Cul medium was observed ( P < 0.002). At room temperature, the Port-A-Cul system is superior to the Copan system with respect to the preservation of fastidious microorganisms and the prevention of the proliferation of facultative organisms.

Despite the fact that bacterial vaginosis (BV) is the most prevalent cause of symptomatic vaginal discharge in the United States1,2 and is a risk factor for preterm birth3 and acquisition of HIV,4 its cause remains unknown and its treatment suboptimal. Not only are initial cure rates of BV poor, but recurrence rates are high.5 Debate exists as to whether recurrences are due to relapse or reinfection. However, 1 hypothesis is that relapse may occur secondary to inadequate treatment of BV-associated organisms, either due to resistance of BV-associated organisms to metronidazole or to inadequate penetration of a biofilm community.6,7 Anecdotally it is more difficult to eradicate BV in some women compared with others. Recent data substantiates this by the finding that women with more complicated vaginal flora reflected by higher Nugent scores (predominantly because of morphotypes consistent with Mobiluncus), are more likely to fail therapy than those with lower scores.8 The presence of Mobiluncus spp. (M. curtisii and M. mulieris) in the vagina is highly specific although not sensitive for the diagnosis of BV.9,10 Of the two, M. curtisii appears to predominate and in vitro is resistant to metronidazole, the most commonly used therapy for BV.11 Therefore, recurrence of BV could be related to failure to clear this organism. Using specimens obtained from a prospective study of the treatment of BV, we determined if there was an association between persistence of M. curtisii and recurrence of BV at 65 to 70 days after completion of initial therapy. Vaginal swabs were collected during a prospective treatment study of BV. Women attending the Jefferson County Health Department STD Clinic with symptomatic BV were enrolled and treated for BV as previously described.8 All women received metronidazole for 7 or 14 days plus or minus azithromycin. Subjects were reevaluated at 21, 35 to 40, and 65 to 70 days after beginning the study. Subjects were discontinued from the study upon recurrence of symptomatic BV but were followed if the BV was asymptomatic. Vaginal swabs for Gram stain and polymerase chain reaction (PCR) were collected at baseline and each follow-up visit. For this substudy we included only those women who had M. curtisii present at baseline as detected by PCR and who completed 3 follow-up visits for the main study. Recurrence of BV at 65 to 70 days was defined as a Nugent score of 7 to 10 at that follow-up visit regardless of symptoms. Persistence of M. curtisii was defined as the organism being present at baseline and at least one of the follow-up visits. Baseline characteristics of the subsample and larger cohort were compared by the chi-square test for categorical variables and the t test for continuous variables. The relationship between the persistence of M. curtisii the development of recurrence was examined by the chi-square test. A stratified analysis was used to examine the relationship between M. curtisii and recurrence controlling for treatment and the Breslow-Day statistic was used to test homogeneity of the strata. All analyses were conducted with SAS Ver. 9.1 and a P value of

In the present study the lower genital tract microbiota in asymptomatic fertile women (n=34) was identified and quantified by culturing vaginal secretions. Also, vaginal and cervical samples were analyzed by a semiquantitative checkerboard DNA-DNA hybridization technique (CDH) based on genomic probes prepared from 13 bacterial species (Bacteroides ureolyticus, Escherichia coli, Fusobacterium nucleatum, Gardnerella vaginalis, Mobiluncus curtisii ss curtisii, Prevotella bivia, Prevotella disiens, Prevotella melaninogenica, Atopobium vaginae, Lactobacillus iners, Staphylococcus aureus ss aureus, Streptococcus anginosus, and Streptococcus agalactiae). The bacterial species found by either culture or CDH were correlated with proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8), secretory leukocyte protease inhibitor (SLPI), and endotoxin in the cervicovaginal samples. Grading the women into healthy, intermediate, or bacterial vaginosis (BV) as based on Gram staining of vaginal smears, the viable counts of lactobacilli (L. gasseri) and of streptococci-staphylococci combined were highest in the intermediate group. In BV, particularly the high concentrations of Actinomyces urogenitalis, Atopobium vaginae, and Peptoniphilus harei were noted (>or=10(11) per ml). The total viable counts correlated with both cervical IL-1 alpha and IL-1 beta. A strong negative correlation was observed between L. iners and total viable counts, G. vaginalis, or cervical IL-1 alpha, while it correlated positively with SLPI. Analysis of vaginal and cervical samples from 26 out of the 34 women by CDH showed that anaerobic bacteria were more frequently detected by CDH compared to culture. By this method, A. vaginae correlated with G. vaginalis, and L. iners with S. aureus. With regard to cytokines, B. ureolyticus correlated with both cervical and vaginal IL-1 alpha as well as with cervical IL-8, while F. nucleatum, S. agalactiae, S. anginosus, or S. aureus correlated with vaginal IL-1 alpha. Furthermore, all Gram-negative bacteria taken together, as measured by CDH, correlated with vaginal endotoxin and inversely with vaginal SLPI. The significance of the results is discussed. In summary, mapping of the identity and quantity of vaginal bacterial species and their association with locally produced host innate immune factors will help in defining various types of abnormal vaginal microbiota, developing new ways of assessing the risk of ascending subclinical infections, and in treating them. CDH appears to be a suitable tool for future analyses of large numbers of clinical samples with an extended number of bacterial probes.

We present the case of a 54-year-old female with life-threatening septicaemia due to Mobiluncus curtisii subsp. curtisii . Her admission was complicated by septic shock, renal failure, disseminated intravascular coagulation, the adult respiratory distress syndrome and spontaneous splenic rupture. The patient survived with full intensive care support and intravenous ceftriaxone. Extra-genital infection with Mobiluncus species is rarely diagnosed and has been confined to breast abscesses and non-life-threatening bacteraemia. A review of extra-genital infections with Mobiluncus species is presented

While bacterial vaginosis (BV) is a polymicrobial syndrome, Mobiluncus spp. are the organisms most highly associated with this condition. It is possible that serum antibody to Mobiluncus spp. could be used as a serological marker for BV. Using immunofluorescence techniques, we studied the prevalence of antibody to M. curtisii among three cohorts-pregnant women, pediatric patients, and sexually inexperienced women. The prevalence of antibody in each of these three groups was 75, 6, and 0%, respectively. Of the three pediatric patients with antibody to Mobiluncus curtisii, two were neonates, and the only class of antibody detected was immunoglobulin G. Among the cohort of pregnant women, the presence of antibody could not be correlated with a clinical history of BV. Serum antibody to M. curtisii could be a useful serological marker for BV. The lack of correlation of antibody positivity to historical information regarding BV suggests that unrecognized or undiagnosed episodes of BV may be common.

Sorbarod biofilms were investigated for their suitability in establishing a continuous growth for the study of bacterial vaginosis. Important organisms in the condition, Gadnerella vaginalis, Mobiluncus curtisii and Lactobacillus acidophilus were studied. In contrast to growth in broth culture, L. acidophilus and G. vaginalis were maintained for at least 96 hours in steady state on the biofilms. The strict anaerobe M. curtisii was maintained in these biofilms for at least 48 hours an indication that anaerobic conditions do exist in these biofilms. The haemolytic activity of G. vaginalis was consistently maintained in the biofilms in contrast to the short-term activity in the broth culture, which matched the bacterial titre. Gram stains of thin sections of paraffin wax embedded Sorbarods showed the micro-colonies as distinct irregular aggregates of organisms adherent to the cellulose matrix for G. vaginalis while L. acidophilus appeared as large latticed networks that formed between the cellulose fibre matrix. The simple Sorbarod system appears to be suitable for studying the growth of bacteria in continuous culture and has a potential for investigating interactions between micro- organisms. Journal of Tropical Microbiology Vol.1(1) 2002: 36-40

OBJECTIVE: To develop a polymerase chain reaction (PCR)-based diagnostic method for bacterial vaginosis using bacterial vaginosis-associated anaerobes. METHODS: A multiple PCR assay was developed using primers specific to 16S ribosomal deoxyribonucleic acid (DNA) ( Mobiluncus mulieris and Mobiluncus curtisii ), nanH ( Bacteroides fragilis ), and an internal spacer region of ribosomal DNA ( Gardnerella vaginalis ). The vaginal swabs from pregnant and nonpregnant women were examined by Gram stain-based Nugent scoring system. One hundred seventy-two samples of 853 Gram stain-interpretable samples were randomly selected and subjected to multiplex PCR assay. RESULTS: The sensitivity of the PCR assay ranged from 10 3 to 10 4 colony-forming units per vaginal swab. The prevalence of the bacterial vaginosis, intermediate, and normal categories was found by Nugent scoring system to be 21.6% (184/853), 26.0% (222/853), and 52.4% (447/853), respectively. By the multiplex PCR-based diagnostic method, 20.3% (35/172) of the samples were identified as bacterial vaginosis. The diagnostic sensitivity, specificity, positive predictive value, and negative predictive value of multiplex PCR in comparison with Gram stain examination were 78.4% (95% confidence interval [CI] 65.1%, 91.6%), 95.6% (95% CI 92.1%, 99.0%), 82.9% (95% CI 70.4%, 95.4%), and 94.2% (95% CI 90.3%, 98.1%), respectively. CONCLUSION: This multiplex PCR can be used as a diagnostic or screening test for bacterial vaginosis.

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) multipliedor =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) multipliedor =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 ofor =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.

Tetracycline-resistant Tet M-negative isolates of Actinomyces viscosus, Eubacterium lentum, Mobiluncus curtisii, and Mobiluncus mulieris were screened with the Tet K, Tet L, and Tet O DNA probes. Ten (71%) of the resistant Mobiluncus strains hybridized with the Tet O probe, two of the three E. lentum strains hybridized with the Tet K probe, and the A. viscosus isolate hybridized with the Tet L probe.

Mobiluncus curtisii was isolated from the blood of a 35-year-old man with a medical history of ulcerative colitis who was admitted unconscious to the Intensive Care Unit (ICU). A CT scan revealed massive intracerebral hemorrhage in the left hemisphere. Temperature remained constant over 38.5 degrees C; therefore, two sets of blood cultures were collected. One anaerobic bottle BacT/ALERT SN (bioMerieux, France) was detected as positive after 5 days of incubation and a Gram stain confirmed a gram variable curved-shaped rod. The patient died 18 h after being admitted to the hospital.

After co-cultivation of Mobiluncus curtisii , an obligate non-sporeforming anaerobe, with free living amebae from the Acanthamoeba spp. under aerobic conditions, internalization, multiplication and persistence of bacterial cells were established for at least 4–6 weeks. Under the same conditions and media without viable amebae, the cells of M. curtisii did not replicate and died in 4–7 days. The infection of amebae occurred with 10 to 100 bacteria per ml of co-cultivation media. In 7–14 days the amount of bacterial cells increased to 1×105–1×106CFU/mL. Electron microscopic examinations revealed bacteria within vacuoles in the amebae and intracellular replication. These results suggest a previously undescribed mechanism for spread, replication and persistence of obligately anaerobe bacteria in the environment and new possible sources, reservoirs and transfer mechanisms of infections caused by obligate anaerobe bacteria.

Using DNA primers based on highly conserved regions of bacterial 16S ribosomal RNA genes, a technique was established for detection of Mobiluncus species by polymerase chain reaction (PCR) and hybridization analysis. Part of the 16S rRNA genes of Mobiluncus mulieris, Mobiluncus curtisii and uncharacterized Mobiluncus strains were analyzed by broad-range PCR amplification and direct DNA sequencing analysis. Sequence comparison of the partial 16S rRNA genes of Mobiluncus curtisii, Mobiluncus mulieris and atypical Mobiluncus strains studied indicated genus and species-specific motifs within the variable regions V3, V4 and V9 of 16S ribosomal DNAs. A Mobiluncus curtisii-specific primer, located within the variable region V3 of the 16S rRNA gene, was designed for Southern blot hybridization analysis of broad-range PCR products. Broad-range amplification combined with a M. curtisii-specific hybridization probe, Mob V3, distinguished between Mobiluncus curtisii, Mobiluncus mulieris, and atypical Mobiluncus strains.

This study consisted of a search for the possible reservoir and mode of spread of the four bacterial vaginosis-associated organisms Mobiluncus mulieris, Mobiluncus curtisii, Mycoplasma hominis, and Gardnerella vaginalis. Their occurrence in rectal, oral, and pharyngeal specimens from women with and without bacterial vaginosis, their male sexual consorts, four homosexual men, and children (altogether, 374 people) was studied. Genital samples were also obtained from all adults. All four organisms were isolated from the rectums of 45 to 62% of women with bacterial vaginosis and 10 to 14% of women without bacterial vaginosis. They also occurred in the rectums of males and children. M. hominis was recovered from the oropharynxes of 12 adults whose sexual consorts had genital occurrences of the organism. Mobiluncus spp. occurred only in the vaginas of women with bacterial vaginosis (97%). The organisms were only infrequently recovered from genital samples from 135 males. Organisms were recovered from the urethras and/or coronal sulci of 10 of 44 male consorts of women with bacterial vaginosis. However, after 2 weeks of condom use during sexual intercourse, only M. hominis remained in the urethra of one man. These findings suggest that the organisms associated with bacterial vaginosis are not spread sexually but colonize the vagina from an endogenous intestinal tract site. The pathophysiological mechanisms leading to bacterial vaginosis in a subpopulation of all women are still unknown.

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.

The susceptibility of 12 strains of Mobiluncus curtisii and 10 strains of M. mulieris to 23 antimicrobial agents and 15 other compounds was determined. All strains were susceptible to chloramphenicol, clindamycin, rifampin, tobramycin, vancomycin, virginiamycin, and all beta-lactam antibiotics tested, including imipenem. One strain of M. mulieris was resistant to erythromycin and josamycin. All were resistant to colistin, cycloserine, nalidixic acid, and neomycin. Tetracycline had variable activity. All M. curtisii strains were resistant to metronidazole and its hydroxy metabolite. Of 10 M. mulieris strains, 5 were resistant to metronidazole and 2 were resistant to its hydroxy metabolite. All 12 M. curtisii and 1 of 10 M. mulieris strains were resistant to tinidazole. M. curtisii and M. mulieris produced two mutually exclusive clusters of MICs when tested against ampicillin, cefoxitin, cephalothin, moxalactam, alizarin red, Evans blue, and sodium fluoride. Gardnerella vaginalis was more susceptible to Nile blue A than was either M. curtisii or M. mulieris. Clindamycin and imipenem may be useful agents in the therapy of metronidazole-resistant bacterial vaginosis. Metronidazole, tinidazole, and Nile blue A may be of value in the development of a selective agar for Mobiluncus species.

A total of 22 strains of curved, anaerobic bacteria from the vaginae of women with bacterial vaginosis were found to be morphologically, biochemically, and genetically distinct from species in previously described genera. We placed these organisms in the genus Mobiluncus gen. nov., which includes curved, anaerobic, motile, rod-shaped bacteria that have multiple subpolar flagella and multilayered gram-variable cell walls, produce succinate and acetate, are stimulated by rabbit serum, and have guanine-plus-cytosine contents of 49 to 52 mol%. Mobiluncus curtisii is the type species (type strain, ATCC 35241). Mobiluncus curtisii strains are small (length, 1.7 μm) and gram variable, are stimulated by arginine, and produce ornithine, citrulline, and ammonia from arginine. Six of these strains migrate through soft (0.25%) agar and are nitrate negative, and these organisms are designated Mobiluncus curtisii subsp. curtisii. Six strains that do not migrate through soft agar and are nitrate positive are designated Mobiluncus curtisii subsp. holmesii (type strain, ATCC 35242). A total of 10 strains are designated Mobiluncus mulieris (type strain, ATCC 35243). These organisms are large (length, 2.9 μm) and gram negative, produce acid (pH 75% within and ≤25% between strains of the two species.

Thirty-two strains of anaerobic curved rods isolated from vaginal secretions and one isolated from seminal fluid were examined. Growth of all strains on solid media was superior to growth in liquid media, and at 37 degrees C they grew both anaerobically and in O2 5% in N2; they also grew anaerobically at 33 degrees C but not at 42 degrees C. No growth factors were identified, but strains grew more profusely at pH values above 5 X 0. The strains were screened in 80 biochemical tests, and for their susceptibility to 30 different antimicrobial agents. Most of the tests did not differentiate between the strains, but they were divided into four groups on the basis of cell morphology, metronidazole susceptibility, beta-galactosidase activity and arginine and hippurate hydrolysis. Group 1 consisted of 19 strains conforming to the species M. curtisi; group 2 consisted of five strains conforming to the species M. mulieris; group 3 consisted of five strains that resembled M. curtisi morphologically, and group 4 consisted of four strains that resembled M. mulieris morphologically, but the strains in the latter two groups reacted differently in at least one of the three major differential biochemical tests. Of three strains of M. curtisi and three of M. mulieris chosen at random, one of M. mulieris had a SDS-PAGE and fast-protein liquid chromatography protein profile indistinguishable from that of M. curtisi. We conclude that further efforts are required to clarify the taxonomic status of the genus Mobiluncus.

Partial reverse transcriptase sequencing of 16S rRNA from Mobiluncus curtisii and M. mulieris clearly indicate that the genus Mobiluncus is not a member of the Bacteroidaceae but belongs to the order Actinomycetales. The highest degree of relationship is found with the genus Actinomyces which is supported by the presence of common physiological properties.

In a bacteriological study of leukorrheal discharges now in progress, curved bacilli have been found in a considerable number of cases. These bacilli are as a rule sparsely scattered among other organisms without definite indication that they play a part in maintaining the leukorrhea. Due to the small number of curved bacilli and the difficulty with which they are isolated, numerous early attempts to obtain pure cultures met with repeated failures. In two later cases, however, morphologically similar bacteria were found in large numbers. One patient suffered from an infection following instrumental abortion; in the other case infection complicated labor at full term. Smears from the first patient revealed an overwhelming number of gram-negative, curved bacilli. Stained specimens of the vaginal and cervical discharges obtained from the patient with puerperal infection contained a great preponderance of apparently identical organisms. There were also present scattered gram-negative, fusiform bacilli, a fair number of gram-positive diplococci, and some gram-negative diplococci. A single set of cultures from the abortion case failed to yield the desired growth and material for later cultures was not available. In anaerobic cultures from the vagina and from the uterus of the patient with puerperal infection, the bacillus was readily isolated.

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.

From patients with bacterial vaginosis motile, anaerobic, comma-shaped bacteria can be isolated, which have recently been placed into the new genus Mobiluncus. In this study, electron microscopy was used to examine the in situ adherence of these motile curved rods to detached epithelial cells (comma cells) in vaginal fluid from two patients with bacterial vaginosis. Thin sections showed that the curved rods attached both directly to the epithelial cell surface and at various distances from it. It is concluded that after initial attachment these motile bacteria can grow at the epithelial cell surface in sessile microcolonies. Ruthenium red staining demonstrated a coating of precipitated glycocalyx material both on the surface of the curved rods and on their flagella. This may indicate that in situ the adherent curved rods were enclosed in a very hydrated matrix of exopolysaccharides. Conspicuous was the ability of the curved rods to attach to the epithelial cell surface via their cell tips. However, in situ no specialized bacterial cell surface structures were seen that might explain this polar attachment. Electron microscopy of pure cultures demonstrated that both Mobiluncus curtisii subsp. curtisii and Mobiluncus mulieris can produce a glycocalyx in vitro.

Twenty-four strains of Mobiluncus mulieris and 27 strains of Mobiluncus curtisii were tested with respect to 6 different biochemical characteristics: arginin-decarboxylase activity, beta-galactosidase activity, synergistic hemolysis with Staphylococcus aureus, hydrolysis of hippurate, migration through soft agar and reduction of nitrate. Antigens of the same strains, prepared by ultrasonication, were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting using polyclonal rabbit antisera against two of the M. mulieris strains and five of the M. curtisii strains. Two different strongly reacting protein antigens could be detected in the M. mulieris strains. These strains could be separated into three groups based on the possession of either of the two antigens or both. In the M. curtisii strains, 10 strongly reacting protein antigens could be detected. Four strains possessed only one of these antigens, one did not possess any, while the remaining strains possessed different sets of 9 of them. Within each species common protein antigens were detected. No antigens were found which were shared by both species. The biochemical characteristics studied could not differentiate between the antigenic groups in any of the species. None of the antigenic subgroups of M. curtisii found in the present study was identical with any of the two subspecies (curtisii and holmesii) which have been proposed.

Sialic acid, n-acetylneuraminic acid, is found in the cell wall of all serotypes of group B Streptococcus and Escherichia coli serotypes K1 and K3. It has been most strongly associated with those strains which cause neonatal meningitis. The purpose of this study is to report the presence of sialic acid in some genital tract bacteria. Bacteria were grown to log phase. Mild-acid hydrolysis of whole organisms released a compound which reacted positively in the periodatethiobarbituric acid (PTA) assay, a colorimetric assay detecting n-acetylneuraminic acid. Surface localisation of sialic acid was supported by the observation of bacterial agglutination with an affinity-purified lectin ( Limulus polyphemus agglutinin) which binds sialic acid residues. Four vaginal isolates of Mobiluncus curtisii and three vaginal isolates of Mobiluncus mulieris strains tested contained a level of sialic acid equivalent to that of the group B Streptococcus control by PTA assay. Four Gardnerella vaginalis strains (three vaginal and two placental isolates) were positive for sialic acid by PTA assay. Nine clinical vaginal isolates of Peptostreptococcus : P. anaerobius (2), P. asaccharolyticus (3), P. magnus (2) and P. tetradius (2) were negative for sialic acid. Two clinical vaginal isolates of Enterococcus faecalis and four diphtheroid isolates were also negative. Eleven clinical vaginal isolates of viridans streptococci including S. intermedius (5) , S. constellatus (4), and S. acidominimus (2) were negative for sialic acid. One of three clinical vaginal isolates of S. acidominimus , one of four clinical vaginal isolates of coagulase negative staphylococci and two of seven clinical vaginal isolates of lactobacilli tested positive for low levels of sialic acid. All strains in which sialic acid was detected by PTA assay showed surface localisation by lectin agglutination. The presence of sialic acid on the surface of Gardnerella and Mobiluncus , two organisms associated with bacterial vaginosis, may be associated with their capacity to colonise or infect the genital tract. Keywords: Sialic acid; Mobiluncus spp.; Gardnerella vaginalis ; vaginal bacteria.

Anaerobic, curved, motile, Gram-negative organism, belonging to a “new” genus have been isolated recently from up to 50% of women with bacterial vaginosis, suggesting that they might play an important role in the pathogenesis of the syndrome. The organisms have been given the genus name Mobiluncus and two species are recognised at present: Mobiluncus curtisii and M.mulieris.

Monoclonal antibodies were raised to the anaerobic curved rods, Mobiluncus curtisii subsp. curtisii NCTC 11656, M. curtisii subsp. holmesii NCTC 11657 and M. mulieris NCTC 11658. Three antibodies reacted with the two subspecies of M. curtisii and, when used in combination against clinical isolates, showed 100% sensitivity and specificity in immunofluorescence studies. Immunoblotting showed that two of these antibodies reacted with an epitope on a protein which had an electrophoretic mobility corresponding to a Mr of 75 Kda in the absence of a reducing agent and 82 Kda in its presence in both type strains and in clinical isolates. The third antibody reacted with an epitope in type strains which had a mobility corresponding to 75 Kda and was unaffected by a reducing agent. However, in clinical isolates the epitope was present on a protein of 75 Kda or 71 Kda, or on both. A fourth antibody showed reactivity with M. mulieris NCTC 11658 alone, but only 6 (24%) of 25 clinical isolates gave positive results by immunofluorescence. The epitope is believed to be present on a protein of greater than 90 Kda. All four antibodies were shown by immunogold staining to be directed against epitopes exposed on the cell surface.

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.

Vaginal anaerobic curved rods isolated from cases of bacterial vaginosis were characterised by morphology, biochemical tests and pyrolysis gas-liquid chromatography (PGLC). Classification of the strains based upon each of the above criteria independently showed division into two similar groups. Group I organisms were short, Gram-variable, comma-shaped rods, ONPG positive and able to hydrolyse arginine and hippurate. Group II organisms were long, Gram-negative, curved rods, ONPG negative and unable to hydrolyse arginine or hippurate. Each group produced a distinct PGLC pattern. These groups corresponded to the valid descriptions of Mobiluncus curtisii and M. mulieris. PGLC patterns of all other genera examined were not similar to those obtained for either group of anaerobic curved rods. The role of Mobiluncus species in disease processes remains obscure: characterisation studies such as this may lead to a better understanding of their significance in human infection.

Motile curved rods seen in vaginal secretions have been isolated on Columbia agar supplemented with 5% human blood and vitamin K. Growth occurred anaerobically and in 5% oxygen but not in more aerobic conditions. There were two distinct groups of these organisms, distinguishable by morphology, biochemical activity and susceptibility to metronidazole. All isolates were sensitive to a wide range of antimicrobial agents, with the exception of nalidixic acid and polymyxin, but one group was resistant to metronidazole. There was little difference between the results of tests of susceptibility to aminoglycosides or to metronidazole performed in anaerobic and microaerophi-lic conditions. Motile curved rods were isolated from 18 of 80 patients with a clinical diagnosis of non-specific vaginitis, but from only two of 39 without the disease.