Mobiluncus Curtisii Research Articles

Penile prostheses harbor biofilms driven by individual variability and manufacturer even in the absence of clinical infection.

Culture-based studies have shown that penile prostheses harbor biofilms in the presence and absence of infection, but these findings have not been adequately validated using contemporary microbiome analytic techniques. The study sought to characterize microbial biofilms of indwelling penile prosthesis devices according to patient factors, device components, manufacturer, and infection status. Upon penile prostheses surgical explantation, device biofilms were extracted, sonicated, and characterized using shotgun metagenomics and culture-based approaches. Device components were also analyzed using scanning electron microscopy. Outcomes included the presence or absence of biofilms, alpha and beta diversity, specific microbes identified and the presence of biofilm, and antibiotic resistance genes on each prosthesis component. The average age of participants from whom devices were explanted was 61 ± 11 years, and 9 (45%) of 20 had a diagnosis of diabetes mellitus. Seventeen devices were noninfected, and 3 were associated with clinical infection. Mean device indwelling time prior to explant was 5.1 ± 5.1 years. All analyzed components from 20 devices had detectable microbial biofilms, both in the presence and absence of infection. Scanning electron microscopy corroborated the presence of biofilms across device components. Significant differences between viruses, prokaryotes, and metabolic pathways were identified between individual patients, device manufacturers, and infection status. Mobiluncus curtisii was enriched in manufacturer A device biofilms relative to manufacturer B device biofilms. Bordetella bronchialis, Methylomicrobium alcaliphilum, Pseudoxanthomonas suwonensis, and Porphyrobacter sp. were enriched in manufacturer B devices relative to manufacturer A devices. The most abundant bacterial phyla were the Proteobacteria, Actinobacteria, and Firmicutes. Glycogenesis, the process of glycogen synthesis, was among the predominant metabolic pathways detected across device components. Beta diversity of bacteria, viruses, protozoa, and pathways did not differ among device components. All components of all penile prostheses removed from infected and noninfected patients have biofilms. The significance of biofilms on noninfected devices remains unknown and merits further investigation. Strengths include the multipronged approach to characterize biofilms and being the first study to include all components of penile prostheses in tandem. Limitations include the relatively few number of infected devices in the series, a relatively small subset of devices included in shotgun metagenomics analysis, and the lack of anaerobic and other expanded conditions for culture. Penile prosthesis biofilms are apparent in the presence and absence of infection, and the composition of biofilms was driven primarily by device manufacturer, individual variability, and infection, while being less impacted by device component.

Vaginal and Cervical Microbiota Composition in Patients with Endometrial Cancer.

According to recent data, changes in the vaginal microbiota could affect the risk of gynaecological cancers. Women suffering from endometrial cancer present significant changes in cervicovaginal microbiota composition. The objective of our study was to characterize the cervicovaginal microbiota of women undergoing hysterectomy due to benign disease, atypical hyperplasia, and endometrial cancer; The study included 96 patients, who undergone surgical treatment due to benign uterine disease, precancerous endometrial lesion, and endometrial cancer. Quantitative and qualitative real-time PCR analysis of DNA isolated from vaginal fornix and endocervical canal samples was performed to detect the 19 most commonly identified microorganisms, including different Lactobacillus spp., Atopobium, Bifidobacterium, Chlamydia, and Gardnerella; At least one of the tested microorganisms was identified in 88.5% of vaginal and 83.3% of cervical samples. Lactobacillus iners was significantly more frequent in patients with benign condition, whereas Dialister pneumosintes and Mobiluncus curtisii was more frequent in cancer patients; Mobiluncus curtisi and Dialister pneumosintes, which were identified as significantly more common in endometrial cancer vaginal samples, may be considered as potential endometrial cancer co-factors which promote/stimulate carcinogenesis. However, the exact mechanism of such activity remains unexplained and requires further investigations.

MP27-07 PENILE PROSTHESES UNIVERSALLY HARBOR BIOFILMS, AND COMPOSITION DIFFERS BY DEVICE TYPE, BUT NOT COMPONENT OR INFECTION STATUS

You have accessJournal of UrologyCME1 Apr 2023MP27-07 PENILE PROSTHESES UNIVERSALLY HARBOR BIOFILMS, AND COMPOSITION DIFFERS BY DEVICE TYPE, BUT NOT COMPONENT OR INFECTION STATUS Glenn Werneburg, Ava Adler, Prajit Khooblall, Hadley Wood, Bradley Gill, Sarah Vij, Kenneth Angermeier, Scott Lundy, Aaron Miller, and Petar Bajic Glenn WerneburgGlenn Werneburg More articles by this author , Ava AdlerAva Adler More articles by this author , Prajit KhooblallPrajit Khooblall More articles by this author , Hadley WoodHadley Wood More articles by this author , Bradley GillBradley Gill More articles by this author , Sarah VijSarah Vij More articles by this author , Kenneth AngermeierKenneth Angermeier More articles by this author , Scott LundyScott Lundy More articles by this author , Aaron MillerAaron Miller More articles by this author , and Petar BajicPetar Bajic More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To characterize microbial biofilms of indwelling penile prostheses by device component, manufacturer and infection status. Understanding biofilms in the presence and absence of infection will guide development of novel materials and coatings resistant to pathogenic biofilm formation – improving strategies for the prevention and treatment of device infection. METHODS: Penile prosthetic devices were removed in 20 patients having mechanical failure, pain, or infection. Device components were subjected to sonication, DNA extraction, next generation sequencing, shotgun metagenomics, and culture-based approaches. Curated reads were compared to known bacterial, viral, fungal, and protozoal databases, then mapped to KEGG and antibiotic resistance gene databases. RESULTS: Biofilms were detected in all 102 prosthetic components. 65% of species were prokaryotic, 15% viral, 15% protozoan, and 0% fungal in origin. Beta-diversity of prokaryotes and viruses did not differ by infection status or device component, but differed by device type (p<0.05) (Figure 1). The most abundant bacterial phyla were Proteobacteria and Actinobacteria. Mobiluncus curtisii was enriched in Manufacturer A (M.A.) device biofilms relative to Manufacturer B (M.B.). Bordetella bronchialis and Methylomicrobium alcaliphilum were enriched in M.B. relative to M.A. devices. The most abundant viruses across device components were the bean 58058 virus and cotesia congregate virus. The angelica virus and magnaporthe oryzae chrisovirus were enriched in M.B. relative to M.A. devices. CONCLUSIONS: Penile prostheses harbored biofilms regardless of device component and infection status (Figure 2). The significance of biofilm differences based on device type remains unknown. Stable, non-pathogenic biofilms may inhibit pathogenic infection and further investigation is needed into prevention of the transition to a state of dysbiosis. Source of Funding: This study was funded by the 2020 Sexual Medicine Society of North America Young Clinicians Research Grant © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e364 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Glenn Werneburg More articles by this author Ava Adler More articles by this author Prajit Khooblall More articles by this author Hadley Wood More articles by this author Bradley Gill More articles by this author Sarah Vij More articles by this author Kenneth Angermeier More articles by this author Scott Lundy More articles by this author Aaron Miller More articles by this author Petar Bajic More articles by this author Expand All Advertisement PDF downloadLoading ...

In vitro bacterial vaginosis biofilm community manipulation using endolysin therapy

Bacterial vaginosis (BV) affects approximately 26% of women of childbearing age globally, presenting with 3–5 times increased risk of miscarriage and two-fold risk of pre-term birth. Antibiotics (metronidazole and clindamycin) are typically employed to treat BV; however the success rate is low due to the formation of recalcitrant polymicrobial biofilms. As a novel therapeutic, promising results have been obtained in vitro using Gardnerella endolysins, although to date their efficacy has only been demonstrated against simple biofilm models.In this study, a four-species biofilm was developed consisting of Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia and Mobiluncus curtisii. Biofilms were grown in NYC III broth and treated using antibiotics and an anti-Gardnerella endolysin (CCB7.1) for 24 h. Biofilm composition, viability and structure were assessed using colony counts, live/dead qPCR and scanning electron microscopy.All species colonised biofilms to varying degrees, with G. vaginalis being the most abundant. Biofilm composition remained largely unchanged when challenged with escalated concentrations of conventional antibiotics. A Gardnerella-targeted endolysin candidate (CCB7.1) showed efficacy against several Gardnerella species planktonically, and significantly reduced viable G. vaginalis within polymicrobial biofilms at 1 to 4X pMIC (p < 0.05 vs. vehicle control).Collectively, this study highlights the resilience of biofilm-embedded pathogens against the currently used antibiotics and provides a polymicrobial model that allows for more effective pre-clinical screening of BV therapies. The Gardnerella-specific endolysin CCB7.1 demonstrated significant activity against G. vaginalis within polymicrobial biofilms, altering the overall community dynamic and composition.

In vitro antimicrobial activity of Bixa orellana L . Leaves extract against anaerobic bacteria associated to bacterial vaginosis and Lactobacillus spp.

Motivation for the study: bacterial vaginosis is a bacterial infection that frequently affects women of reproductive age. The treatment is based on synthetic antimicrobials. Bixa orellana L. possesses antimicrobial properties and could represent a potential non-synthetic therapeutic alternative. Main findings: in vitro results suggest that, methanolic extract of Bixa orellana L. leaves possesses potential antimicrobial properties against bacteria associated to bacterial vaginosis. Implications: to identify new sources with therapeutic potential, and to promote research, discovery, and characterization of non-synthetic antimicrobials. To describe the in vitro antimicrobial activity of the methanolic extract of Bixa orellana L. leaves against anaerobic bacteria associated to bacterial vaginosis and Lactobacillus spp. Eight ATCC reference strains; Gardnerella vaginalis, Prevotella bivia, Peptococcus niger, Peptostreptococcus anaerobius, Mobiluncus curtisii, Atopobium vaginae, Veillonella parvula, and Lactobacillus crispatus, and twenty-two clinical isolates; eleven Gardnerella vaginalis and eleven Lactobacillus strains, were included in the study. The antimicrobial susceptibility was determined by the agar diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by using agar dilution and a modified dilution plating method, respectively. All ATCC reference strains showed high levels of susceptibility to the extract, except P. vibia, V. parvula and L. crispatus. Interestingly, all G. vaginalis clinical isolates and the G. vaginalis ATTC strain were the most susceptible to the extract, given their low MIC (1.0 - 2.0 mg/mL) and MBC (1.0 - 4.0 mg/mL) values, whereas, the Lactobacillus spp. clinical isolates and the L. crispatus ATCC strain were the least susceptible bacteria given their high MIC (32.0 mg/mL) and MBC (≥ 32.0 mg/mL) values. In vitro experiments suggest that the extract possesses selective antimicrobial properties given its high activity against bacterial vaginosis-associated anaerobic bacteria and low activity against Lactobacillus species.

Tetanus Presented as Acute Abdomen

Tetanus is commonly seen in the emergency department with a puncture wound. This is an unusual presentation of tetanus without an open wound. A 59 year old male with a past history of hypertension, hepatitis C, prior injection drug use, and homeless presented to the emergency department with altered mental status and hyperthermia with fever of 105 F on a hot July afternoon. Patient was found in his car sleeping. Initially, the patient declined medical care, but was later found unresponsive with a methadone bottle next to him. Given Narcan on field with improvement of mental status. Patient developed acute ridged abdominal pain. A CT scan raised concern for acute abdomen, with portal venous gas, and intestinal pneumatosis. Surgery consulted and was concerned for ischemic colon with perforation. Vancomycin, ceftriaxone and metronidazole were given. Emergency exploratory laparotomy was performed with a small serosal injury on colon repaired with suture. Blood culture came back positive for Clostridium tetani and Mobiluncus curtisii. Infectious disease consulted. Felt tetanus cannot be ruled out. Patient was given tetanus immune globulin (TIG), metronidazole and later tetanus vaccine. Patient has fully recovered, tolerates oral diets, and has been discharged to a nursing facility without any neurological deficit. Tetanus is a neurological disorder caused by the tetanus toxins produced by clostridium tetani, which is a gram positive obligate anaerobes commonly found in soil. It is relevant to note that this bacterium can also be found inside the gut of animals and humans. The disease is well characterized by ancient Greeks, and common symptoms include trismus (“lockjaw”), opisthotonos, and a board-like rigid abdomen. Incubation is usually 7-21 days and this disease is usually prevented by vaccination. This case is unusual because there is no open wound and PE is limited due to patient’s overdose on opiates (methadone). Possible cause for this particular case may be that since there was no open wound, the blood could have been seeded by the perforated bowel because they may also be found in the gut. The patient was given metronidazole as part of Tx for acute abdomen, which happens to also be a treatment for tetanus. Diseases can be presented atypically. This case illustrates the importance of differential diagnosis when considering treatments in any pathology.

The bacterial microbiota of Hunner lesion interstitial cystitis/bladder pain syndrome.

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. 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. 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. 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.

Vaginal Microbiome Composition in Early Pregnancy and Risk of Spontaneous Preterm and Early Term Birth Among African American Women.

ObjectiveTo evaluate the association between the early pregnancy vaginal microbiome and spontaneous preterm birth (sPTB) and early term birth (sETB) among African American women.MethodsVaginal samples collected in early pregnancy (8-14 weeks’ gestation) from 436 women enrolled in the Emory University African American Vaginal, Oral, and Gut Microbiome in Pregnancy Study underwent 16S rRNA gene sequencing of the V3-V4 region, taxonomic classification, and community state type (CST) assignment. We compared vaginal CST and abundance of taxa for women whose pregnancy ended in sPTB (N = 44) or sETB (N= 84) to those who delivered full term (N = 231).ResultsNearly half of the women had a vaginal microbiome classified as CST IV (Diverse CST), while one-third had CST III (L. iners dominated) and just 16% had CST I, II, or V (non-iners Lactobacillus dominated). Compared to vaginal CST I, II, or V (non-iners Lactobacillus dominated), both CST III (L. iners dominated) and CST IV (Diverse) were associated with sPTB with an adjusted odds ratio (95% confidence interval) of 4.1 (1.1, infinity) and 7.7 (2.2, infinity), respectively, in multivariate logistic regression. In contrast, no vaginal CST was associated with sETB. The linear decomposition model (LDM) based on amplicon sequence variant (ASV) relative abundance found a significant overall effect of the vaginal microbiome on sPTB (p=0.034) but not sETB (p=0.320), whereas the LDM based on presence/absence of ASV found no overall effect on sPTB (p=0.328) but a significant effect on sETB (p=0.030). In testing for ASV-specific effects, the LDM found that no ASV was significantly associated with sPTB considering either relative abundance or presence/absence data after controlling for multiple comparisons (FDR 10%), although in marginal analysis the relative abundance of Gardnerella vaginalis (p=0.011), non-iners Lactobacillus (p=0.016), and Mobiluncus curtisii (p=0.035) and the presence of Atopobium vaginae (p=0.049), BVAB2 (p=0.024), Dialister microaerophilis (p=0.011), and Prevotella amnii (p=0.044) were associated with sPTB. The LDM identified the higher abundance of 7 ASVs and the presence of 13 ASVs, all commonly residents of the gut, as associated with sETB at FDR < 10%.ConclusionsIn this cohort of African American women, an early pregnancy vaginal CST III or IV was associated with an increased risk of sPTB but not sETB. The relative abundance and presence of distinct taxa within the early pregnancy vaginal microbiome was associated with either sPTB or sETB.

Classification and Regression Trees for Bacterial Vaginosis Diagnosis in Pregnant Women Based on High-Throughput Quantitative PCR

Bacterial vaginosis (BV) diagnosis in pregnancy is based on the Nugent score, which consists of semiquantitation of bacterial morphotypes. Limited data exist concerning molecular-based diagnosis in asymptomatic pregnant women. Using high-throughput quantitative PCR, 34 microorganisms were screened in asymptomatic pregnant women and compared with the Nugent score. Three-hundred and four vaginal samples had a Nugent score <7 (69.9%) and 131, a Nugent score ≥7 (30.1%), consistent with BV. More pregnant women with BV share Atopobiumvaginae, bacterial vaginosis associated bacteria-2, Gardnerella spp., Mobiluncus curtisii, Mo. mulieris, Mycoplasma hominis, Ureaplasma urealyticum, Prevotella bivia, Megasphaera 1, and Megasphaera 2 in their vaginal sample. Fewer pregnant women with BV share Lactobacillus crispatus, L. gasseri, L. jensenii, and Enterococcus faecalis in their vaginal sample (P<0.001). Classification and regression tree analysis was performed to determine which combinations of detected bacteria optimally diagnose BV in this population. A set of only four bacteria of 34 microorganisms (A. vaginae, Gardnerella spp., L. crispatus, and P. bivia) was the best combination to identify BV in a cohort of asymptomatic pregnant women, with a sensitivity of 77.1%, and specificity of 97.0% compared with the Nugent score. The quantitative PCR in the present study responds to the limits of the Nugent score by implementing an easily reproducible quantitative assay to assess the absence of BV in pregnancy.

Evaluation of different culture media to support in vitro growth and biofilm formation of bacterial vaginosis-associated anaerobes.

BackgroundBacterial 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.AimTo 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.MethodsA 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.ResultsSignificant 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.ConclusionsOur 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.

Structure determination of CAMP factor of Mobiluncus curtisii and insights into structural dynamics

Bacterial vaginosis (BV) is a common type of vaginal inflammation caused by a proliferation of pathogenic bacteria, among which Mobiluncus curtisii. In our previous studies on M. curtisii genome, we identified the presence of a genomic fragment encoding a 25 kDa pore-forming toxin, the CAMP factor, which is known to be involved in the synergistic lysis of erythrocytes namely CAMP reaction. However, whether this hypothetical gene product has hemolytic activity is unknown. Moreover, its relative structure and function are not yet solved. Here we found that the M. curtisii CAMP factor is a monomer at pH 4.4 and oligomer at pH > 4.6. Hemolysis assays showed that M. curtisii CAMP factor could lyse sheep red blood cells efficiently in pH 5.4–7.4. Negative staining electron microscope analysis of the CAMP factor revealed ring-like structures at pH above 4.6. Additionally, the crystal structure of M. curtisii CAMP factor, determineded at 1.85 Å resolution, reveals a 5 + 3 helix motif. Further functional analysis suggested that the structural rearrangement of the N-terminal domain might be required for protein function. In conclusion, this structure-function relationship study of CAMP factor provides a new perspective of the M. curtisii role in BV development.

Identification of Gardnerella vaginalis and Atopobium vaginae in Women With Bacterial Vaginosis in Northern Iran

Background Bacterial vaginosis is the most common lower genital tract infection among women of reproductive age. Its causative agent is often the bacterium Gardnerella vaginalis, which exists in synergism with other mostly anaerobic bacteria such as Atopobium vaginae, Mobiluncus curtisii, and Megasphaera type I. Methods A total of 211 women, who referred to obstetrics and gynecology clinics in Rasht City for suspected vaginal infections, were tested for possible bacterial vaginosis. The presence of G. vaginalis and A. vaginae was verified by 5 different laboratory methods based on the Amsel criteria. These methods included determination of pH, whiff test, and observation of clue cells in a direct smear. Polymerase chain reaction using specific primers was performed on the extracted DNA from the vaginal specimens. Results Sixty cases of bacterial vaginosis, of the 211 women tested for suspected vaginal infections, were confirmed using Amsel criteria. In 44% (29/66) and 29% (19/66) of these bacterial vaginosis cases, the presence of G. vaginalis and A. vaginae was confirmed by polymerase chain reaction, respectively. Conclusions Gardnerella vaginalis had a very high prevalence in the 18- to 35-year-old women suffering from bacterial vaginosis in this region of Iran. Presumably, these cases were recommended for appropriate treatment, but the wider issue is about prevention in this age group and appropriate public health messages to raise awareness.

Classification and phylogenetic analysis of the genus Mobiluncus based on matrix-assisted laser de-sorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and whole genome sequen-cing

Objective To identify Mobiluncus species with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing and to analyze antibiotic resistance genes and phylogenetic relationships among Mobiluncus species with whole genome sequencing. Methods Twenty-five Mobiluncus strains were isolated after anaerobic culture of 65 vaginal secretion samples of patients with bacterial vaginosis (BV) and identified to species level by MALDI-TOF MS and 16S rRNA gene sequencing. The whole genome DNA of each strain was extracted for next-generation sequencing. SPAdes was used to assembly genomes. Resistance genes were searched in ARGD database. The core genome of all isolates was analyzed by Harvest to construct phylogenetic tree. Results MALDI-TOF MS could only identify Mobiluncus curtisii, while 16S rRNA gene sequencing could identify both Mobiluncus mulieris and Mobiluncus curtisii. Results of the whole genome sequencing showed that tetracycline resistance gene tet(o) and macrolides resistance gene erm(x) were the two predominant acquired resistance genes of Mobiluncus with a positive rate of 84.7% and 61.5% respectively. Intra-species relationships of the two Mobiluncus species were close, but a distant phylogenetic relationship was found between the two species. Conclusion This study shows that MALDI-TOF MS can′t be used to identify Mobiluncus mulieris at present. Mobiluncus strains have potential resistance to tetracycline and macrolides. Intra-species evolution of Mobiluncus is slow, which indicates that there is no growing trend towards new species for the time being. Key words: Mobiluncus; Bacterial vaginosis (BV); MALDI-TOF MS; Whole genome sequencing; Resistance gene

Survival of vaginal microorganisms in three commercially available transport systems

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 h (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 maintained 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 and stability of vaginal microorganisms was maintained better in the Cary-Blair based transport system (Starswab®) than in the two Amies based transport systems.

Bacterial vaginosis: a synthesis of the literature on etiology, prevalence, risk factors, and relationship with chlamydia and gonorrhea infections.

Bacterial vaginosis (BV) is a common vaginal disorder in women of reproductive age. Since the initial work of Leopoldo in 1953 and Gardner and Dukes in 1955, researchers have not been able to identify the causative etiologic agent of BV. There is increasing evidence, however, that BV occurs when Lactobacillus spp., the predominant species in healthy vaginal flora, are replaced by anaerobic bacteria, such as Gardenella vaginalis, Mobiluncus curtisii, M. mulieris, other anaerobic bacteria and/or Mycoplasma hominis. Worldwide, it estimated that 20–30 % of women of reproductive age attending sexually transmitted infection (STI) clinics suffer from BV, and that its prevalence can be as high as 50–60 % in high-risk populations (e.g., those who practice commercial sex work (CSW). Epidemiological data show that women are more likely to report BV if they: 1) have had a higher number of lifetime sexual partners; 2) are unmarried; 3) have engaged in their first intercourse at a younger age; 4) have engaged in CSW, and 5) practice regular douching. In the past decade, several studies have provided evidence on the contribution of sexual activity to BV. However, it is difficult to state that BV is a STI without being able to identify the etiologic agent. BV has also emerged as a public health problem due to its association with other STIs, including: human immunodeficiency virus (HIV), herpes simplex virus type 2 (HSV-2), Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). The most recent evidence on the association between BV and CT/NG infection comes from two secondary analyses of cohort data conducted among women attending STI clinics. Based on these studies, women with BV had a 1.8 and 1.9-fold increased risk for NG and CT infection, respectively. Taken together, BV is likely a risk factor or at least an important contributor to subsequent NG or CT infection in high-risk women. Additional research is required to determine whether this association is also present in other low-risk sexually active populations, such as among women in the US military. It is essential to conduct large scale cross-sectional or population-based case-control studies to investigate the role of BV as a risk factor for CT/NG infections. These studies could lead to the development of interventions aimed at reducing the burden associated with bacterial STIs worldwide.

Natural antimicrobials subtilosin and lauramide arginine ethyl ester synergize with conventional antibiotics clindamycin and metronidazole against biofilms of Gardnerella vaginalis but not against biofilms of healthy vaginal lactobacilli.

The purpose of this study was to evaluate the ability of clindamycin and metronidazole to synergize with natural antimicrobials against biofilms of bacterial vaginosis (BV)-associated Gardnerella vaginalis. Minimum bactericidal concentrations for biofilm cells (MBCs-B) were determined for each antimicrobial. The MBCs-B of lauramide arginine ethyl ester (LAE), subtilosin, clindamycin and metronidazole were 50, 69.5, 20 and 500 μg mL(-1), respectively. A checkerboard assay and isobologram were used to analyze the type of interactions between these antimicrobials. The combination of metronidazole with natural antimicrobials did not inhibit planktonic lactobacilli. Clindamycin with either LAE or with subtilosin was inhibitory for planktonic but not for biofilm-associated lactobacilli. All tested antimicrobial combinations were inhibitory for BV-associated Mobiluncus curtisii and Peptostreptococcus anaerobius. LAE and subtilosin synergized with clindamycin and metronidazole against biofilms of G. vaginalis but not biofilm-associated vaginal lactobacilli. The biofilms of BV-associated pathogens can be controlled by synergistically acting combinations of conventional antibiotics and natural antimicrobials which will help better management of current antibiotics, especially considering robust bacterial resistance. Our findings create a foundation for a new strategy in the effective control of vaginal infections.

Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR

Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 107cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 104cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 108.8 and 108.9cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections.

Combination of microbiological culture and multiplex PCR increases the range of vaginal microorganisms identified in cervical cancer patients at high risk for bacterial vaginosis and vaginitis.

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. 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. A one-year hospital-based study was conducted in 2011/2012. Cervical swabs from 130 patients were examined by microbiological culture and multiplex PCR. 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. 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.

Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction.

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, Megasphaera type I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs) 1, 2, and 3, Sneathia sanguinegens, and Mycoplasma 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.

Microflora of the penile skin-lined neovagina of transsexual women

BackgroundThe microflora of the penile skin-lined neovagina in male-to-female transsexuals is a recently created microbial niche which thus far has been characterized only to a very limited extent. Yet the knowledge of this microflora can be considered as essential to the follow-up of transsexual women. The primary objective of this study was to map the neo-vaginal microflora in a group of 50 transsexual women for whom a neovagina was constructed by means of the inverted penile skin flap technique. Secondary objectives were to describe possible correlations of this microflora with multiple patients' characteristics, such as sexual orientation, the incidence of vaginal irritation and malodorous vaginal discharge.ResultsBased on Gram stain the majority of smears revealed a mixed microflora that had some similarity with bacterial vaginosis (BV) microflora and that contained various amounts of cocci, polymorphous Gram-negative and Gram-positive rods, often with fusiform and comma-shaped rods, and sometimes even with spirochetes. Candida cells were not seen in any of the smears.On average 8.6 species were cultured per woman. The species most often found were: Staphylococcus epidermidis, Streptococcus anginosus group spp., Enterococcus faecalis, Corynebacterium sp., Mobiluncus curtisii and Bacteroides ureolyticus. Lactobacilli were found in only one of 30 womenThere was no correlation between dilatation habits, having coitus, rinsing habits and malodorous vaginal discharge on the one hand and the presence of a particular species on the other. There was however a highly significant correlation between the presence of E. faecalis on the one hand and sexual orientation and coitus on the other (p = 0.003 and p = 0.027 respectively).Respectively 82%, 58% and 30% of the samples showed an amplicon after amplification with M. curtisii, Atopobium vaginae and Gardnerella vaginalis primer sets.ConclusionOur study is the first to describe the microflora of the penile skin-lined neovagina of transsexual women. It reveals a mixed microflora of aerobe and anaerobe species usually found either on the skin, in the intestinal microflora or in a BV microflora.