Amygdalobacter indicium gen. nov., sp. nov., and Amygdalobacter nucleatus sp. nov., gen. nov.: novel bacteria from the family Oscillospiraceae isolated from the female genital tract.

Four obligately anaerobic Gram-positive bacteria representing one novel genus and two novel species were isolated from the female genital tract. Both novel species, designated UPII 610-JT and KA00274T, and an additional isolate of each species were characterized utilizing biochemical, genotypic and phylogenetic analyses. All strains were non-motile and non-spore forming, asaccharolytic, non-cellulolytic and indole-negative coccobacilli. Fatty acid methyl ester analysis for UPII 610-JT and KA00274T and additional isolates revealed C16 : 0, C18 : 0, C18:1ω9c and C18:2ω6,9c to be the major fatty acids for both species. UPII 610-JT had a 16S rRNA gene sequence similarity of 99.4 % to an uncultured clone sequence (AY724740) designated as Bacterial Vaginosis Associated Bacterium 2 (BVAB2). KA00274T had a 16S rRNA gene sequence similarity of 96.5 % to UPII 610-JT. Whole genomic DNA mol% G+C content was 42.2 and 39.3 % for UPII 610-JT and KA00274T, respectively. Phylogenetic analyses indicate these isolates represent a novel genus and two novel species within the Oscillospiraceae family. We propose the names Amygdalobacter indicium gen. nov., sp. nov., for UPII 610-JT representing the type strain of this species (=DSM 112989T, =ATCC TSD-274T) and Amygdalobacter nucleatus gen. nov., sp. nov., for KA00274T representing the type strain of this species (=DSM 112988T, =ATCC TSD-275T).

Impact of Dapivirine and Placebo Vaginal Rings on the Microbiota of Adolescent, Lactating, and Postmenopausal Females.

BACKGROUND: A 25-mg dapivirine vaginal ring has been demonstrated to reduce risk of human immunodeficiency virus (HIV) acquisition in nonpregnant adult women. In this secondary analysis of studies conducted in US adolescent, lactating, and postmenopausal females, vaginal microbiota was assessed prior to and after ring use, and between dapivirine and placebo ring users. METHODS: Vaginal fluid swabs were collected before and after product use for the evaluation of microbiota using Nugent criteria, quantitative culture, and quantitative polymerase chain reaction. RESULTS: Vaginal ring use did not impact bacterial vaginosis prevalence among the 3 populations and was associated with minimal shifts in microbiota. Adolescents in both arms demonstrated an increased prevalence of Lactobacillus crispatus and a decrease in quantity of Megasphaera lornae. Postmenopausal active and placebo ring users demonstrated an increased prevalence of lactobacilli and non-albicans yeast, while dapivirine ring users demonstrated an increased prevalence of Candida albicans and increased quantity of group B Streptococcus and non-albicans yeasts. Prevotella species were increased in lactating women, whereas Prevotella timonensis increased in prevalence and concentration among adolescent and postmenopausal females and Prevotella bivia increased in prevalence among adolescent dapivirine ring users. CONCLUSIONS: Dapivirine vaginal ring use was associated with minimal changes in the vaginal microbiota that are likely not clinically significant.

Megasphaera lornae sp. nov., Megasphaera hutchinsoni sp. nov., and Megasphaera vaginalis sp. nov.: novel bacteria isolated from the female genital tract.

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6T, KA00182T and BV3C16-1T. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus Megasphaera. UPII 199-6T and KA00182T had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as Megasphaera type 1 and Megasphaera type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6T exhibited sequence identities ranging from 92.9 to 93.6 % with validly named Megasphaera isolates and KA00182T had 16S rRNA gene sequence identities ranging from 92.6-94.2 %. BV3C16-1T was most closely related to Megasphaera cerevisiae with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6T, KA00182T and BV3C16-1T were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named Megasphaera species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C12 : 0, C16 : 0, C16 : 0 dimethyl acetal (DMA) and summed feature 5 (C15 : 0 DMA and/or C14 : 0 3-OH) in UPII 199-6T; C16 : 0 and C16 : 1 cis 9 in KA00182T; C12 : 0; C14 : 0 3-OH; and summed feature 5 in BV3C16-1T. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6T, KA00182T and BV3C16-1T represent novel species within the genus Megasphaera. We propose the following names: Megasphaera lornae sp. nov. for UPII 199-6T representing the type strain of this species (=DSM 111201T=ATCC TSD-205T), Megasphaera hutchinsoni sp. nov. for KA00182T representing the type strain of this species (=DSM 111202T=ATCC TSD-206T) and Megasphaera vaginalis sp. nov. for BV3C16-1T representing the type strain of this species (=DSM 111203T=ATCC TSD-207T).

Impact of contraceptive initiation on vaginal microbiota.

BACKGROUND: Data evaluating the impact of contraceptives on the vaginal microbiome are limited and inconsistent. OBJECTIVE: We hypothesized that women initiating copper intrauterine device use would have increased bacterial vaginosis and bacterial vaginosis-associated microbes with use compared to women initiating and using hormonal contraceptive methods. STUDY DESIGN: Vaginal swabs (N = 1047 from 266 participants seeking contraception) for Nugent score determination of bacterial vaginosis and quantitative polymerase chain reaction analyses for assessment of specific microbiota were collected from asymptomatic, healthy women aged 18-35 years in Harare, Zimbabwe, who were confirmed to be free of nonstudy hormones by mass spectrometry at each visit. Contraception was initiated with an injectable (depot medroxyprogesterone acetate [n = 41], norethisterone enanthate [n = 44], or medroxyprogesterone acetate and ethinyl estradiol [n = 40]), implant (levonorgestrel [n = 45] or etonogestrel [n = 48]), or copper intrauterine device (n = 48) and repeat vaginal swabs were collected after 30, 90, and 180 days of continuous use. Self-reported condom use was similar across all arms at baseline. Quantitative polymerase chain reaction was used to detect Lactobacillus crispatus, L jensenii, L gasseri/johnsonii group, L vaginalis, L iners, Gardnerella vaginalis, Atopobium vaginae, and Megasphaera-like bacterium phylotype I from swabs. Modified Poisson regression and mixed effects linear models were used to compare marginal prevalence and mean difference in quantity (expressed as gene copies/swab) prior to and during contraceptive use. RESULTS: Bacterial vaginosis prevalence increased in women initiating copper intrauterine devices from 27% at baseline, 35% at 30 days, 40% at 90 days, and 49% at 180 days (P = .005 compared to marginal prevalence at enrollment). Women initiating hormonal methods had no change in bacterial vaginosis prevalence over 180 days. The mean increase in Nugent score was 1.2 (95% confidence interval, 0.5-2.0; P = .001) in women using copper intrauterine devices. Although the frequency and density of beneficial lactobacilli did not change among intrauterine device users over 6 months, there was an increase in the log concentration of G vaginalis (4.7, 5.2, 5.8, 5.9; P = .046) and A vaginae (3.0, 3.8, 4.6, 5.1; P = .002) between baseline and 30, 90, and 180 days after initiation. Among other contraceptive groups, women using depot medroxyprogesterone acetate had decreased L iners (mean decrease log concentration = 0.8; 95% confidence interval, 0.3-1.5; P = .004) and there were no significant changes in beneficial Lactobacillus species over 180 days regardless of contraceptive method used. CONCLUSION: Copper intrauterine device use may increase colonization by bacterial vaginosis-associated microbiota, resulting in increased prevalence of bacterial vaginosis. Use of most hormonal contraception does not alter vaginal microbiota.

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 species colonizing the vagina of healthy women are not associated with race.

The vaginal microbiota of 36 white versus 25 black asymptomatic women were compared using both cultivation-dependent and -independent identification. Significant differences by race were found in colonization and density of bacterial species. However, exclusion of 12 women with bacterial vaginosis by Nugent criteria resulted in no significant differences by race.

Mageeibacillus indolicus gen. nov., sp. nov.: a novel bacterium isolated from the female genital tract.

Three isolates of a bacterium recovered from human endometrium using conventional culture methods were characterized biochemically and subjected to 16S rRNA gene sequencing and phylogenetic analysis. Isolates were non-motile, obligately anaerobic, non-spore forming, asaccharolytic, non-cellulolytic, indole positive, Gram positive rods. Cell wall fatty acid profiling revealed C14:0, C16:0, C18:2 ω6, 9c, C18:1 ω9c and C18:0 to be the major fatty acid composition. The DNA mol % G+C was determined to be 44.2%. 16S rRNA gene sequence analysis revealed only 91% sequence similarity with the closest cultivated bacterial isolate, Saccharofermentans acetigenes. Based on genotypic and phenotypic data, all three isolates are considered to be members of the same species and data suggest it represents a novel genus and species in the order Clostridiales with an association with Clostridium rRNA cluster III within the family Ruminococcaceae. We propose the name, Mageeibacillus indolicus gen. nov., sp. nov. The type strain is BAA-2120(T) and CCUG 59143(T).

Incidence and epidemiology of Streptococcus pseudoporcinus in the genital tract.

Streptococcus pseudoporcinus, a beta-hemolytic microorganism first isolated from the female gastrourinary tract in 2006, cross-reacts with serogrouping kits for group B Streptococcus (GBS) and could be misidentified in the laboratory. The epidemiologic characteristics of this species have not been reported previously, but this organism is thought to be rare. Paired vaginal and rectal samples were collected from 663 nonpregnant women enrolled in a phase II clinical vaccine trial of a GBS type III capsular polysaccharide-protein conjugate vaccine, and isolates initially identified as S. pseudoporcinus were collected for further testing. A total of 120 isolates of S. pseudoporcinus were recovered from 36 unique individuals with 5.4% of 663 women having this organism recovered at least once during follow-up. All of these isolates cross-reacted with a commercially available GBS serogrouping kit. Women colonized with isolates confirmed as S. pseudoporcinus by genotypic and phenotypic methodologies were compared to women who were not colonized to determine whether there were any significant factors associated with acquisition of S. pseudoporcinus. Acquisition of S. pseudoporcinus vaginally and/or rectally was 36 per 846.0 women-years of follow-up for an annual incidence of 4 per 100 woman-years of follow-up. Acquisition of S. pseudoporcinus was independently associated with black women, being 30 to 40 years of age, recent Trichomonas vaginalis infection, primary or recurrent genital herpes, having bacterial vaginosis by Nugent criteria, and having had two or more male sexual partners since the last visit. This study suggests that S. pseudoporcinus is not rare, especially among black women, and could be misidentified as GBS.

Limitations of the dye-based method for determining vaginal applicator use in microbicide trials.

BACKGROUND: A dye-based method for determining applicator usage in microbicide trials has been developed to assess whether applicators have been exposed to vaginal fluid. Our objective was to evaluate this method on polypropylene HTI applicators that are being widely used in several effectiveness trials of microbicides. METHODS: Study participants enrolled in a clinical trial assessing SPL7013 (VivaGel) inserted gel intravaginally twice daily for 14 days and returned used and unused applicators. Before staining, smears were prepared from each participant-inserted applicator, Gram stained and assessed independently for the presence of vaginal cells and bacteria. Of the 169 participant-inserted applicators, 168 (99%) had vaginal cells identified by Gram stain. PARTICIPANT: Inserted applicators were stained with a 0.05% FD & C blue dye No. 1 solution and compared with 70 inserted positive control applicators and 70 unused negative control applicators. Intravaginally inserted applicators should stain turquoise, whereas unused applicators should not retain any stain. The individual responsible for labeling and preparing the applicators did not serve as an evaluator. RESULTS: : Under optimized conditions, the sensitivity and specificity ranged from 81% to 95% and 86% to 93%, respectively for single use and unused applicators. The dye-based method was only 47% to 77% sensitive for participant-inserted applicators obtained from women using gel twice daily. CONCLUSION: The dye test for HTI polypropylene applicators had a sensitivity of 47% to 95%, depending on the evaluator and whether gel was present in the vagina. The sensitivity was decreased with multiple gel applications. The dye-based method cannot be recommended for HTI polypropylene applicators to monitor product adherence.

Quantitative survival of aerobic and anaerobic microorganisms in Port-A-Cul and Copan transport systems.

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 degrees C. Both transporters maintained the viability of organisms better at 4 degrees C than at 24 degrees C. Prevotella bivia and Prevotella corporis had a loss of viability in both transporters at both temperatures. However, at 24 degrees 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 degrees 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.

Antimicrobial resistance associated with the treatment of bacterial vaginosis.

OBJECTIVE: This study was undertaken to evaluate antimicrobial susceptibility of vaginal anaerobic bacteria before and after treatment of bacterial vaginosis. STUDY DESIGN: A randomized clinical trial of 119 nonpregnant women with bacterial vaginosis receiving either intravaginal metronidazole for 5 days or clindamycin for 3 days was performed. Women had 1 baseline and 3 follow-up visits at which quantitative vaginal cultures were performed. Anaerobic isolates underwent antimicrobial susceptibility testing. RESULTS: Complete susceptibility data was available on 95 women (47 metronidazole and 48 clindamycin). Of 1059 anaerobic bacterial isolates, less than 1% demonstrated resistance to metronidazole. In contrast, 17% demonstrated baseline clindamycin resistance, and 53% demonstrated resistance to clindamycin after therapy. Women exposed to clindamycin (but not metronidazole) had high frequencies (80%) of clindamycin-resistant anaerobic bacteria that persisted for 90 days after treatment. CONCLUSION: Treatment of bacterial vaginosis with clindamycin is associated with marked evidence of antimicrobial resistance among vaginal anaerobic bacteria. This may increase the vaginal reservoir of macrolide-resistant bacteria.