Recruiting transgender men in the Southeastern United States for genital microbiome research: Lessons learned.

BACKGROUND: Transgender men (TGM) are underrepresented in genital microbiome research. Our prospective study in Birmingham, AL investigated genital microbiota changes over time in TGM initiating testosterone, including the development of incident bacterial vaginosis (iBV). Here, we present lessons learned from recruitment challenges encountered during the conduct of this study. METHODS: Inclusion criteria were assigned female sex at birth, TGM or non-binary identity, age ≥18 years, interested in injectable testosterone but willing to wait 7 days after enrollment before starting, and engaged with a testosterone-prescribing provider. Exclusion criteria were recent antibiotic use, HIV/STI infection, current vaginal infection, pregnancy, or past 6 months testosterone use. Recruitment initiatives included community advertisements via flyers, social media posts, and referrals from local gender health clinics. RESULTS: Between February 2022 and October 2023, 61 individuals contacted the study, 17 (27.9%) completed an in-person screening visit, and 10 (58.8%) of those screened were enrolled. The primary reasons for individuals failing study screening were having limited access to testosterone-prescribing providers, already being on testosterone, being unwilling to wait 7 days to initiate testosterone therapy, or desiring the use of topical testosterone. Engagement of non-White TGM was also minimal. CONCLUSION: Despite robust study inquiry by TGM, screening and enrollment challenges were faced including engagement by TGM not yet in care and specific study eligibility criteria. Excitement among TGM for research representation should be leveraged in future work by engaging transgender community stakeholders at the inception of study development, particularly regarding feasibility of study inclusion and exclusion criteria, as well as recruitment of TGM of color. These results also highlight the need for more clinical resources for prescribing gender-affirming hormone therapy, especially in the Southeastern US.

Microbiota-Short Chain Fatty Acid Relationships Underlie Clinical Heterogeneity and Identify Key Microbial Targets in Irritable Bowel Syndrome (IBS).

Background: Identifying microbial targets in irritable bowel syndrome (IBS) and other disorders of gut-brain interaction (DGBI) is challenging due to the dynamic nature of microbiota-metabolite-host interactions. SCFA are key microbial metabolites that modulate intestinal homeostasis and may influence IBS pathophysiology. We aimed to assess microbial features associated with short chain fatty acids (SCFA) and determine if features varied across IBS subtypes and endophenotypes. Among 96 participants who were screened, 71 completed the study. We conducted in-depth investigations of stool microbial metagenomes, stool SCFA, and measurable IBS traits (stool bile acids, colonic transit, stool form) in 41 patients with IBS (IBS with constipation [IBS-C] IBS with diarrhea [IBS-D]) and 17 healthy controls. We used partial canonical correspondence analyses (pCCA), conditioned on transit, to quantify microbe-SCFA associations across clinical groups. To explore relationships between microbially-derived SCFA and IBS traits, we compared gut microbiome-encoded potential for substrate utilization across groups and within a subset of participants selected by their stool characteristics as well as stool microbiomes of patients with and without clinical bile acid malabsorption. Results: Overall stool microbiome composition and individual taxa abundances differed between clinical groups. Microbes-SCFA associations differed across groups and revealed key taxa including Dorea sp. CAG:317 and Bifidobacterium pseudocatenulatum in IBS-D and Akkermansia muciniphila and Prevotella copri in IBS-C that that may drive subtype-specific microbially-mediated mechanisms. Strongest microbe-SCFA associations were observed in IBS-D and several SCFA-producing species surprisingly demonstrated inverse correlations with SCFA. Fewer bacterial taxa were associated with acetate to butyrate ratios in IBS compared to health. In participants selected by stool form, we demonstrated differential abundances of microbial genes/pathways for SCFA metabolism and degradation of carbohydrates and mucin across groups. SCFA-producing taxa were reduced in IBS-D patients with BAM. Conclusion: Keystone taxa responsible for SCFA production differ according to IBS subtype and traits and the IBS microbiome is characterized by reduced functional redundancy. Differences in microbial substrate preferences are also linked to bowel functions. Focusing on taxa that drive SCFA profiles and stool form may be a rational strategy for identifying relevant microbial targets in IBS and other DGBI.

Association of Chlamydia trachomatis burden with the vaginal microbiota, bacterial vaginosis, and metronidazole treatment.

Bacterial vaginosis (BV), a dysbiosis of the vaginal microbiota, is a common coinfection with Chlamydia trachomatis (Ct), and BV-associated bacteria (BVAB) and their products have been implicated in aiding Ct evade natural immunity. Here, we determined if a non-optimal vaginal microbiota was associated with a higher genital Ct burden and if metronidazole, a standard treatment for BV, would reduce Ct burden or aid in natural clearance of Ct infection. Cervicovaginal samples were collected from women at enrollment and, if testing positive for Ct infection, at a follow-up visit approximately one week later. Cervical Ct burden was assessed by inclusion forming units (IFU) and Ct genome copy number (GCN), and 16S rRNA gene sequencing was used to determine the composition of the vaginal microbiota. We observed a six-log spectrum of IFU and an eight-log spectrum of GCN in our study participants at their enrollment visit, but BV, as indicated by Amsel's criteria, Nugent scoring, or VALENCIA community state typing, did not predict infectious and total Ct burden, although IFU : GCN increased with Amsel and Nugent scores and in BV-like community state types. Ct burden was, however, associated with the abundance of bacterial species in the vaginal microbiota, negatively with Lactobacillus crispatus and positively with Prevotella bivia. Women diagnosed with BV were treated with metronidazole, and Ct burden was significantly reduced in those who resolved BV with treatment. A subset of women naturally cleared Ct infection in the interim, typified by low Ct burden at enrollment and resolution of BV. Abundance of many BVAB decreased, and Lactobacillus increased, in response to metronidazole treatment, but no changes in abundances of specific vaginal bacteria were unique to women who spontaneously cleared Ct infection.

A novel Gardnerella, Prevotella, and Lactobacillus standard that improves accuracy in quantifying bacterial burden in vaginal microbial communities.

Bacterial vaginosis (BV) is the most common vaginal dysbiosis. In this condition, a polymicrobial biofilm develops on vaginal epithelial cells. Accurately quantifying the bacterial burden of the BV biofilm is necessary to further our understanding of BV pathogenesis. Historically, the standard for calculating total bacterial burden of the BV biofilm has been based on quantifying Escherichia coli 16S rRNA gene copy number. However, E. coli is improper for measuring the bacterial burden of this unique micro-environment. Here, we propose a novel qPCR standard to quantify bacterial burden in vaginal microbial communities, from an optimal state to a mature BV biofilm. These standards consist of different combinations of vaginal bacteria including three common BV-associated bacteria (BVAB) Gardnerella spp. (G), Prevotella spp. (P), and Fannyhessea spp. (F) and commensal Lactobacillus spp. (L) using the 16S rRNA gene (G:P:F:L, G:P:F, G:P:L and 1G:9L). We compared these standards to the traditional E. coli (E) reference standard using known quantities of mock vaginal communities and 16 vaginal samples from women. The E standard significantly underestimated the copy numbers of the mock communities, and this underestimation was significantly greater at lower copy numbers of these communities. The G:P:L standard was the most accurate across all mock communities and when compared to other mixed vaginal standards. Mixed vaginal standards were further validated with vaginal samples. This new G:P:L standard can be used in BV pathogenesis research to enhance reproducibility and reliability in quantitative measurements of BVAB, spanning from the optimal to non-optimal (including BV) vaginal microbiota.

Characterization of Vaginal Microbial Community Dynamics in the Pathogenesis of Incident Bacterial Vaginosis, a Pilot Study.

BACKGROUND: Despite more than 60 years of research, the etiology of bacterial vaginosis (BV) remains controversial. In this pilot study, we used shotgun metagenomic sequencing to characterize vaginal microbial community changes before the development of incident BV (iBV). METHODS: A cohort of African American women with a baseline healthy vaginal microbiome (no Amsel criteria, Nugent score 0-3 with no Gardnerella vaginalis morphotypes) were followed for 90 days with daily self-collected vaginal specimens for iBV (≥2 consecutive days of a Nugent score of 7-10). Shotgun metagenomic sequencing was performed on select vaginal specimens from 4 women, every other day for 12 days before iBV diagnosis. Sequencing data were analyzed through Kraken2 and bioBakery 3 workflows, and specimens were classified into community state types. Quantitative polymerase chain reaction was performed to compare the correlation of read counts with bacterial abundance. RESULTS: Common BV-associated bacteria such as G. vaginalis , Prevotella bivia , and Fannyhessea vaginae were increasingly identified in the participants before iBV. Linear modeling indicated significant increases in G. vaginalis and F . vaginae relative abundance before iBV, whereas the relative abundance of Lactobacillus species declined over time. The Lactobacillus species decline correlated with the presence of Lactobacillus phages. We observed enrichment in bacterial adhesion factor genes on days before iBV. There were also significant correlations between bacterial read counts and abundances measured by quantitative polymerase chain reaction. CONCLUSIONS: This pilot study characterizes vaginal community dynamics before iBV and identifies key bacterial taxa and mechanisms potentially involved in the pathogenesis of iBV.

Impact of testosterone use on the vaginal microbiota of transgender men, including susceptibility to bacterial vaginosis: study protocol for a prospective, observational study.

INTRODUCTION: The effect of testosterone (T) therapy on the vaginal microbiota of transgender men (TGM) is not well characterised, although one cross-sectional study comparing the vaginal microbiota of cisgender women to TGM on T≥1 year found that, in 71% of the TGM, the vaginal microbiota was less likely to be Lactobacillus-dominated and more likely to be enriched with >30 other bacterial species, many associated with bacterial vaginosis (BV). This prospective study aims to investigate changes in the composition of the vaginal microbiota over time in TGM who retain their natal genitalia (ie, vagina) and initiate T. In addition, we will identify changes in the vaginal microbiota preceding incident BV (iBV) in this cohort while investigating behavioural factors, along with hormonal shifts, which may be associated with iBV. METHODS AND ANALYSIS: T-naïve TGM who have not undergone gender-affirming genital surgery with normal baseline vaginal microbiota (ie, no Amsel criteria, normal Nugent Score with no Gardnerella vaginalis morphotypes) will self-collect daily vaginal specimens for 7 days prior to initiating T and for 90 days thereafter. These specimens will be used for vaginal Gram stain, 16S rRNA gene sequencing and shotgun metagenomic sequencing to characterise shifts in the vaginal microbiota over time, including development of iBV. Participants will complete daily diaries on douching, menses and behavioural factors including sexual activity during the study. ETHICS AND DISSEMINATION: This protocol is approved through the single Institutional Review Board mechanism by the University of Alabama at Birmingham. External relying sites are the Louisiana State University Health Sciences Center, New Orleans Human Research Protection Program and the Indiana University Human Research Protection Program. Study findings will be presented at scientific conferences and peer-reviewed journals as well as shared with community advisory boards at participating gender health clinics and community-based organisations servicing transgender people. REGISTRATION DETAILS: Protocol # IRB-300008073.

Sexual behavior shapes male genitourinary microbiome composition.

The origin, composition, and significance of the distal male urethral microbiome are unclear, but vaginal microbiome dysbiosis is linked to new sex partners and several urogynecological syndromes. We characterized 110 urethral specimens from men without urethral symptoms, infections, or inflammation using shotgun metagenomics. Most urethral specimens contain characteristic lactic acid bacteria and Corynebacterium spp. In contrast, several bacteria associated with vaginal dysbiosis were present only in specimens from men who reported vaginal intercourse. Sexual behavior, but not other evaluated behavioral, demographic, or clinical variables, strongly associated with inter-specimen variance in urethral microbiome composition. Thus, the male urethra supports a simple core microbiome that is established independent of sexual exposures but can be re-shaped by vaginal sex. Overall, the results suggest that urogenital microbiology and sexual behavior are inexorably intertwined, and show that the male urethra harbors female urogenital pathobionts.

A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion.

Many obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.

Characterization of Virulence-Associated Traits in Mycoplasma penetrans Strains Acting as Likely Etiological Agents of Idiopathic Nongonococcal Urethritis.

Mycoplasma penetrans is an emerging pathogen with a reduced genome. This bacterium has only previously been cultured from individuals with chronic immunodeficiencies. Here we report the characteristics of 4 M. penetrans isolates from the urine of immunocompetent males with nongonococcal urethritis, in comparison with strain HF-2 from an immunocompromised patient. Several features exhibited distinct differences between these isolates and HF-2. Unlike HF-2, all 4 were resistant to azithromycin. They exhibited greater sialic acid-dependent binding to erythrocytes, gliding motility speed, and H2O2 production than HF-2. All new isolates produced thinner capsules than HF-2. Invasiveness varied, with some isolates being more invasive than HF-2 and some less invasive. Cytotoxicity to HeLa cells was similar to HF-2, and all strains could clear extracellular traps produced by innate immune cells. We conclude that subtle differences among M. penetrans strains may be critical for this organism to establish an infection in an otherwise healthy individual.

Esophageal Microbiome in Healthy Children and Esophageal Eosinophilia.

OBJECTIVES: There is limited knowledge about the role of esophageal microbiome in pediatric esophageal eosinophilia (EE). We aimed to characterize the esophageal microbiome in pediatric patients with and without EE. METHODS: In the present prospective study, esophageal mucosal biopsies were obtained from 41 children. Of these, 22 had normal esophageal mucosal biopsies ("healthy"), 6 children had reflux esophagitis (RE), 4 had proton pump inhibitor (PPi)-responsive esophageal eosinophilia (PPi-REE), and 9 had eosinophilic esophagitis (EoE). The microbiome composition was analyzed using 16S rRNA gene sequencing. The age median (range) in years for the healthy, RE, PPi-REE, and EoE group were 10 (1.5-18), 6 (2-15), 6.5 (5-15), and 9 (1.5-17), respectively. RESULTS: The bacterial phylum Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria were the most predominant. The Epsilonproteobacteria, Betaproteobacteria, Flavobacteria, Fusobacteria, and Sphingobacteria class were underrepresented across groups. The Vibrionales was predominant in healthy and EoE group but lower in RE and PPi-REE groups. The genus Streptococcus, Rahnella, and Leptotrichia explained 29.65% of the variation in the data with an additional 10.86% variation in the data was explained by Microbacterium, Prevotella, and Vibrio genus. The healthy group had a higher diversity and richness index compared to other groups, but this was not statistically different. CONCLUSIONS: The pediatric esophagus has an abundant and diverse microbiome, both in the healthy and diseased states. The healthy group had a higher, but not significantly different, diversity and richness index compared to other groups.