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Item Aetiology and prevalence of mixed-infections and mono-infections in non-gonococcal urethritis in men: a case-control study(BMJ, 2020-06) Jordan, Stephen J.; Toh, Evelyn; Williams, James A.; Fortenberry, Lora; LaPradd, Michelle L.; Katz, Barry P.; Batteiger, Byron E.; Nelson, David E.; Batteiger, Teresa A.; Medicine, School of MedicineObjectives: Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) cause the majority of non-gonococcal urethritis (NGU). The role of Ureaplasma urealyticum (UU) in NGU is unclear. Prior case-control studies that examined the association of UU and NGU may have been confounded by mixed infections and less stringent criteria for controls. The objective of this case-control study was to determine the prevalence and aetiology of mixed infections in men and assess if UU monoinfection is associated with NGU. Methods: We identified 155 men with NGU and 103 controls. Behavioural and clinical information was obtained and men were tested for Neisseria gonorrhoeae and CT, MG, UU and Trichomonas vaginalis (TV). Men who were five-pathogen negative were classified as idiopathic urethritis (IU). Results: Twelve per cent of NGU cases in which a pathogen was identified had mixed infections, mostly UU coinfections with MG or CT; 27% had IU. In monoinfected NGU cases, 34% had CT, 17% had MG, 11% had UU and 2% had TV. In controls, pathogens were rarely identified, except for UU, which was present in 20%. Comparing cases and controls, NGU was associated with CT and MG monoinfections and mixed infections. UU monoinfection was not associated with NGU and was almost twice as prevalent in controls. Men in both the case and control groups who were younger and who reported no prior NGU diagnosis were more likely to have UU (OR 0.97 per year of age, 95% CI 0.94 to 0.998 and OR 6.3, 95% CI 1.4 to 28.5, respectively). Conclusions: Mixed infections are common in men with NGU and most of these are UU coinfections with other pathogens that are well-established causes of NGU. UU monoinfections are not associated with NGU and are common in younger men and men who have never previously had NGU. Almost half of NGU cases are idiopathic.Item Associations Between Dysmenorrhea Symptom-Based Phenotypes and Vaginal Microbiome: A Pilot Study(Wolters Kluwer, 2021) Chen, Chen X.; Carpenter, Janet S.; Gao, Xiang; Toh, Evelyn; Dong, Qunfeng; Nelson, David E.; Mitchell, Caroline; Fortenberry, J. DennisBACKGROUND: Dysmenorrhea is highly prevalent; it places women at risk for other chronic pain conditions. There is a high degree of individual variability in menstrual pain severity, the number of painful sites, and co-occurring gastrointestinal symptoms. Distinct dysmenorrhea symptom-based phenotypes were previously identified, but the biological underpinnings of these phenotypes are less known. One underexplored contributor is the vaginal microbiome. The vaginal microbiota differs significantly among reproductive-age women and may modulate as well as amplify reproductive tract inflammation, which may contribute to dysmenorrhea symptoms. OBJECTIVES: The objective of this study was to examine associations between dysmenorrhea symptom-based phenotypes and vaginal microbiome compositions on- and off-menses. METHODS: We conducted a prospective, longitudinal, pilot study of 20 women (aged 15-24 years) grouped into three dysmenorrhea symptom-based phenotypes: "mild localized pain," "severe localized pain," and "severe multiple pain and gastrointestinal symptoms." Over one menstrual cycle, participants provided vaginal swabs when they were on- and off-menses. We assayed the vaginal microbiome using 16S rRNA gene sequencing. Permutational multivariate analysis of variance tests were used to compare microbiome compositions across phenotypes, with heat maps generated to visualize the relative abundance of bacterial taxa. RESULTS: The vaginal microbiome compositions (n = 40) were different across the three phenotypes. After separating the on-menses (n = 20) and off-menses (n = 20) specimens, the statistically significant difference was seen on-menses, but not off-menses. Compared to the "mild localized pain" phenotype, participants in the "multiple severe symptoms" phenotype had a lower lactobacilli level and a higher abundance of Prevotella, Atopobium, and Gardnerella when on-menses. We also observed trends of differences across phenotypes in vaginal microbiome change from off- to on-menses. DISCUSSION: The study provides proof-of-concept data to support larger studies on associations between dysmenorrhea symptom-based phenotypes and vaginal microbiome that might lead to new intervention targets and/or biomarkers for dysmenorrhea. This line of research has the potential to inform precision dysmenorrhea treatment that can improve women's quality of life.Item Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation(American Society for Microbiology, 2016-09-19) Muramatsu, Matthew K.; Brothwell, Julie A.; Steinman, Barry D.; Putman, Timothy E.; Rockey, Daniel D.; Nelson, David E.; Department of Microbiology & Immunology, IU School of MedicineChlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells.Item Characterization of Proximal Small Intestinal Microbiota in Patients With Suspected Small Intestinal Bacterial Overgrowth: A Cross-Sectional Study(Lippincott, Williams & Wilkins, 2019-08) Shin, Andrea S.; Gao, Xiang; Bohm, Matthew; Lin, Huaiying; Gupta, Anita; Nelson, David E.; Toh, Evelyn; Teagarden, Sean; Siwiec, Robert; Dong, Qunfeng; Wo, John M.; Medicine, School of MedicineOBJECTIVES: The composition of the small intestinal microbiota has not yet been characterized thoroughly using culture-independent techniques. We compared small intestinal microbial communities in patients with and without small intestinal bacterial overgrowth (SIBO) using culture-dependent and culture-independent bacterial identification approaches. METHODS: Small bowel aspirate and mucosal samples were collected from patients with suspected SIBO. The aspirates were cultured to diagnose SIBO, defined as ≥10 colony-forming units/mL coliform or ≥10 colony-forming units/mL upper aerodigestive tract bacteria. Bacteria in the aspirates and mucosa were identified using 16S rRNA gene sequencing. We compared small intestinal microbiome composition between groups with and without a culture-based SIBO diagnosis. RESULTS: Analysis of the aspirate and mucosal microbial communities from 36 patients revealed decreased α-diversity but no differences in β-diversity in patients with SIBO compared with those without SIBO. There were no significant differences in the relative abundance of individual taxa from the aspirates or mucosa after adjustment for false discovery rate between patients with and without SIBO. Subgroup analysis revealed significant differences in mucosal β-diversity between the coliform and upper aerodigestive tract subgroups. Relative abundances of a mucosal Clostridium spp. (P = 0.05) and an aspirate Granulicatella spp. (P = 0.02) were higher in coliform SIBO vs non-SIBO subgroups. The microbial composition and relative abundance of multiple taxa significantly differed in the mucosal and aspirate specimens. DISCUSSION: Culture-based results of small bowel aspirates do not correspond to aspirate microbiota composition but may be associated with species richness of the mucosal microbiota.Item Chlamydia muridarum Genital and Gastrointestinal Infection Tropism Is Mediated by Distinct Chromosomal Factors(American Society for Microbiology, 2018-06-21) Morrison, Sandra G.; Giebel, Amanda M.; Toh, Evelyn C.; Spencer, Horace J., III; Nelson, David E.; Morrison, Richard P.; Microbiology and Immunology, School of MedicineSome members of the genus Chlamydia, including the human pathogen Chlamydia trachomatis, infect multiple tissues, including the genital and gastrointestinal (GI) tracts. However, it is unknown if bacterial targeting to these sites is mediated by multifunctional or distinct chlamydial factors. We previously showed that disruption of individual large clostridial toxin homologs encoded within the Chlamydia muridarum plasticity zone were not critical for murine genital tract infection. Here, we assessed whether cytotoxin genes contribute to C. muridarum GI tropism. Infectivity and shedding of wild-type (WT) C. muridarum and three mutants containing nonsense mutations in different cytotoxin genes, tc0437, tc0438, and tc0439, were compared in mouse genital and GI infection models. One mutant, which had a nonsense mutation in tc0439, was highly attenuated for GI infection and had a GI 50% infectious dose (ID50) that was 1,000 times greater than that of the WT. GI inoculation with this mutant failed to elicit anti-chlamydial antibodies or to protect against subsequent genital tract infection. Genome sequencing of the tc0439 mutant revealed additional chromosomal mutations, and phenotyping of additional mutants suggested that the GI attenuation might be linked to a nonsense mutation in tc0600 The molecular mechanism underlying this dramatic difference in tissue-tropic virulence is not fully understood. However, isolation of these mutants demonstrates that distinct chlamydial chromosomal factors mediate chlamydial tissue tropism and provides a basis for vaccine initiatives to isolate chlamydia strains that are attenuated for genital infection but retain the ability to colonize the GI tract and elicit protective immune responses.Item Chlamydia muridarum infection of macrophages elicits bactericidal nitric oxide production via reactive oxygen species and cathepsin B(IAI, 2015-08) Rajaram, Krithika; Nelson, David E.; Department of Microbiology and Immunology, IU School of MedicineThe ability of certain species of Chlamydia to inhibit the biogenesis of phagolysosomes permits their survival and replication within macrophages. The survival of macrophage-adapted chlamydiae correlates with the multiplicity of infection (MOI), and optimal chlamydial growth occurs in macrophages infected at an MOI of ≤1. In this study, we examined the replicative capacity of Chlamydia muridarum in the RAW 264.7 murine macrophage cell line at different MOIs. C. muridarum productively infected these macrophages at low MOIs but yielded few viable elementary bodies (EBs) when macrophages were infected at a moderate (10) or high (100) MOI. While high MOIs caused cytotoxicity and irreversible host cell death, macrophages infected at a moderate MOI did not show signs of cytotoxicity until late in the infectious cycle. Inhibition of host protein synthesis rescued C. muridarum in macrophages infected at a moderate MOI, implying that chlamydial growth was blocked by activated defense mechanisms. Conditioned medium from these macrophages was antichlamydial and contained elevated levels of interleukin 1β (IL-1β), IL-6, IL-10, and beta interferon (IFN-β). Macrophage activation depended on Toll-like receptor 2 (TLR2) signaling, and cytokine production required live, transcriptionally active chlamydiae. A hydroxyl radical scavenger and inhibitors of inducible nitric oxide synthase (iNOS) and cathepsin B also reversed chlamydial killing. High levels of reactive oxygen species (ROS) led to an increase in cathepsin B activity, and pharmacological inhibition of ROS and cathepsin B reduced iNOS expression. Our data demonstrate that MOI-dependent TLR2 activation of macrophages results in iNOS induction via a novel ROS- and cathepsin-dependent mechanism to facilitate C. muridarum clearance.Item Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells(American Society for Microbiology, 2016-12-13) Haldar, Arun K.; Piro, Anthony S.; Finethy, Ryan; Espenschied, Scott T.; Brown, Hannah E.; Giebel, Amanda M.; Frickel, Eva-Maria; Nelson, David E.; Coers, Jörn; Department of Microbiology and Immunology, IU School of MedicineThe cytokine gamma interferon (IFN-γ) induces cell-autonomous immunity to combat infections with intracellular pathogens, such as the bacterium Chlamydia trachomatis. The present study demonstrates that IFN-γ-primed human cells ubiquitinate and eliminate intracellular Chlamydia-containing vacuoles, so-called inclusions. We previously described how IFN-γ-inducible immunity-related GTPases (IRGs) employ ubiquitin systems to mark inclusions for destruction in mouse cells and, furthermore, showed that the rodent pathogen Chlamydia muridarum blocks ubiquitination of its inclusions by interfering with mouse IRG function. Here, we report that ubiquitination of inclusions in human cells is independent of IRG and thus distinct from the murine pathway. We show that C. muridarum is susceptible to inclusion ubiquitination in human cells, while the closely related human pathogen C. trachomatis is resistant. C. muridarum, but not C. trachomatis, inclusions attract several markers of cell-autonomous immunity, including the ubiquitin-binding protein p62, the ubiquitin-like protein LC3, and guanylate-binding protein 1. Consequently, we find that IFN-γ priming of human epithelial cells triggers the elimination of C. muridarum, but not C. trachomatis, inclusions. This newly described defense pathway is independent of indole-2,3-dioxygenase, a known IFN-γ-inducible anti-Chlamydia resistance factor. Collectively, our observations indicate that C. trachomatis evolved mechanisms to avoid a human-specific, ubiquitin-mediated response as part of its unique adaptation to its human host.Item Comparison of Whole and Acellular Bronchoalveolar Lavage to Oral Wash Microbiomes. Should Acellular Bronchoalveolar Lavage Be the Standard?(American Thoracic Society, 2014-01) Twigg, Homer L.; Nelson, David E.; Day, Richard Brandon; Gregory, Richard L.; Dong, Qunfeng; Rong, Ruichen; Knox, Kenneth; Crothers, Kristina; Sodergren, Erica; Weinstock, George; Department of Medicine, IU School of MedicineItem Cutaneous Burn Injury Modulates Urinary Antimicrobial Peptide Responses and the Urinary Microbiome(Lippincott, Williams & Wilkins, 2017-06) Plichta, Jennifer K.; Holmes, Casey J.; Nienhouse, Vanessa; Puszynski, Michelle; Gao, Xiang; Dong, Qunfeng; Lin, Huaiying; Sinacore, James; Zilliox, Michael; Toh, Evelyn; Nelson, David E.; Gamelli, Richard L.; Radek, Katherine A.; Microbiology and Immunology, School of MedicineOBJECTIVES: Characterization of urinary bacterial microbiome and antimicrobial peptides after burn injury to identify potential mechanisms leading to urinary tract infections and associated morbidities in burn patients. DESIGN: Retrospective cohort study using human urine from control and burn subjects. SETTING: University research laboratory. PATIENTS: Burn patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Urine samples from catheterized burn patients were collected hourly for up to 40 hours. Control urine was collected from "healthy" volunteers. The urinary bacterial microbiome and antimicrobial peptide levels and activity were compared with patient outcomes. We observed a significant increase in urinary microbial diversity in burn patients versus controls, which positively correlated with a larger percent burn and with the development of urinary tract infection and sepsis postadmission, regardless of age or gender. Urinary psoriasin and β-defensin antimicrobial peptide levels were significantly reduced in burn patients at 1 and 40 hours postadmission. We observed a shift in antimicrobial peptide hydrophobicity and activity between control and burn patients when urinary fractions were tested against Escherichia coli and Enterococcus faecalis urinary tract infection isolates. Furthermore, the antimicrobial peptide activity in burn patients was more effective against E. coli than E. faecalis. Urinary tract infection-positive burn patients with altered urinary antimicrobial peptide activity developed either an E. faecalis or Pseudomonas aeruginosa urinary tract infection, suggesting a role for urinary antimicrobial peptides in susceptibility to select uropathogens. CONCLUSIONS: Our data reveal potential links for urinary tract infection development and several morbidities in burn patients through alterations in the urinary microbiome and antimicrobial peptides. Overall, this study supports the concept that early assessment of urinary antimicrobial peptide responses and the bacterial microbiome may be used to predict susceptibility to urinary tract infections and sepsis in burn patients.Item Cutaneous Burn Injury Promotes Shifts in the Bacterial Microbiome in Autologous Donor Skin: Implications for Skin Grafting Outcomes(Wolters Kluwer, 2017-10) Plichta, Jennifer K.; Gao, Xiang; Lin, Huaiying; Dong, Qunfeng; Toh, Evelyn; Nelson, David E.; Gamelli, Richard L.; Grice, Elizabeth A.; Radek, Katherine A.; Microbiology and Immunology, School of MedicineINTRODUCTION: The cutaneous microbiome maintains skin barrier function, regulates inflammation, and stimulates wound-healing responses. Burn injury promotes an excessive activation of the cutaneous and systemic immune response directed against commensal and invading pathogens. Skin grafting is the primary method of reconstructing full-thickness burns, and wound infection continues to be a significant complication. METHODS: In this study, the cutaneous bacterial microbiome was evaluated and subsequently compared to patient outcomes. Three different full-thickness skin specimens were assessed: control skin from non-burned subjects; burn margin from burn patients; and autologous donor skin from the same cohort of burn patients. RESULTS: We observed that skin bacterial community structure of burn patients was significantly altered compared with control patients. We determined that the unburned autologous donor skin from burn patients exhibits a microbiome similar to that of the burn margin, rather than unburned controls, and that changes in the cutaneous microbiome statistically correlate with several post-burn complications. We established that Corynebacterium positively correlated with burn wound infection, while Staphylococcus and Propionibacterium negatively correlated with burn wound infection. Both Corynebacterium and Enterococcus negatively correlated with the development of sepsis. CONCLUSIONS: This study identifies distinct differences in the cutaneous microbiome between burn subjects and unburned controls, and ascertains that select bacterial taxa significantly correlate with several comorbid complications of burn injury. These preliminary data suggest that grafting donor skin exhibiting bacterial dysbiosis may augment infection and/or graft failure and sets the foundation for more in-depth and mechanistic analyses in presumably "healthy" donor skin from patients requiring skin grafting procedures.