David E. Nelson

Objective evidence of urethral inflammation is required to diagnose urethritis in men. Clinically, this is established by confirming urethral discharge on examination in symptomatic patients. Alternatively, laboratory approaches rely on identifying polymorphonuclear neutrophils (PMNs) from a urethral swab specimen or leukocyte esterase (LE) activity in urine. However, physical examination-based diagnosis may be subjective, collection of a urethral swab is invasive, and often painful, and LE has poor sensitivity. To address these limitations, we evaluated the white blood cell (WBC) parameter of the Sysmex UF-5000 automated urinary flow cytometer for the detection of urethral inflammation and clinician-diagnosed urethritis. Using 1,000 remnant male urine specimens from a sexual health clinic, we measured urinary WBC counts (WBC/μL) using the UF-5000, LE by urinalysis, and abstracted corresponding clinical data and urethral Gram stain results from the electronic medical record. Urinary WBC counts measured by the UF-5000 were significantly higher in men with urethral symptoms, LE ≥1+ status, a clinical diagnosis of urethritis, and strongly correlated with elevated and increasing urethral Gram stain PMN density. The UF-5000 demonstrated excellent diagnostic discrimination, yielding areas under the receiver operating characteristic curve (AUC) of 0.86 for Gram stain-defined urethral inflammation, and 0.90 for clinician-defined urethritis. Optimized thresholds provided >95% sensitivity for ruling out infection and >95% specificity for ruling it in, outperforming standard LE testing. In conclusion, the UF-5000 automated urine flow cytometer offers a robust, non-invasive alternative for male urethritis diagnosis. This approach could expand diagnostic reach—particularly for men who decline urethral swab collection—and warrants prospective clinical validation. IMPORTANCE: Male urethritis diagnostics are limited by the poor sensitivity of leukocyte esterase (LE) testing or the invasive nature of urethral swab collection for Gram stain microscopy. These limitations may create barriers to patient care and delay urethritis diagnosis. Automated urinary flow cytometry may overcome these limitations, but few studies exist. We analyzed 1,000 remnant male urine specimens to evaluate the Sysmex UF-5000 white blood cell (WBC) parameter for detecting urethral inflammation. Elevated urinary WBC counts strongly correlated with elevated WBCs on urethral Gram stain smear microscopy and positive LE. The UF-5000 outperformed LE for ruling in and ruling out urethral inflammation and clinical urethritis. Automated urinary flow cytometry is a powerful, non-invasive diagnostic approach that warrants prospective evaluation in point-of-care clinical studies.

Chlamydia are obligate intracellular bacterial pathogens that infect a wide range of vertebrate hosts. Despite having highly conserved genomes, closely related Chlamydia species can exhibit distinct host and tissue tropisms. The host tropisms of the human pathogen Chlamydia trachomatis and the closely related mouse pathogen Chlamydia muridarum are influenced by their ability to evade host immune responses, particularly those mediated by interferon gamma. However, there is evidence that tissue tropism is driven by additional poorly understood host and Chlamydia factors. In this study, we used a forward genetic approach to investigate the mechanisms that mediate C. muridarum tissue tropism. We conducted a tropism screen using a randomly mutagenized C. muridarum library and murine cell lines representing different tissues. We identified a mutant isolate whose growth was restricted in murine rectal and oviduct epithelial cells in an interferon gamma-independent manner. This phenotype was mapped to a missense mutation in tc0237, a gene that mediates the affinity of C. muridarum for cultured human epithelial cells. Our analysis of growth dynamics showed that the tc0237 mutant exhibits a developmental delay in rectal epithelial cells. Together, these results suggest that TC0237 plays a role in C. muridarum tissue tropism.

We investigated the relationship between Chlamydia trachomatis load and anti-chlamydial antibody using first-catch urine from 108 men with nongonococcal urethritis. Men who reported prior chlamydia had elevated anti-chlamydial IgA and lower loads, but load and antibody were not correlated. Load positively correlated with anti-chlamydial IgA only in men who did not self-report prior chlamydia and with anti-chlamydial IgG regardless of infection history. Overall, we document genotype-specific differences in C. trachomatis load, clarify that partial immunity associated with repeat infections is not antibody mediated, and demonstrate a method to monitor humoral immune responses to C. trachomatis in men using noninvasively collected urine.

Background: Understanding the impact of testosterone on the vaginal microbiota of transgender men (TGM) and nonbinary people assigned female sex at birth over time is imperative as vaginal dysbiosis and incident bacterial vaginosis (iBV) may cause bothersome genital symptoms and increase human immunodeficiency virus/sexually transmitted infection (HIV/STI) acquisition risk. We investigated shifts in the composition of the vaginal microbiota over time in TGM initiating testosterone for gender-affirming hormone therapy, including development of vaginal dysbiosis and iBV. Methods: Participants ages ≥18 years, assigned female sex at birth and reporting TGM or nonbinary identity, interested in starting injectable testosterone, demonstrating optimal vaginal microbiota, with no current STI(s) were enrolled. Participants self-collected daily vaginal specimens for 7 days prior to testosterone initiation and 90 days thereafter for vaginal Gram staining and 16S rRNA gene sequencing. Episodes of vaginal dysbiosis and iBV were defined as Nugent scores ≥4 or ≥7, respectively, each for ≥2 consecutive days. Results: Between February 2022 and November 2023, 9 participants enrolled, 89% (8/9) developed ≥1 episode(s) of vaginal dysbiosis after testosterone initiation, and 56% (5/9) developed iBV. Among those who developed iBV, most did so between days 20 and 40 after testosterone initiation. Community state type (CST) I was found most often in participants who did not develop iBV and CST IV-B most often in participants who developed iBV. Sexual activity and menses also appeared to influence the development of iBV. Conclusions: The majority of participants developed vaginal dysbiosis including iBV. Additional studies with larger sample sizes are needed to further elucidate how testosterone impacts the vaginal microbiota.

Tail-specific proteases (Tsp) are members of a widely distributed family of serine proteases that commonly target and process periplasmic proteins in Gram-negative bacteria. The obligately intracellular, Gram-negative Chlamydia encode a highly conserved Tsp homolog whose target and function are unclear. We identified a Chlamydia muridarum mutant with a nonsense mutation in tsp . Differentiation of the tsp mutant elementary bodies into vegetative reticulate bodies was delayed at 37°C and completely blocked at 40°C. Tsp localized to C. muridarum cells but was not detected outside the inclusion, suggesting that it targets chlamydial rather than host proteins. The abundance of key chlamydia outer membrane complex and virulence-related proteins differed in wild-type and tsp mutant elementary bodies, consistent with the possibility that Tsp regulates developmental cycle progression. The altered abundances of chlamydial structural and virulence factors could explain why the mutant, but not an isogenic recombinant with wild-type tsp , was highly attenuated in a mouse intravaginal infection model. Thus, chlamydial Tsp is required for timely differentiation of elementary bodies into reticulate bodies in vitro and is an essential virulence factor in vivo .

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.

Chlamydiae are obligate intracellular pathogens that rely on secreted effector proteins to establish their intracellular niche. In this issue of the Journal of Bacteriology , Yanatori et al. describe a screen for Chlamydia pneumoniae effectors, performed in Chlamydia trachomatis , which identified several new proteins that are translocated during infection (I).

Multiple species of obligate intracellular bacteria in the genus Chlamydia are important veterinary and/or human pathogens. These pathogens all share similar biphasic developmental cycles and transition between intracellular vegetative reticulate bodies and infectious elementary forms, but vary substantially in their host preferences and pathogenic potential. A lack of tools for genetic engineering of these organisms has long been an impediment to the study of their biology and pathogenesis. However, the refinement of approaches developed in C. trachomatis over the last 10 years, and adaptation of some of these approaches to other Chlamydia spp. in just the last few years, has opened exciting new possibilities for studying this ubiquitous group of important pathogens.

DNA replication is essential for the growth and development of Chlamydia trachomatis, however it is unclear how this process contributes to and is controlled by the pathogen’s biphasic lifecycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature sensitive dnaE mutant (dnaE^ts) that exhibits a ∼100-fold reduction in genome copy number at the non-permissive temperature (40°C), but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in dnaE^ts at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. dnaE^ts formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernable peptidoglycan and polymerized MreB, the major cell division organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion reversed when dnaE^ts was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE also restored dnaE^ts genome replication and inclusion expansion at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe’s replicative and infectious forms. SIGNIFICANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and non-infectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen's infectious cycle has not been characterized. We show that genome replication is dispensable for EB to RB conversion, but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe's biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.