Testing for extragenital Neisseria gonorrhoeae and Chlamydia trachomatis: At-home pharyngeal and rectal self-swabs are non-inferior to those completed in healthcare settings.

INTRODUCTION: The rates of gonorrhea and chlamydia have been increasing in the years preceding the COVID19 pandemic. Because most gonorrhea and chlamydia infections are located in the oropharynx and rectum for men who have sex with men (MSM), and because at-home self-collected swabs for these infections are not licensed by Health Canada or the United States Food and Drug Administration, decreased accessed to in-person care during and since the COVID19 pandemic potentially means missed case findings. OBJECTIVES: To evaluate the performance of at-home self-collected pharyngeal and rectal swabs for gonorrhea and chlamydia nucleic acid amplification testing. METHODOLOGY: All persons who contacted our Sexual Health Clinic and who had a clinical indication to complete oral and/or rectal swabs for gonorrhea and chlamydia were invited to complete at-home swabs in advance of their scheduled appointments. We mailed swabs and instructions to those who consented. Participants brought these swabs to their scheduled in clinic appointments, where we repeated the same swabs. All matching swabs were sent to the laboratory for analysis to determine concordance. RESULTS: From September 8, 2022 to July 18, 2023, we enrolled 296 eligible participants who provided 1184 swabs. For analysis, cancelled specimens and specimens with invalid results were excluded, leaving 1032 swabs for comparison. We identified 66 STI diagnoses in 47 unique participants. Overall accuracy was high (exceeding 99%), except for rectal chlamydia, which was 96.0%. While the performance of self-swabs for chlamydia was lower compared to gonorrhea, at-home swabs identified six chlamydia infections that were missed by in-clinic collected swabs (two pharyngeal, four rectal). Removing these six cases as "false positives" increased overall accuracy for chlamydia detection to 99.7% (pharyngeal) and 97.8% (rectal). CONCLUSION: Self-collected at-home swabs had good performance acceptable for gonorrhea and chlamydia nucleic acid amplification testing.

Association between bacterial vaginosis, Chlamydia trachomatis infection and tubal factor infertility in Bukavu, Democratic Republic of Congo.

BACKGROUND: Tubal factor infertility (TFI) is common in sub-Saharan Africa and often secondary to pelvic inflammatory disease (PID). Anaerobes associated with bacterial vaginosis (BV) are also found in PIDs widely dominated by Chlamydia trachomatis (C. trachomatis), whose role in TFI is better demonstrated than that of BV. OBJECTIVES: To determine the prevalence of BV and C. trachomatis and to investigate the association between BV, C. trachomatis and TFI. METHODS: We included 137 patients treated for infertility between January 2020 and November 2021. Cases were defined as women with infertility aged 18-45 years presenting with TFI (n = 52), and controls as infertile women in the same age groups without TFI (n = 85). Data on social habits, life style and infertility parameters were collected, and we performed screening for BV and C. trachomatis. Multiple regression was used to measure associations. RESULTS: The prevalence of BV and C. trachomatis was 42.3% (58/137) and 23.4% (32/137), respectively. BV (61.5% vs 30.6%, p<0.001) and C. trachomatis (48.1 vs 8.2%, p<0.001) were more frequent in cases of TFI. BV and C. trachomatis increased the risk of TFI approximately 4-fold [aOR: 3.77 (1.61-8.83), p=0.002] and 14-fold [aOR: 13.77 (4.59-41.27), p<0.001], respectively. CONCLUSION: BV and C. trachomatis infection are strongly associated with TFI in Bukavu. Prevention and screening should be implemented to reduce the risk of TFI.

Hijacking host cell vesicular transport: New insights into the nutrient acquisition mechanism of Chlamydia.

Chlamydia infection is an important cause of public health diseases, and no effective vaccine is currently available. Owing to its unique intracellular lifestyle, Chlamydia requires a variety of nutrients and substrates from host cells, particularly sphingomyelin, cholesterol, iron, amino acids, and the mannose-6-phosphate receptor, which are essential for inclusion development. Here, we summarize the recent advances in Chlamydia nutrient acquisition mechanism by hijacking host cell vesicular transport, which plays an important role in chlamydial growth and development. Chlamydia obtains the components necessary to complete its intracellular developmental cycle by recruiting Rab proteins (major vesicular trafficking regulators) and Rab effector proteins to the inclusion, interfering with Rab-mediated multivesicular trafficking, reorienting the nutrition of host cells, and reconstructing the intracellular niche environment. Consequently, exploring the role of vesicular transport in nutrient acquisition offers a novel perspective on new approaches for preventing and treating Chlamydia infection.

Chlamydia muridarum Associated Pulmonary and Urogenital Disease and Pathology in a Colony of Enzootically Infected Il12rb2 Deficient and Stat1 Knockout Mice.

Chlamydia muridarum (Cm), an intracellular bacterium of historical importance, was recently rediscovered as moderately prevalent in research mouse colonies. Cm was first reported as a causative agent of severe pneumonia in mice about 80 y ago, and while it has been used experimentally to model Chlamydia trachomatis infection of humans, there have been no further reports of clinical disease associated with natural infection. We observed clinical disease and pathology in 2 genetically engi- neered mouse (GEM) strains, Il12rb2 KO and STAT1 KO, with impaired interferon-γ signaling and Th1 CD4+ T cell responses in a colony of various GEM strains known to be colonized with and shedding Cm. Clinical signs included poor condition, hunched posture, and poor fecundity. Histopathology revealed disseminated Cm with lesions in pulmonary, gastrointestinal, and urogenital tissues. The presence of Cm was confirmed using both immunohistochemistry for Cm major outer membrane protein-1 antigen and in situ hybridization using a target probe directed against select regions of Cm strain Nigg. Cm was also found in association with a urothelial papilloma in one mouse. These cases provide additional support for excluding Cm from research mouse colonies.

Chlamydia trachomatis and Neisseria gonorrhoeae rectal infections: Interplay between rectal microbiome, HPV infection and Torquetenovirus.

Men having sex with men (MSM) represent a key population, in which sexually transmitted rectal infections (STIs) caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and high-risk HPV (HR-HPV) are very common and linked to significant morbidity. Investigating the anorectal microbiome associated with rectal STIs holds potential for deeper insights into the pathogenesis of these infections and the development of innovative control strategies. In this study, we explored the interplay at the rectal site between C. trachomatis, N. gonorrhoeae, HR-HPV infection, and the anorectal microbiome in a cohort of 92 MSM (47 infected by CT and/or NG vs 45 controls). Moreover, we assessed the presence of Torquetenovirus (TTV), a non-pathogenic endogenous virus, considered as a possible predictor of immune system activation. We found a high prevalence of HR-HPV rectal infections (61%), especially in subjects with a concurrent CT/NG rectal infection (70.2%) and in people living with HIV (84%). In addition, we observed that TTV was more prevalent in subjects with CT/NG rectal infections than in non-infected ones (70.2% vs 46.7%, respectively). The anorectal microbiome of patients infected by CT and/or NG exhibited a reduction in Escherichia, while the presence of TTV was significantly associated with higher levels of Bacteroides. We observed a positive correlation of HR-HPV types with Escherichia and Corynebacterium, and a negative correlation with the Firmicutes phylum, and with Prevotella, Oscillospira, Sutterella. Our findings shed light on some of the dynamics occurring within the rectal environment involving chlamydial/gonococcal infections, HPV, TTV, and the anorectal microbiome. These data could open new perspectives for the control and prevention of STIs in MSM.

Novel typing scheme reveals emergence and genetic diversity of Chlamydia pecorum at the local management scale across two koala populations.

To overcome shortcomings in discriminating Chlamydia pecorum strains infecting the koala (Phascolarctos cinereus) at the local level, we developed a novel genotyping scheme for this pathogen to inform koala management at a fine-scale subpopulation level. We applied this scheme to two geographically distinct koala populations in New South Wales, Australia: the Liverpool Plains and the Southern Highlands to South-west Sydney (SHSWS). Our method provides greater resolution than traditional multi-locus sequence typing, and can be used to monitor strain emergence, movement, and divergence across a range of fragmented habitats. Within the Liverpool Plains population, suspected recent introduction of a novel strain was confirmed by an absence of genetic diversity at the earliest sampling events and limited diversity at recent sampling events. Across the partially fragmented agricultural landscape of the Liverpool Plains, diversity within a widespread sequence type suggests that this degree of fragmentation may hinder but not prevent spread. In the SHSWS population, our results suggest movement of a strain from the south, where diverse strains exist, into a previously Chlamydia-free area in the north, indicating the risk of expansion towards an adjacent Chlamydia-negative koala population in South-west Sydney. In the south of the SHSWS where koala subpopulations appear segregated, we found evidence of divergent strain evolution. Our tool can be used to infer the risks of strain introduction across fragmented habitats in population management, particularly through practices such as wildlife corridor constructions and translocations.

Irgm proteins attenuate inflammatory disease in mouse models of genital Chlamydia infection.

Chlamydiae are obligate intracellular bacterial pathogens that may cause genital pathology via induction of destructive host immune responses. Human-adapted Chlamydia trachomatis causes inflammatory disease in human hosts but is easily cleared in mice, and mouse-adapted Chlamydia muridarum establishes a productive and pathogenic infection in murine hosts. While numerous anti-chlamydial host resistance factors have been discovered in mice and humans alike, little is known about host factors promoting host fitness independent of host resistance. Here, we show that interferon-inducible immunity-related GTPase M (Irgm) proteins function as such host factors ameliorating infection-associated sequalae in the murine female genital tract, thus characterizing Irgm proteins as mediators of disease tolerance. Specifically, we demonstrate that mice deficient for all three murine Irgm paralogs (pan-Irgm-/-) are defective for cell-autonomous immunity to C. trachomatis, which correlates with an early and transient increase in bacterial burden and sustained hyperinflammation in vivo. In contrast, upon infection of pan-Irgm-/- mice with C. muridarum, bacterial burden is unaffected, yet genital inflammation and scarring pathology are nonetheless increased, demonstrating that Irgm proteins can promote host fitness without altering bacterial burden. Additionally, pan-Irgm-/- mice display increased granulomatous inflammation in genital Chlamydia infection, implicating Irgm proteins in the regulation of granuloma formation and maintenance. These findings demonstrate that Irgm proteins regulate pathogenic immune responses to Chlamydia infection in vivo, establishing an effective infection model to examine the immunoregulatory functions and mechanisms of Irgm proteins. IMPORTANCE: In response to genital Chlamydia infection, the immune system mounts a proinflammatory response to resist the pathogen, yet inflammation must be tightly controlled to avoid collateral damage and scarring to host genital tissue. Variation in the human IRGM gene is associated with susceptibility to autoinflammatory diseases but its role in ameliorating inflammatory diseases caused by infections is poorly defined. Here, we use mice deficient for all three murine Irgm paralogs to demonstrate that Irgm proteins not only provide host resistance to Chlamydia infections but also limit associated inflammation in the female genital tract. In particular, we find that murine Irgm expression prevents granulomatous inflammation, which parallels inflammatory diseases associated with variants in human IRGM. Our findings therefore establish genital Chlamydia infection as a useful model to study the roles for Irgm proteins in both promoting protective immunity and limiting pathogenic inflammation.

Chronic sexually acquired reactive arthritis secondary to Chlamydiatrachomatis.

Reactive arthritis is included in the spectrum of seronegative spondyloarthritides, occurring secondary to triggers of genitourinary and gastrointestinal tract infections. We describe two cases of sexually acquired reactive arthritis secondary to genital infection by Chlamydia trachomatis, diagnosed by in-house polymerase chain reaction performed on the first void urine. Both patients were managed with a combined approach of short course antibiotics, immunosuppressive agents, biologicals and surgical intervention.

Pharmacokinetics of single dose doxycycline in the rectum, vagina, and urethra: implications for prevention of bacterial sexually transmitted infections.

BACKGROUND: Clinical trials showed a single oral dose of doxycycline taken after sex protects against STIs among men who have sex with men (MSM) but not women. Pharmacokinetic data at vaginal, rectal and penile sites of STI exposure are lacking. We examined vaginal, rectal and urethral doxycycline concentrations in men and women to better inform STI prevention. METHODS: Doxycycline pharmacokinetics in male and female participants 18-59 years of age were evaluated in blood and urine and on rectal and vaginal swabs collected at 1, 2, 4, 8, 24, 48, 72, 96 and 168 h after receiving a 200 mg oral doxycycline dose in a non-randomised single dose open label single centre study in Atlanta, Georgia. Rectal, vaginal, and cervical biopsies and male urethral swabs were collected 24 h after dosing (Trial registration: NCT04860505). Doxycycline was measured by liquid chromatography-mass spectrometry. FINDINGS: Eleven male and nine female participants participated in the study. Doxycycline concentrations on rectal and vaginal swabs collected up to 96 h after dosing were approximately twice those of plasma and remained above minimum inhibitory concentrations (MICs) for at least four, three, and two days for Chlamydia trachomatis, Treponema pallidum, and tetracycline-sensitive Neisseria gonorrhoeae, respectively. Geometric mean doxycycline concentrations in male urethral secretions (1.166 µg/mL; 95% CI 0.568-2.394 µg/mL), male rectal (0.596 µg/g; 0.442-0.803 µg/g), vaginal (0.261 µg/g; 0.098-0.696 µg/g) and cervical tissue (0.410 µg/g; 0.193-0.870 µg/g) in biopsies collected 24 h after dosing exceeded MICs. Plasma and urine doxycycline levels defined adherence markers up to four and seven days postdosing, respectively. No adverse events were reported in this study. INTERPRETATION: Doxycycline efficiently distributes to the rectum, vagina and urethra. Findings can help explain efficacy of STI prevention by doxycycline. FUNDING: Funded by CDC intramural funds, CDC contract HCVJCG-2020-45044 (to CFK).

IFNγ insufficiency during mouse intra-vaginal Chlamydia trachomatis infection exacerbates alternative activation in macrophages with compromised CD40 functions.

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is a leading cause of preventable blindness. We have developed in vivo mouse models of acute and chronic C. trachomatis genital infection to explore the significance of macrophage-directed response in mediating immune activation/suppression. Our findings reveal that during chronic and repeated C. trachomatis infections, Th1 response is abated while Treg response is enhanced. Additionally, an increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is observed during chronic infection. We have also observed that M2 macrophages with low CD40 expression promote Th2 and Treg differentiation leading to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to an M2 phenotype. C. trachomatis infection prevents the increase in CD40 expression as observed in western blots and flow cytometric analysis. Insufficient IFNγ, as observed during chronic infection, leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFNγ responsiveness in macrophages via hampering IFNγRI and IFNγRII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFNγ supplementation. M2 macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th2 and Treg differentiation which could not be reverted even by IFNγ supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFNγ, thus promoting immunosuppression. In summary, C. trachomatis modulates the innate immune cells, attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages.

Chlamydia retesting remains low among young women in Australia: an observational study using sentinel surveillance data, 2018-2022.

BACKGROUND: Chlamydia remains the most notified bacterial sexually transmissible infection in Australia with guidelines recommending testing for re-infection at 3months post treatment. This paper aimed to determine chlamydia retesting and repeat positivity rates within 2-4months among young women in Australia, and to evaluate what factors increase or decrease the likelihood of retesting. METHODS: Chlamydia retesting rates among 16-29-year-old women were analysed from Australian Collaboration for Coordinated Enhanced Sentinel Surveillance of sexually transmissible infection and bloodborne virus (ACCESS) sentinel surveillance data (n =62 sites). Among women with at least one positive test between 1 January 2018 and 31 August 2022, retesting counts and proportions within 2-4months were calculated. Logistic regression was performed to assess factors associated with retesting within 2-4months. RESULTS: Among 8758 women who were positive before 31 August 2022 to allow time for follow up, 1423 (16.2%) were retested within 2-4months, of whom 179 (12.6%) tested positive. The odds of retesting within 2-4months were 25% lower if tested in a coronavirus disease 2019 (COVID-9) pandemic year (2020-2022) (aOR=0.75; 95% CI 0.59-0.95). Among 9140 women with a positive test before 30 November 2022, 397 (4.3%) were retested too early (within 7days to 1month) and 81 (20.4%) of those were positive. CONCLUSIONS: Chlamydia retesting rates remain low with around a sixth of women retested within 2-4months in line with guidelines. Re-infection is common with around one in eight retesting positive. An increase in retesting is required to reduce the risk of reproductive complications and onward transmission.

Seroprevalence and Risk Factors of Chlamydia Infection in Pigs in Hunan Province, Southern China, 2017-2018.

Background: Chlamydia is a Gram-negative obligate intracellular bacterium that is pathogenic for humans and a large variety of veterinary animal species. However, there is no continuous monitoring of chlamydia infection data in pigs in Hunan province, southern China. Therefore, in order to evaluate the seroprevalence and identify risk factors associated with Chlamydia infection in pigs within this region, a comprehensive study was conducted. Methods: A total of 3848 serum samples were collected from pigs (from farmers and companies) between May 2017 and August 2018. The presence of specific antibodies against Chlamydia was determined through the employment of the indirect hemagglutination assay (IHA). Results: The overall seroprevalence of Chlamydia was determined to be 26.90% (1038/3848, 95% confidence interval: 25.60-28.40). By employing statistical analysis using SPSS software (p < 0.05), factors such as altitude, sampling regions, and rearing systems of pigs were identified as potential risk factors for Chlamydia infection. Conclusion: These findings elucidate a substantial prevalence of Chlamydia in pigs within the mountainous region of Hunan province, southern China, thereby highlighting a potential risk to human health. These results underscore the need for proactive measures and targeted interventions to mitigate the transmission of Chlamydia in porcine populations, safeguarding both animal welfare and public health.

Immune signature of Chlamydia vaccine CTH522/CAF®01 translates from mouse-to-human and induces durable protection in mice.

The clinical development of an effective Chlamydia vaccine requires in-depth understanding of how well protective pre-clinical immune signatures translate to humans. Here, we report a comparative immunological characterization of CTH522/CAF®01 in female mice and humans. We find a range of immune signatures that translate from mouse to human, including a Th1/Th17 cytokine profile and antibody functionality. We identify vaccine-induced T cell epitopes, conserved among Chlamydia serovars, and previously found in infected individuals. Using the mouse model, we show that the common immune signature protected against ascending infection in mice, and vaccine induced antibodies could delay bacterial ascension to the oviduct, as well as development of pathology, in a T cell depleted mouse model. Finally, we demonstrate long-lasting immunity and protection of mice one year after vaccination. Based on the results obtained in the present study, we propose to further investigate CTH522/CAF®01 in a phase IIb study.

Metabolic dormancy in Chlamydia trachomatis treated with different antibiotics.

Diseases caused by Chlamydia spp. are often associated with persistent infections. Chlamydial persistence is commonly associated with a unique non-infectious intracellular developmental form, termed an aberrant form. Although infectious chlamydiae can be cultured consistently in cells stressed to aberrancy, their role in persistence is not clear. Recovery from antibiotic stress was explored as a model to determine how survival of non-aberrant chlamydiae, in the presence of fully inhibitory drug concentrations, may participate in persistence. Assays included incubation in quinolones, tetracyclines, or chloramphenicol for differing lengths of time, followed by an extended recovery period in antibiotic-free media. Culturable elementary bodies were not detected during treatment with each antibiotic, but viable and culturable Chlamydia trachomatis emerged after the drug was removed. Time-lapse imaging of live, antibiotic-treated infected cells identified metabolically dormant developmental forms within cells that emerged to form typical productive inclusions. The effects of the increasing concentration of most tested antibiotics led to predictable inhibitory activity, in which the survival rate decreased with increasing drug concentration. In contrast, in fluoroquinolone-treated cells, there was a paradoxical increase in productive development that was directly correlated with drug concentration and inversely associated with aberrant form production. This model system uncovers a unique chlamydial persistence pathway that does not involve the chlamydial aberrant form. The association between productive latency and metabolic dormancy is consistent with models for many bacterial species and may lead to a different interpretation of mechanisms of chlamydial persistence in patients.IMPORTANCEThe life history of most pathogens within the genus Chlamydia relies on lengthy persistence in the host. The most generally accepted model for Chlamydia spp. persistence involves an unusual developmental stage, termed the aberrant form, which arises during conditions that mimic a stressful host environment. In this work, we provide an alternate model for chlamydial persistence in the face of antibiotic stress. This model may be relevant to antibiotic treatment failures in patients infected with C. trachomatis.

Prevalence, risk factors and genotyping of chlamydia trachomatis from endocervical specimens of infertile women at a tertiary care hospital, Vietnam.

BACKGROUND: To our knowledge, the prevalence, risk factors and distribution of C. trachomatis genotypes are rarely mentioned in Vietnam. This study aimed to find the prevalence, risk factors and distribution of C. trachomatis genotypes in infertile Vietnamese women. METHODS: Endocervical swabs were collected from infertile women at the National Hospital of Obstetrics and Gynecology, Vietnam, between January 2020 and December 2021. All samples were analyzed for C. trachomatis presence by Cobas 4800 CT/NG Test. Sequencing methods of ompA gene were used to determine the C. trachomatis genotypes. An approximately 1200 bp ompA fragment was aligned with reference sequences from GenBank to identify the corresponding genotype. RESULTS: The prevalence of endocervical C. trachomatis infection was 15.6% of 761 participants. Factors independently associated with CT infection among infertile women, obtained by multivariate analysis, included abnormal vaginal discharge, cervicitis, lower abdominal pain, a history of ectopic pregnancy, having more than one sex partner, and age at first intercourse. Among the samples, genotype E (25.93%) was most frequently found, followed by genotypes D/Da (22.23%), F (13.58%), G/Ga (12.35%), J (12.35%), H (6.17%), K (3.70%), B/Ba (2.47%), and I/Ia (1.23%), respectively. Genotype F was related to types of infertility, and genotype H was associated with a history of miscarriage. CONCLUSIONS: The present study indicated a high prevalence of C. trachomatis in infertile Vietnamese women. The most common genotypes found in this population were E, D, and F. Our findings suggest that routine screening is necessary for early detection and performance of infection control methods.

Elimination of Chlamydia muridarum from the female reproductive tract is IL-12p40 dependent, but independent of Th1 and Th2 cells.

Chlamydia vaccine approaches aspire to induce Th1 cells for optimal protection, despite the fact that there is no direct evidence demonstrating Th1-mediated Chlamydia clearance from the female reproductive tract (FRT). We recently reported that T-bet-deficient mice can resolve primary Chlamydia infection normally, undermining the potentially protective role of Th1 cells in Chlamydia immunity. Here, we show that T-bet-deficient mice develop robust Th17 responses and that mice deficient in Th17 cells exhibit delayed bacterial clearance, demonstrating that Chlamydia-specific Th17 cells represent an underappreciated protective population. Additionally, Th2-deficient mice competently clear cervicovaginal infection. Furthermore, we show that sensing of IFN-γ by non-hematopoietic cells is essential for Chlamydia immunity, yet bacterial clearance in the FRT does not require IFN-γ secretion by CD4 T cells. Despite the fact that Th1 cells are not necessary for Chlamydia clearance, protective immunity to Chlamydia is still dependent on MHC class-II-restricted CD4 T cells and IL-12p40. Together, these data point to IL-12p40-dependent CD4 effector maturation as essential for Chlamydia immunity, and Th17 cells to a lesser extent, yet neither Th1 nor Th2 cell development is critical. Future Chlamydia vaccination efforts will be more effective if they focus on induction of this protective CD4 T cell population.

Regulation of chlamydial spreading from the small intestine to the large intestine by IL-22-producing CD4+ T cells.

Following an oral inoculation, Chlamydia muridarum descends to the mouse large intestine for long-lasting colonization. However, a mutant C. muridarum that lacks the plasmid-encoded protein pGP3 due to an engineered premature stop codon (designated as CMpGP3S) failed to do so even following an intrajejunal inoculation. This was because a CD4+ T cell-dependent immunity prevented the spread of CMpGP3S from the small intestine to the large intestine. In the current study, we found that mice deficient in IL-22 (IL-22-/-) allowed CMpGP3S to spread from the small intestine to the large intestine on day 3 after intrajejunal inoculation, indicating a critical role of IL-22 in regulating the chlamydial spread. The responsible IL-22 is produced by CD4+ T cells since IL-22-/- mice were rescued to block the CMpGP3S spread by donor CD4+ T cells from C57BL/6J mice. Consistently, CD4+ T cells lacking IL-22 failed to block the spread of CMpGP3S in Rag2-/- mice, while IL-22-competent CD4+ T cells did block. Furthermore, mice deficient in cathelicidin-related antimicrobial peptide (CRAMP) permitted the CMpGP3S spread, but donor CD4+ T cells from CRAMP-/- mice were still sufficient for preventing the CMpGP3S spread in Rag2-/- mice, indicating a critical role of CRAMP in regulating chlamydial spreading, and the responsible CRAMP is not produced by CD4+ T cells. Thus, the IL-22-producing CD4+ T cell-dependent regulation of chlamydial spreading correlated with CRAMP produced by non-CD4+ T cells. These findings provide a platform for further characterizing the subset(s) of CD4+ T cells responsible for regulating bacterial spreading in the intestine.

Neisseria gonorrhoeae drives Chlamydia trachomatis into a persistence-like state during in vitro co-infection.

Chlamydia trachomatis and Neisseria gonorrhoeae are the most prevalent bacterial sexually transmitted infections (STIs) globally. Despite frequent co-infections in patients, few studies have investigated how mono-infections may differ from co-infections. We hypothesized that a symbiotic relationship between the pathogens could account for the high rates of clinical co-infection. During in vitro co-infection, we observed an unexpected phenotype where the C. trachomatis developmental cycle was impaired by N. gonorrhoeae. C. trachomatis is an obligate intracellular pathogen with a unique biphasic developmental cycle progressing from infectious elementary bodies (EB) to replicative reticulate bodies (RB), and back. After 12 hours of co-infection, we observed fewer EBs than in a mono-infection. Chlamydial genome copy number remained equivalent between mono- and co-infections. This is a hallmark of Chlamydial persistence. Chlamydial persistence alters inclusion morphology but varies depending on the stimulus/stress. We observed larger, but fewer, Chlamydia during co-infection. Tryptophan depletion can induce Chlamydial persistence, but tryptophan supplementation did not reverse the co-infection phenotype. Only viable and actively growing N. gonorrhoeae produced the inhibition phenotype in C. trachomatis. Piliated N. gonorrhoeae had the strongest effect on C. trachomatis, but hyperpiliated or non-piliated N. gonorrhoeae still produced the phenotype. EB development was modestly impaired when N. gonorrhoeae were grown in transwells above the infected monolayer. C. trachomatis serovar L2 was not impaired during co-infection. Chlamydial impairment could be due to cytoskeletal or osmotic stress caused by an as-yet-undefined mechanism. We conclude that N. gonorrhoeae induces a persistence-like state in C. trachomatis that is serovar dependent.

Chlamydia muridarum infection causes bronchointerstitial pneumonia in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice.

The murine bacterial pathogen Chlamydia muridarum (Cm) has been used to study human Chlamydia infections in various mouse models. CD4+ T-cells, natural killer cells, and interferon-gamma (IFN-γ)-mediated immunity are important to control experimentally induced Cm infections. Despite its experimental use, natural infection by Cm has not been documented in laboratory mice since the 1940s. In 2022, the authors reported the discovery of natural Cm infections in numerous academic institutional laboratory mouse colonies around the globe. To evaluate the impact of Cm infection in severely immunocompromised mice, 19 NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were cohoused with Cm shedding, naturally infected immunocompetent mice and/or their soiled bedding for 4 weeks and subsequently euthanized. Clinical disease, characterized by lethargy, dyspnea, and weight loss, was observed in 11/19 NSG mice, and 16/18 NSG mice had neutrophilia. All mice exhibited multifocal to coalescing histiocytic and neutrophilic bronchointerstitial pneumonia (17/19) or bronchiolitis (2/19) with intraepithelial chlamydial inclusions (CIs). Immunofluorescence showed CIs were often associated with bronchiolar epithelium. CIs were frequently detected by immunohistochemistry in tracheal and bronchiolar epithelium (19/19), as well as throughout the small and large intestinal epithelium without lesions (19/19). In a subset of cases, Cm colonized the surface epithelium in the nasopharynx (16/19), nasal cavity (7/19), and middle ear canal (5/19). Endometritis and salpingitis with intraepithelial CI were identified in a single mouse. These findings demonstrate that Cm infection acquired through direct contact or soiled bedding causes significant pulmonary pathology and widespread intestinal colonization in NSG mice.

Unique microbial diversity, community composition, and networks among Pacific Islander endocervical and vaginal microbiomes with and without Chlamydia trachomatis infection in Fiji.

IMPORTANCE: Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterium globally. Endocervical and vaginal microbiome interactions are rarely examined within the context of Ct or among vulnerable populations. We evaluated 258 vaginal and 92 paired endocervical samples from Fijian women using metagenomic shotgun sequencing. Over 37% of the microbiomes could not be classified into sub-community state types (subCSTs). We, therefore, developed subCSTs IV-D0, IV-D1, IV-D2, and IV-E-dominated primarily by Gardnerella vaginalis-to improve classification. Among paired microbiomes, the endocervix had a significantly higher alpha diversity and, independently, higher diversity for high-risk human papilloma virus (HPV) genotypes compared to low-risk and no HPV. Ct-infected endocervical networks had smaller clusters without interactions with potentially beneficial Lactobacillus spp. Overall, these data suggest that G. vaginalis may generate polymicrobial biofilms that predispose to and/or promote Ct and possibly HPV persistence and pathogenicity. Our findings expand on the existing repertoire of endocervical and vaginal microbiomes and fill in knowledge gaps regarding Pacific Islanders.