Sexually transmitted infections (STIs) of the cervicovaginal mucosa are among the most common global infections. Current monolayer cell culture and animal models fail to reproduce the multilevel complexity required to investigate host-microbiota-pathogen relationships simultaneously and/or with sufficient physiological relevance. To address this limitation, we have developed a microphysiologic system (MPS) that models human cervical tissue and its microbiota and is susceptible to infection by two prominent genital pathogens, Chlamydia trachomatis and Neisseria gonorrhoeae. Notably, this MPS platform recapitulates essential dynamic, polymicrobial, immune, and pathogenic features of chlamydial and gonococcal infections as they occur in humans. The low-cost MPS device requires no specialized equipment or specific expertise and was experimentally validated across multiple nonengineering, remotely located laboratories, demonstrating its transferability and reproducibility. The platform provides a tool for research into genital infections in a system that closely mimics the cervical epithelium, an important advance over existing models.

Sexually transmitted infections (STIs) of the cervicovaginal mucosa are among the most common global infections. Clinical studies have revealed that susceptibility to STIs and the subsequent host responses they elicit are frequently associated with vaginal microbiota compositions that facilitate infection. Current monolayer cell culture and animal models fail to reproduce the multilevel complexity required to investigate these relationships simultaneously and/or with sufficient physiological relevance. To address this limitation, we have developed a microphysiologic system (MPS) that models human cervical tissue, its microbiota, and is susceptible to infection by two prominent genital pathogens, Chlamydia trachomatis and Neisseria gonorrhoeae. Significantly, this MPS platform recapitulates essential dynamic, polymicrobial, immune, and pathogenic features of chlamydial and gonococcal infections as they occur in humans. The low-cost MPS device requires no specialized equipment or specific expertise and was experimentally validated for both chlamydial and gonococcal infections across multiple non-engineering, remotely located laboratories, demonstrating its transferability and reproducibility. The MPS platform described herein provides a novel tool for expanded research into genital infections in a reconstituted system that closely mimics the cervical epithelium, a significant advance over existing models.

Chlamydia trachomatis (CT) is the most commonly reported sexually transmitted infection in the United States. Untreated urogenital infection in women can result in adverse sequelae such as pelvic inflammatory disease and infertility. Despite national screening and treatment guidelines, rates continue to rise; because most infections are asymptomatic, the actual prevalence of CT infection is likely significantly higher than reported. Spontaneous clearance of CT in women (in the absence of antibiotic treatment) has been described in multiple epidemiologic studies. Given the serious consequences and high prevalence of CT infection, there is growing interest in understanding this phenomenon and factors that may promote CT clearance in women. Spontaneous CT clearance is likely the result of complex interactions between CT, the host immune system, and the vaginal microbiota (i.e., the communities of bacteria inhabiting the vagina), which has been implicated in CT acquisition. Herein, we briefly review current literature regarding the role of each of these factors in spontaneous CT clearance, identify knowledge gaps, and discuss future directions and possible implications for the development of novel interventions that may protect against CT infection, facilitate clearance, and prevent reproductive sequelae.

The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum Chlamydiae (Chlamydiota) consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

Background We previously observed that the nine-member family of autotransported polymorphic membrane proteins (Pmps) of Chlamydia trachomatis is variably expressed in cell culture. Additionally, C. trachomatis -infected patients display variable Pmp-specific serum antibody profiles indirectly suggesting expression of unique Pmp profiles is an adaptive response to host-specific stimuli during infection. Here, we propose that the host response to Pmps and other outer surface proteins may correlate with disease severity. Methods This study tests this hypothesis using an ELISA that measures serum IgG antibodies specific for the nine C. trachomatis Pmp subtypes and four immunodominant antigens (MOMP, OmcB, GroEL, ClpP) in 265 participants of the Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort. Results More C. trachomatis -infected females displayed high Pmp-specific antibody levels (cut-off Indexes) than males (35.9-40.7% of females vs . 24.2-30.0% of males), with statistical significance for PmpC, F and H (P<0.05). Differences in Pmp-specific antibody profiles were not observed between C. trachomatis -infected females with a clinical diagnosis of pelvic inflammatory disease (PID) and those without. However, a statistically significant association between high levels of OmcB-specific antibody and a PID diagnosis (P<0.05) was observed. Conclusions Using antibody levels as an indirect measure of antigen expression, our results suggest that gender- and/or site-specific (cervix in females vs . urethra in males) stimuli control pmp expression in infected patients. They also support the possible existence of immune biomarkers of chlamydial infection associated with disease and underline the need for high resolution screening in human serum.

The International Committee on Systematics of Prokaryotes (ICSP) Subcommittee on the Taxonomy of Chlamydiae (STC) was founded in 2010 to solve major taxonomy and nomenclature issues regarding members of the order Chlamydiales. The ICSP-STC clarifies how species and Candidatus species should be reported. When based on genomic data, the genome sequence (at least 80% complete) should reveal the presence of core genes [1], with the taxonomically informative genes defined by Pillonel et al. [2] as the preferred core subset.

Two-dimensional (2D) cell culture systems have provided controlled, reproducible means to analyze host-pathogen interactions. Although inexpensive, straightforward, and requiring very short time commitment, these models recapitulate neither the functionality of multi-layered cell types nor the microbial diversity of an infected human. Animal models have commonly been used to recreate the complexity of human infections. However, extensive modifications are commonly required to recreate interactions that resemble those in the human reproductive tract microbiologically and physiologically. Three-dimensional (3D) cell culture models have emerged as alternative means of reproducing key elements of human infections at a fraction of the cost of animal models and on a scale that allows for replicative experiments to be readily performed. Here we describe a new 3D model that utilizes transwells with epithelial cells seeded apically and a basolateral extra cellular matrix (ECM)-like layer containing collagen and fibroblasts. In this system, basal feeding creates a liquid/air interface on the apical side. The model produced tissues with close morphologic and physiological resemblance to human cervical and vaginal epithelia, including observable levels of mucus produced by cervical cells. Infection by both Chlamydia trachomatis and Neisseria gonorrhoeae was demonstrated as well as the growth of bacterial species observed in the human vaginal microbiota, enabling controlled mechanistic analyses of the interactions between host cells, vaginal microbiota and STI pathogens. Future experiments may include immune cells to mimic more closely the genital environment. Finally, the modular set up of the model makes it fully applicable to the analysis of non-genital host-microbiome-pathogen interactions. IMPORTANCE Infected sites in humans are a complex mix of host and microbial cell types interacting with each other to perform specific and necessary functions. The ability to understand the mechanism(s) that facilitate these interactions, and interactions with external factors is paramount to being able to develop preventative therapies. Models that attempt to faithfully replicate the complexity of these interactions are time intensive, costly, and not conducive to high throughput analysis. Two-dimensional (2D) models that have been used as a platform to understand these interactions, while cost effective, are generally limiting in experimental flexibility and structural/physiological relevance. Our three-dimensional (3D) models of the cervicovaginal epithelium can facilitate analysis of interactions between the host epithelium, sexually transmitted pathogens and bacteria present in the vaginal microbiota. Due to the modular design, additional cell types and environmental modulators can be introduced to the system to provide added complexity, approaching conditions in the infected human host.

The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus.

We characterized the composition and structure of the vaginal microbiota in a cohort of 149 women with genital Chlamydia trachomatis infection at baseline who were followed quarterly for 9 months after antibiotic treatment. At time of diagnosis, the vaginal microbiota was dominated by Lactobacillus iners or a diverse array of bacterial vaginosis-associated bacteria including Gardnerella vaginalis. Interestingly, L. iners-dominated communities were most common after azithromycin treatment (1 g monodose), consistent with the observed relative resistance of L. iners to azithromycin. Lactobacillus iners-dominated communities have been associated with increased risk of C. trachomatis infection, suggesting that the impact of antibiotic treatment on the vaginal microbiota could favor reinfections. These results provide support for the dual need to account for the potential perturbing effect(s) of antibiotic treatment on the vaginal microbiota, and to develop strategies to protect and restore optimal vaginal microbiota.

Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) are two highly prevalent bacterial sexually transmitted infections (STIs) with a significant rate of co-infection in some populations. Vaginal metabolites are influenced by resident vaginal microbiota, affect susceptibility to sexually transmitted infections (STIs), and may impact local inflammation and patient symptoms. Examining the vaginal metabolome in the context of CT mono (CT+) and CT/MG co-infection (CT+/MG+) may identify biomarkers for infection or provide new insights into disease etiology and pathogenesis. Yet, the vaginal metabolome in the setting of CT infection is understudied and the composition of the vaginal metabolome in CT/MG co-infected women is unknown. Therefore, in this analysis, we used an untargeted metabolomic approach combined with 16S rRNA gene amplicon sequencing to characterize the vaginal microbiota and metabolomes of CT+, CT+/MG+, and uninfected women. We found that CT+ and CT+/MG+ women had distinct vaginal metabolomic profiles as compared to uninfected women both before and after adjustment for the vaginal microbiota. This study provides important foundational data documenting differences in the vaginal metabolome between CT+, CT+/MG+ and uninfected women. These data may guide future mechanistic studies that seek to provide insight into the pathogenesis of CT and CT/MG infections.

The ability to introduce targeted genetic modifications in microbial genomes has revolutionized our ability to study the role and mode of action of individual bacterial virulence factors. Although the fastidious lifestyle of obligate intracellular bacterial pathogens poses a technical challenge to such manipulations, the last decade has produced significant advances in our ability to conduct molecular genetic analysis in Chlamydia trachomatis, a major bacterial agent of infertility and blindness. Similar approaches have not been established for the closely related veterinary Chlamydia spp., which cause significant economic damage, as well as rare but potentially life-threatening infections in humans. Here we demonstrate the feasibility of conducting site-specific mutagenesis for disrupting virulence genes in C. caviae, an agent of guinea pig inclusion conjunctivitis that was recently identified as a zoonotic agent in cases of severe community-acquired pneumonia. Using this approach, we generated C. caviae mutants deficient for the secreted effector proteins IncA and SinC. We demonstrate that C. caviae IncA plays a role in mediating fusion of the bacteria-containing vacuoles inhabited by C. caviae. Moreover, using a chicken embryo infection model, we provide first evidence for a role of SinC in C. caviae virulence in vivo.

The mechanism(s) by which Lactobacillus-dominated cervicovaginal microbiota provide a barrier to Chlamydia trachomatis infection remain(s) unknown. Here we evaluate the impact of different Lactobacillus spp. identified via culture-independent metataxonomic analysis of C. trachomatis-infected women on C. trachomatis infection in a three-dimensional (3D) cervical epithelium model. Lactobacillus spp. that specifically produce d(-) lactic acid were associated with long-term protection against C. trachomatis infection, consistent with reduced protection associated with Lactobacillus iners, which does not produce this isoform, and with decreased epithelial cell proliferation, consistent with the observed prolonged protective effect. Transcriptomic analysis revealed that epigenetic modifications involving histone deacetylase-controlled pathways are integral to the cross talk between host and microbiota. These results highlight a fundamental mechanism whereby the cervicovaginal microbiota modulates host functions to protect against C. trachomatis infection.IMPORTANCE The vaginal microbiota is believed to protect women against Chlamydia trachomatis, the etiologic agent of the most prevalent sexually transmitted infection (STI) in developed countries. The mechanism underlying this protection has remained elusive. Here, we reveal the comprehensive strategy by which the cervicovaginal microbiota modulates host functions to protect against chlamydial infection, thereby providing a novel conceptual mechanistic understanding. Major implications of this work are that (i) the impact of the vaginal microbiota on the epithelium should be considered in future studies of chlamydial infection and other STIs and (ii) a fundamental understanding of the cervicovaginal microbiota's role in protection against STIs may enable the development of novel microbiome-based therapeutic strategies to protect women from infection and improve vaginal and cervical health.

IntroductionChlamydia trachomatis (CT) is a common sexually transmitted pathogen with significant reproductive health implications. Data are mounting that the bacterial communities that reside within the vagina, collectively termed the vaginal microbiota, aid in defense against sexually transmitted infections. Disruptions in the vaginal microbiota, such as during episodes of bacterial vaginosis, may increase susceptibility to infection. Herein, we describe the clinical core protocol for a NIH NIAID Cooperative Research Center titled Eco-Pathogenomic of Chlamydial Reproductive Tract Infection. The primary goals of the project are to describe the interrelationships between the urogenital microenvironment, the properties of the pathogen and immunologic responses of the host in men and women, and their association with clinical outcomes of CT infection in women.MethodsMen and women with confirmed genital CT infections were recruited to a number of study protocols, including cross-sectional and longitudinal sub-studies. Participants completed a demographic and sexual health questionnaire and underwent a physical exam at baseline. In the longitudinal study arms, biologic samples were collected daily, weekly, and monthly to determine the relationships between the vaginal microbiota, prevalent CT infection, re-infection and treatment.DiscussionThe biological samples and the demographic and history information collected throughout this study will be used for various analyses evaluating genomics, metabolomics and host immune responses in the context of CT infection.

Chlamydiaceae are obligate intracellular bacterial pathogens for humans and animals. A recent study highlighted that a Chlamydiaceae intermediary between C. psittaci and C. abortus can infect hawks. Here, an isolate was obtained upon passage of cloacal and conjunctival sac material collected from a female hatch-year red-shouldered hawk (Buteo lineatus) in cultured cells. The diseased bird, one of 12 birds housed in a rehabilitation center, developed conjunctivitis and later died. Swabs from both sites tested positive for Chlamydia using the QuickVue Chlamydia test. The isolate, named RSHA, tested negative in qPCR assays specific for C. psittaci and C. abortus, respectively. Analysis of the 16S rRNA, 23S rRNA and whole genome sequences as well as MLST, ANIb and TETRA values reveal that C. psittaci and C. abortus are the closest relatives of RSHA. However, the overall results strongly suggest a phylogenetic intermediate position between these two species. Therefore, we propose the introduction of a new species designated Chlamydia buteonis with RSHAT as the type strain.

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Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

Vaginal metabolites are influenced by resident vaginal microbiota, may affect susceptibility to sexually transmitted infections and could impact local inflammation, symptoms, and risk of ascending infection. Chlamydia trachomatis (CT) is the most prevalent bacterial STI, and rates of co-infection with Mycoplasma genitalium (MG) in certain populations are high. We sought to compare the vaginal metabolome in CT+, CT+/MG+ and uninfected women.

Introduction Epidemiological studies have demonstrated that the vaginal microbiota can significantly impact the risk of acquiring sexually transmitted infections. The human vagina often contains Lactobacillus spp., which produce lactic acid and create an acidic environment (pH 3.5–4) thought to reduce vaginal STIs. Unlike high d-lactate producers, Lactobacillus spp. that produce low amounts or no d-lactate, while achieving low pH do not reduce Chlamydia trachomatis infectivity. Further, exposure to culture supernatants from d-lactate producing Lactobacillus spp. reduces epithelial cell proliferation. We tested if low proliferation affects infection. Methods A 3D model of A2EN cervical epithelial cells was exposed to lactic acid (D, L or D/L) at concentrations that produce pH 7, 5.5 and 4 or to several Lactobacillus spp. conditioned media (LCM) and infected with C. trachomatis serovar L2. Lysates from these A2EN cells were used to infect HeLa cells, and IFUs counted to determine infectivity. 2D A2EN cells were exposed to lactic acid, proliferation chemical inhibitors or LCM followed by infection with C. trachomatis L2. Proliferation and infectivity were evaluated by microscopy. Results At pH 4, d-lactate and LCMs from high d-lactate producing vaginal Lactobacillus spp. afforded maximal protection compared to l-lactate. Interestingly, high infectivity was observed with HCl at pH 4, indicating that pH alone is not responsible for this protection. Exposure to d-lactate or LCMs reduced cell proliferation. Chemical cell proliferation inhibitors dramatically reduced C. trachomatis infectivity. Conclusion These results suggest a differential role for vaginal Lactobacillus spp. in protecting against C. trachomatis infections and potentially other STIs. This protection is driven by the production of d-lactate, which acts on epithelial cells by inhibiting cell proliferation, which appears to be required for infection.

Introduction We present the following tri-factorial hypothesis: 1. All members of the Chlamydiaceae have evolved primarily as commensals of the digestive tract of their host(s) with fecal-oral transmission (FOT) as the principal route of dissemination to new hosts. In communities where fecal-oral transmission is reduced (e.g., via global sanitation), the occurrence of chlamydiae in the digestive tract of their host is reduced.2. Chlamydia trachomatis is a commensal microorganism of the human gastro-intestinal (GI) tract, and an opportunistic pathogen in the genital and respiratory tracts, and the conjunctiva. Under conditions of reduced FOT, direct contact (e.g., sexual) is the primary mode of transmission. 3. C. trachomatis is efficiently transmitted to the GI tract of new hosts via oral sex. The practice of oral sex has ‘reintroduced’ C. trachomatis to the human GI tract in communities where FOT was previously reduced. Methods Circumstantial, historical and recent evidence from humans and animals that support the hypothesis is reviewed. Imaging of mCherry expressing Chlamydia muridarum in the murine GI tract was obtained. Results and Conclusion: Tenets 1 and 2 imply a paradigm shift to reflect a revision of the status of C. trachomatis from that of a principal pathogen to that of a commensal organism that causes opportunistic infection at mucosal epithelia other than its preferred GI site. High frequency on/off switching of the expression of autotransported polymorphic membrane proteins, the unique properties of peptidoglycan and lipo-oligosaccharide, and observed extruded inclusions in the GI tract of C. muridarum-infected mice may facilitate chlamydial survival and colonisation of the GI tract.Tenet 3 implies that orally inoculated chlamydiae that survive in and colonise the GI tract, may reach the rectum and chronically, or episodically, infect the female genital tract eventually causing/contributing to tubal pathology and infertility. The global hypothesis therefore raises the provocative question: does oral sex cause or contribute to female infertility?

Based on recent, historical, and circumstantial evidence, we present a multifactorial hypothesis that has potential direct implications on the epidemiology and management of chlamydial infection and disease in humans. We propose that (1) like its veterinary relatives, the oculogenital pathogen Chlamydia trachomatis evolved as a commensal organism of the human gastrointestinal (GI) tract primarily transmissible via the fecal-oral route; (2) in the modern era, C. trachomatis causes "opportunistic" infection at non-GI sites under conditions driven by improved sanitation/hygiene and reduced fecal-oral transmission; and (3) the rise in the practice of oral sex is contributing to the increased prevalence of C. trachomatis in the human GI tract. Infectious organisms produced in the GI tract and reaching the rectum may then chronically contaminate and infect the female urogenital tract, thereby potentially contributing to the most serious sequelae of chlamydial infection in women: pelvic inflammatory disease, ectopic pregnancy, and tubal factor infertility.