Chlamydia trachomatis and Candida albicans are common inhabitants of the female genital tract. Candida albicans can impact the viability and pathogenesis of some bacteria. Previously, we investigated physical interactions between Ch. trachomatis elementary bodies (EBs) and Ca. albicans. This work indicated that EBs bind to Ca. albicans and become noninfectious by 24 h post-binding. Here, we continue our investigation of these interkingdom, polymicrobial interactions. Candida albicans adheres to bacteria or host surfaces via agglutinin-like sequence or heat shock 70 (Ssa) proteins. Chlamydia trachomatis EBs did not bind Ca. albicans Ssa2 deficient strains as efficiently as wild-type or complemented strains, indicating a role for this protein in chlamydial adherence to Candida. Additionally, Ca. albicans β-glucans inhibit chlamydial infection when exposure occurs during EB adsorption onto cervical cells. Laminarin, a β-glucan agonist of the C-type lectin receptor Dectin-1, inhibited chlamydial infection in both cervical epithelial cells and mice when exposure occurred prior to, during, or immediately following EB inoculation. Conversely, a Dectin-1 antagonist laminarin did not inhibit infection in vitro, suggesting that β-glucan inhibition of Ch. trachomatis requires C-type lectin receptor signaling. Overall, our data demonstrate that β-glucans from multiple species, including Ca. albicans, inhibit Chlamydia via stimulation of host-signaling pathways.

Introduction. Azelastine hydrochloride, a second-generation histamine H1 receptor (H1R) antagonist, exhibits anti-chlamydial effects against Chlamydia trachomatis (CT) in HeLa cells (genital infection model).Hypothesis/Gap Statement. Non-antibiotic pharmaceutical interactions with CT are an understudied field and the anti-chlamydial effects of azelastine are a potential interaction requiring further elucidation.Aim. To explore the underlying anti-chlamydial mechanisms of azelastine.Methodology. We assessed the specificity of azelastine for the chlamydial species and host cell type, the timing of azelastine application and whether the anti-chlamydial effects could be reproduced with different H1R-modulating compounds.Results. We observed similar anti-chlamydial azelastine effects for Chlamydia muridarum as well as for an ocular CT strain in human conjunctival epithelial cells (ocular infection model). Pre-incubating host cells with azelastine before infection mildly reduced chlamydial inclusion numbers and infectivity. Incubation of cells with azelastine initiated concomitantly with the chlamydial infection, or initiated several hours post-infection, reduced inclusion size, number and infectivity, and altered chlamydial morphology. These effects were strongest when azelastine was added shortly after or with the infection. Azelastine effects were not alleviated by increased concentrations of culture medium nutrients. Additionally, we did not observe anti-chlamydial effects when incubating cultures either with a different H1R antagonist or agonist, indicating that azelastine effects are probably H1R-independent.Conclusion. Accordingly, we conclude that azelastine anti-chlamydial effects are not restricted to a specific chlamydial species, strain or culture model, and are probably not mediated by H1R antagonism. Thus, it appears likely that off-target mechanisms of azelastine may explain our observations.

Our work builds upon the limited understanding of C. muridarum / N. gonorrhoeae coinfection. Previously, N. gonorrhoeae infection of mice with acute (actively replicating) vaginal C. muridarum infection was shown to increase recovery of viable vaginal N. gonorrhoeae and vaginal PMNs, with no effect on C. muridarum vaginal shedding (R. A. Vonck et al., Infect Immun 79:1566–1577, 2011).

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) cause most bacterial sexually transmitted infections (STIs) worldwide. Epidemiological studies have shown high percentages of co-infections with CT/NG and indicate that NG co-infection can reactivate CT shedding during persistent chlamydial infection. These data also suggest that biological interaction between the two bacteria may increase susceptibility or transmissibility. CT is an obligate intracellular bacterium with a developmental cycle that alternates between two forms: infectious elementary bodies (EBs) which invade the epithelium and non-infectious reticulate bodies (RBs) which divide and replicate inside the inclusion. Adverse environmental conditions can interrupt chlamydial development, with a consequent temporary halt in RB division, reduction in infectious EB production and formation of enlarged chlamydiae (aberrant bodies, ABs) - characterizing chlamydial persistence. When the stressor is removed, the chlamydial developmental cycle is restored, together with production of infectious EBs. The beta-lactam amoxicillin (AMX) induces chlamydial persistence, both in vitro and in mice. We investigated the impact of penicillinase-producing NG strain (PPNG) on AMX-persistent chlamydial infection utilizing our recently developed, contact-independent in vitro model of co-infection. We hypothesized that co-infection with PPNG could prevent and/or reverse AMX-induced chlamydial persistence. Our results showed that PPNG can ameliorate AMX-persistence in two chlamydial species, CT and C. muridarum (CM), providing novel evidence for a range of Chlamydia/NG interactions.

ABSTRACT Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) cause most bacterial sexually transmitted infections (STIs) worldwide. CT/NG co-infection is more common than expected due to chance, suggesting CT/NG interaction. However, CT/NG co-infection remains largely unstudied. Obligate intracellular CT has a characteristic biphasic developmental cycle consisting of two bacterial forms, infectious elementary bodies (EBs) and non-infectious, replicating reticulate bodies (RBs), which reside within host-derived, membrane-bound intracellular inclusions. Diverse stressors cause divergence from the normal chlamydial developmental cycle to an aberrant state called chlamydial persistence. Persistence can be induced by host-specific factors such as intracellular nutrient deprivation or cytokine exposure, and exogenous factors such as beta-lactam exposure, which disrupts RB to EB conversion. Persistent chlamydiae are atypical in appearance and, as such, are called aberrant bodies (ABs), but remain viable. The primary hallmark of persistence is reversibility of this temporary non-infectious state; upon removal of the stressor, persistent chlamydiae re-enter normal development, and production of infectious EBs resumes. The beta-lactam amoxicillin (AMX) has been shown to induce chlamydial persistence in a murine vaginal infection model, using the mouse pathogen C. muridarum (CM) to model human CT infection. This remains, to date, the sole experimentally tractable in vivo model of chlamydial persistence. Recently, we found that penicillinase-producing NG (PPNG) can alleviate AMX-induced CT and CM persistence in vitro. We hypothesized that PPNG vaginal co-infection would also alleviate AMX-induced CM persistence in mice. To evaluate this hypothesis, we modified the CM/AMX persistence mouse model, incorporating CM/PPNG co-infection. Contradicting our hypothesis, and recent in vitro findings, PPNG vaginal co-infection failed to alleviate AMX-induced CM persistence.

Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) are the most common bacterial sexually transmitted infections (STIs) worldwide. The primary site of infection for both bacteria is the epithelium of the endocervix in women and the urethra in men; both can also infect the rectum, pharynx and conjunctiva. Ct/Ng co-infections are more common than expected by chance, suggesting Ct/Ng interactions increase susceptibility and/or transmissibility. To date, studies have largely focused on each pathogen individually and models exploring co-infection are limited. We aimed to determine if Ng co-infection influences chlamydial infection and development and we hypothesized that Ng-infected cells are more susceptible to chlamydial infection than uninfected cells. To address this hypothesis, we established an in vitro model of Ct/Ng co-infection in cultured human cervical epithelial cells. Our data show that Ng co-infection elicits an anti-chlamydial effect by reducing chlamydial infection, inclusion size, and subsequent infectivity. Notably, the anti-chlamydial effect is dependent on Ng viability but not extracellular nutrient depletion or pH modulation. Though this finding is not consistent with our hypothesis, it provides evidence that interaction of these bacteria in vitro influences chlamydial infection and development. This Ct/Ng co-infection model, established in an epithelial cell line, will facilitate further exploration into the pathogenic interplay between Ct and Ng.

Water-filtered infrared A (wIRA) alone or in combination with visible light (VIS) exerts anti-chlamydial effects in vitro and in vivo in acute infection models. However, it has remained unclear whether reduced irradiation duration and irradiance would still maintain anti-chlamydial efficacy. Furthermore, efficacy of this non-chemical treatment option against persistent (chronic) chlamydial infections has not been investigated to date. To address this knowledge gap, we evaluated 1) irradiation durations of 5, 15 or 30 min in genital and ocular Chlamydia trachomatis acute infection models, 2) irradiances of 100, 150 or 200 mW/cm2 in the acute genital infection model and 3) anti-chlamydial activity of wIRA and VIS against C. trachomatis serovar B and E with amoxicillin (AMX)- or interferon γ (IFN-γ)-induced persistence. Reduction of irradiation duration reduced anti-chlamydial efficacy. Irradiances of 150 to 200 mW/cm2, but not 100 mW/cm2, induced anti-chlamydial effects. For persistent infections, wIRA and VIS irradiation showed robust anti-chlamydial activity independent of the infection status (persistent or recovering), persistence inducer (AMX or IFN-γ) or chlamydial strain (serovar B or E). This study clarifies the requirement of 30 min irradiation duration and 150 mW/cm2 irradiance to induce significant anti-chlamydial effects in vitro, supports the use of irradiation in the wIRA and VIS spectrum as a promising non-chemical treatment for chlamydial infections and provides important information for follow-up in vivo studies. Notably, wIRA and VIS exert anti-chlamydial effects on persistent chlamydiae which are known to be refractory to antibiotic treatment.

Introduction . Chlamydia trachomatis (Ct) is an obligate intracellular bacterium, causing a range of diseases in humans. Interactions between chlamydiae and antibiotics have been extensively studied in the past.Hypothesis/Gap statement: Chlamydial interactions with non-antibiotic drugs have received less attention and warrant further investigations. We hypothesized that selected cytokine inhibitors would alter Ct growth characteristics in HeLa cells.Aim. To investigate potential interactions between selected cytokine inhibitors and Ct development in vitro.Methodology. The CCR5 receptor antagonist maraviroc (Mara; clinically used as HIV treatment), the triterpenoid celastrol (Cel; used in traditional Chinese medicine) and the histamine H1 receptor antagonist azelastine (Az; clinically used to treat allergic rhinitis and conjunctivitis) were used in a genital in vitro model of Ct serovar E infecting human adenocarcinoma cells (HeLa).Results. Initial analyses revealed no cytotoxicity of Mara up to 20 µM, Cel up to 1 µM and Az up to 20 µM. Mara exposure (1, 5, 10 and 20 µM) elicited a reduction of chlamydial inclusion numbers, while 10 µM reduced chlamydial infectivity. Cel 1 µM, as well as 10 and 20 µM Az, reduced chlamydial inclusion size, number and infectivity. Morphological immunofluorescence and ultrastructural analysis indicated that exposure to 20 µM Az disrupted chlamydial inclusion structure. Immunofluorescence evaluation of Cel-incubated inclusions showed reduced inclusion sizes whilst Mara incubation had no effect on inclusion morphology. Recovery assays demonstrated incomplete recovery of chlamydial infectivity and formation of structures resembling typical chlamydial inclusions upon Az removal.Conclusion. These observations indicate that distinct mechanisms might be involved in potential interactions of the drugs evaluated herein and highlight the need for continued investigation of the interaction of commonly used drugs with Chlamydia and its host.

Microbial interactions represent an understudied facet of human health and disease. In this study, the interactions that occur between Chlamydia trachomatis and the opportunistic fungal pathogen, Candida albicans were investigated. Candida albicans is a common component of the oral and vaginal microbiota responsible for thrush and vaginal yeast infections. Normally, Candida exist in the body as yeast. However, disruptions to the microbiota create conditions that allow expanded growth of Candida, conversion to the hyphal form, and tissue invasion. Previous studies have shown that a myriad of outcomes can occur when Candida albicans interacts with pathogenic bacteria. To determine if C. trachomatis physically interacts with C. albicans, we incubated chlamydial elementary bodies (EB) in medium alone or with C. albicans yeast or hyphal forms for 1 h. Following incubation, the samples were formaldehydefixed and processed for immunofluorescence assays using anti-chlamydial MOMP or anti-chlamydial LPS antibodies. Replicate samples were replenished with culture medium and incubated at 35 degrees C for 0-120 h prior to fixation for immunofluorescence analysis or collection for EB infectivity assays. Data from this study indicates that both C. trachomatis serovar E and C. muridarum EB bind to C. albicans yeast and hyphal forms. This interaction was not blocked by pre-incubation of EB with the Candida cell wall components, mannan or beta-glucans, suggesting that EB interact with a Candida cell wall protein or other structure. Bound EB remained attached to C. albicans for a minimum of 5 days (120 h). Infectivity assays demonstrated that EB bound to C. albicans are infectious immediately following binding (0h). However, once bound to C. albicans, EB infectivity decreased at a faster rate than EB in medium alone. At 6h post binding, 40% of EB incubated in medium alone remained infectious compared to only 16% of EB bound to C. albicans. Likewise, pre-incubation of EB with laminarin, a soluble preparation of beta-glucan, alone or in combination with other fungal cell wall components significantly decreases chlamydial infectivity in HeLa cells. These data indicate that interactions between EB and C. albicans inhibit chlamydial infectivity, possibly by physically blocking EB interactions with host cell receptors.

To emphasize key gaps in knowledge impacting efforts to control single infection and co-infections with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), the most common bacterial sexually transmitted infections (STIs) worldwide. Clinical and epidemiological studies describe gaps in understanding about female rectal CT infection, screening effectiveness, pelvic inflammatory disease, and influence of the microbiome. For NG, gaps in knowledge include factors increasing incidence in men who have sex with men, correlations between treatment and antibiotic resistance, the role of pharyngeal infection, and microbiome influence. CT/NG co-infections are poorly understood, and adequate models to explore pathophysiological consequences of co-infection urgently needed. The sole existing CT/NG co-infection mouse model showed that CT/NG interactions in vivo modulate host response and NG load/shedding—encouraging further consideration of this model and potential alternatives. We stress key challenges in controlling these important STIs. Appropriate, quality-assured animal models are essential to improve understanding of the pathogenic interplay in CT/NG co-infections.

Purpose of Review Interactions between microorganisms can alter subsequent disease outcomes. Human papilloma virus (HPV) and Chlamydia trachomatis (CT) establish human genital co-infections, and CT infection is a co-factor for HPV-induced cervical cancer. This review focuses upon (i) data indicating that clinically significant interactions occur and (ii) proposed mechanisms underlying these outcomes. Recent Findings Epidemiological surveys indicate that (i) simultaneous HPV/CT genital co-infections are common; (ii) CT co-infection accelerates HPV-induced cytopathology; and (iii) HPV infection facilitates CT infection. Single-infection studies suggest specific molecular mechanisms by which co-infection alters clinical outcomes, including (i) HPV E6/E7 protein modification of host cell pathways enhances CT replication or immune evasion and (ii) CT-mediated host cell or neutrophil dysfunctions promote HPV-mediated neoplasia. Summary There are multiple avenues for future dissection of HPV/CT interactions. Moreover, the known and potential health consequences of co-infection highlight the need for improving current HPV vaccines and developing an effective CT vaccine.

Chlamydia trachomatis/HSV-2 vaginal co-infections are seen clinically, suggesting that these sexually transmitted pathogens may interact. We previously established an intravaginal Chlamydia muridarum/HSV-2 super-infection model and observed that chlamydial pre-infection protects mice from a subsequent lethal HSV-2 challenge. However, the mechanism of protection remains unknown. The type I interferon, IFN-β, binds to the type I interferon receptor (IFNR), elicits a host cellular antiviral response and inhibits HSV replication in vitro and in vivo. Previous studies have demonstrated that C. muridarum infection stimulates genital tract (GT) IFN-β production; therefore, we hypothesized that chlamydial pre-infection protects mice from HSV-2 challenge via the IFN-β/IFNR-induced antiviral response. To test this prediction, we quantified IFN-β levels in vaginal swab samples. Detection of IFN-β in C. muridarum singly infected, but not in mock-infected animals, prompted the use of the super-infection model in IFNR knockout (IFNR-/-) mice. We observed that C. muridarum pre-infection reduces HSV-2-induced mortality by 40% in wild-type mice and by 60% IFNR-/- mice. Severity of HSV-2 disease symptoms and viral shedding was also similarly reduced by C. muridarum pre-infection. These data indicate that, while chlamydial infection induces GT production of IFN-β, type I IFN-induced antiviral responses are likely not required for the observed protective effect.

Nuclear factor kappa B (NFκB) is an inflammatory transcription factor that plays an important role in the host immune response to infection. The potential for chlamydiae to activate NFκB has been an area of interest, however most work has focused on chlamydiae impacting human health. Given that inflammation characteristic of chlamydial infection may be associated with severe disease outcomes or contribute to poor overall fitness in farmed animals, we evaluated the ability of porcine chlamydiae to induce NFκB activation in vitro. C. pecorum infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host de novo protein synthesis. C. suis and C. trachomatis showed less capacity for NFκB activation compared to C. pecorum, suggesting a species-specific variation in NFκB activation. At 24 hpi, C. pecorum induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. C. pecorum-dependent secretion of interleukin 6 was also detected in the culture supernatant of infected cells at 24 hpi, and this effect, too, was unchanged by penicillin-induced chlamydial stress. Taken together, these results suggest that NFκB participates in the early inflammatory response to C. pecorum and that stressed chlamydiae can promote inflammation.

Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singly-infected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen, but more than 70% of patients fail to seek treatment due to the asymptomatic nature of these infections. Women suffer from numerous complications from chronic chlamydial infections, which include pelvic inflammatory disease and infertility. We previously demonstrated in culture that host cell nectin-1 knockdown significantly reduced chlamydial titers and inclusion size. Here, we sought to determine whether nectin-1 was required for chlamydial development in vivo by intravaginally infecting nectin-1-/- mice with Chlamydia muridarum and monitoring chlamydial shedding by chlamydial titer assay. We observed a significant reduction in chlamydial shedding in female nectin-1-/- mice compared to nectin-1+/+ control mice, an observation that was confirmed by PCR. Immunohistochemical staining in mouse cervical tissue confirmed that there are fewer chlamydial inclusions in Chlamydia-infected nectin-1-/- mice. Notably, anorectal chlamydial infections are becoming a substantial health burden, though little is known regarding the pathogenesis of these infections. We therefore established a novel male murine model of rectal chlamydial infection, which we used to determine whether nectin-1 is required for anorectal chlamydial infection in male mice. In contrast to the data from vaginal infection, no difference in rectal chlamydial shedding was observed when male nectin-1+/+ and nectin-1-/- mice were compared. Through the use of these two models, we have demonstrated that nectin-1 promotes chlamydial infection in the female genital tract but does not appear to contribute to rectal infection in male mice. These models could be used to further characterize tissue and sex related differences in chlamydial infection.

The Chlamydiaceae are widespread pathogens of both humans and animals. Chlamydia trachomatis infection causes blinding trachoma and reproductive complications in humans. Chlamydia pneumoniae causes human respiratory tract infections and atypical pneumonia. Chlamydia suis infection is associated with conjunctivitis, diarrhea, and failure to gain weight in domestic swine. Chlamydial infections in humans and domesticated animals are generally controlled by antibiotic treatment-particularly macrolides (usually azithromycin) and tetracyclines (tetracycline and doxycycline). Tetracycline-containing feed has also been used to limit infections and promote growth in livestock populations, although its use has decreased because of growing concerns about antimicrobial resistance development. Because Sandoz and Rockey published an elegant review of chlamydial anti-microbial resistance in 2010, we will review the following: (i) antibiotic resistance in C. suis, (ii) recent evidence for acquired resistance in human chlamydial infections, and (iii) recent non-genetic mechanisms of antibiotic resistance that may contribute to treatment failure.

Altered gut microbial diversity has been associated with several chronic disease states, including heart failure. Stimulation of the vagus nerve, which innervates the heart and abdominal organs, is proving to be an effective therapeutic in heart failure. We hypothesized that cervical vagus nerve stimulation (VNS) could alter fecal flora and prevent aberrations observed in fecal samples from heart failure animals. To determine whether microbial abundances were altered by pressure overload (PO), leading to heart failure and VNS therapy, a VNS pulse generator was implanted with a stimulus lead on either the left or right vagus nerve before creation of PO by aortic constriction. Animals received intermittent, open-loop stimulation or sham treatment, and their heart function was monitored by echocardiography. Left ventricular end-systolic and diastolic volumes, as well as cardiac output, were impaired in PO animals compared with baseline. VNS mitigated these effects. Metagenetic analysis was then performed using 16S rRNA sequencing to identify bacterial genera present in fecal samples. The abundance of 10 genera was significantly altered by PO, 8 of which were mitigated in animals receiving either left-or right-sided VNS. Metatranscriptomics analyses indicate that the abundance of genera that express genes associated with ATP-binding cassette transport and amino sugar/nitrogen metabolism was significantly changed following PO. These gut flora changes were not observed in PO animals subjected to VNS. These data suggest that VNS prevents aberrant gut flora following PO, which could contribute to its beneficial effects in heart failure patients.