Computational prediction of crucial genes involved in gonorrhea infection and neoplastic cell transformation: A multiomics approach.

Neisseria gonorrheae, the causative agent of genitourinary infections, has been associated with asymptomatic or recurrent infections and has the potential to form biofilms and induce inflammation and cell transformation. Herein, we aimed to use computational analysis to predict novel associations between chronic inflammation caused by gonorrhea infection and neoplastic transformation. Prioritization and gene enrichment strategies based on virulence and resistance genes utilizing essential genes from the DEG and PANTHER databases, respectively, were performed. Using the STRING database, protein‒protein interaction networks were constructed with 55 nodes of bacterial proteins and 72 nodes of proteins involved in the host immune response. MCODE and cytoHubba were used to identify 12 bacterial hub proteins (murA, murB, murC, murD, murE, purN, purL, thyA, uvrB, kdsB, lpxC, and ftsH) and 19 human hub proteins, of which TNF, STAT3 and AKT1 had high significance. The PPI networks are based on the connectivity degree (K), betweenness centrality (BC), and closeness centrality (CC) values. Hub genes are vital for cell survival and growth, and their significance as potential drug targets is discussed. This computational study provides a comprehensive understanding of inflammation and carcinogenesis pathways that are activated during gonorrhea infection.

Prevalence of Four Sexually Transmitted Infection Pathogens Detected in Urine and Genital Tract Secretion in Hangzhou, China.

BACKGROUND: Sexually transmitted infections (STIs) increase gradually and have become a public health problem in the world. UU, CT, NG, and MG are four common STI pathogens. Our retrospective study analyzed the clinical situation and the laboratory data of patients infected with the four pathogens. The prevalence of the four pathogens, detected in urine and genital tract secretion, was studied in Hangzhou, China. METHODS: A total of 3,168 male and female patients were randomly selected from February 2023 to February 2024. Urine and genital secretions were collected, and four STI pathogens were controlled for detection. Data were collected from the hospital's electronic medical records, and SPSS 25.0 software was used to perform a statistical analysis. RESULTS: Among 3,168 patients, a total of 1,527 were detected as positive, and the positive rate was 48.20%. The age of patients ranged from 13 - 98 years, with an average age of 45.6. The total of patients consisted of 2,191 males and 977 females, which had a significant difference (p < 0.05). Specimens were mainly collected from the Department of Dermatovenerology, Urological Surgery, Obstetrics and Gynecology, and so on. The positive rate was statistically different between male and female patients (p < 0.05). Single infection performed a main role and accounted for 79.57% of all of the positive patients. In the ≤ 20 age group, the positive rate was the highest and was as high as 77.65%. In detail, single infection caused by UU dominated, especially in the 21 - 30 age group. Double infection caused by UU and CT and triple infection caused by UU, CT, and NG were the majority, both especially in the 21 - 30 age group. There were significant differences in the positive rates in the different age groups and in the four pathogens (p < 0.05). Quadruple infection was very rare and had only been detected in one patient. CONCLUSIONS: The prevalence of the four pathogens in Hangzhou was different from other regions. More male than female patients, more single than multiple infections, and more single and multiple infections occurring in young people were the features in Hangzhou. The study would provide reference for prevention, diagnosis, and treatment of STI.

The effect of daily usage of Listerine Cool Mint mouthwash on the oropharyngeal microbiome: a substudy of the PReGo trial.

Introduction. ListerineÒ is a bactericidal mouthwash widely used to prevent oral health problems such as dental plaque and gingivitis. However, whether it promotes or undermines a healthy oral microbiome is unclear.Hypothesis/Gap Statement. We hypothesized that the daily use of Listerine Cool Mint would have a significant impact on the oropharyngeal microbiome.Aim. We aimed to assess if daily usage of Listerine Cool Mint influenced the composition of the pharyngeal microbiome.Methodology. The current microbiome substudy is part of the Preventing Resistance in Gonorrhoea trial. This was a double-blind single-centre, crossover, randomized controlled trial of antibacterial versus placebo mouthwash to reduce the incidence of gonorrhoea/chlamydia/syphilis in men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP). Fifty-nine MSM taking HIV PrEP were enrolled. In this crossover trial, participants received 3 months of daily Listerine followed by 3 months of placebo mouthwash or vice versa. Oropharyngeal swabs were taken at baseline and after 3 months use of each mouthwash. DNA was extracted for shotgun metagenomic sequencing (Illumina Inc.). Non-host reads were taxonomically classified with MiniKraken and Bracken. The alpha and beta diversity indices were compared between baseline and after each mouthwash use. Differentially abundant bacterial taxa were identified using ANOVA-like differential expression analysis.Results. Streptococcus was the most abundant genus in most samples (n = 103, 61.7 %) with a median relative abundance of 31.5% (IQR 20.6-44.8), followed by Prevotella [13.5% (IQR 4.8-22.6)] and Veillonella [10.0% (IQR 4.0-16.8)]. Compared to baseline, the composition of the oral microbiome at the genus level (beta diversity) was significantly different after 3 months of Listerine (P = 0.006, pseudo-F = 2.29) or placebo (P = 0.003, pseudo-F = 2.49, permutational multivariate analysis of variance) use. Fusobacterium nucleatum and Streptococcus anginosus were significantly more abundant after Listerine use compared to baseline.Conclusion. Listerine use was associated with an increased abundance of common oral opportunistic bacteria previously reported to be enriched in periodontal diseases, oesophageal and colorectal cancer, and systemic diseases. These findings suggest that the regular use of Listerine mouthwash should be carefully considered.

Antimicrobial resistance determinants in the oropharyngeal microbiome of 'men having sex with men' attending an sexually transmitted infection clinic.

BACKGROUND: 'Men having sex with men' (MSM) represent a key population with a significant prevalence of pharyngeal Neisseria gonorrhoeae (NG) infections and a high rate of antimicrobial resistance genes in the pharyngeal microbiome. As NG can acquire antibiotic resistance genes from other commensal oropharyngeal bacteria, monitoring the prevalence of these resistance determinants is critical to curtail the spread of NG-resistant strains. PURPOSE AND RESEARCH DESIGN: Here, we assessed the distribution of five resistance genes (pen (A), mtr (R), gyr (A), par (C), msr (D)) in the oropharynx of 164 MSM, attending an Outpatient clinic for STI screening. RESULTS: The most frequently detected resistance gene was msr (D) (88.4%), followed by gyr (A) (67.1%). The distribution of resistance genes was not influenced by pharyngeal gonorrhea nor by the HIV status, whereas a younger age was associated with mtr (R) presence (p = .008). Subjects using mouthwash exhibited significantly lower levels of mtr (R) (p = .0005). Smoking habit was associated with a higher prevalence of par (C) (p = .02). A noteworthy association was observed between the presence of msr (D) gene and the use of antibiotics (p = .014). CONCLUSIONS: Our findings reveal an enrichment of antimicrobial resistance genes in the oropharynx of MSM. These insights could aid in the development of screening programs and antimicrobial stewardship initiatives targeting populations at heightened risk of pharyngeal gonorrhea.

Characterization of Neisseria gonorrhoeae colonization of macrophages under distinct polarization states and nutrients environment.

Neisseria gonorrhoeae (Ng) is a uniquely adapted human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. Ng has developed numerous mechanisms to avoid and actively suppress innate and adaptive immune responses. Ng successfully colonizes and establishes topologically distinct colonies in human macrophages and avoids phagocytic killing. During colonization, Ng manipulates the actin cytoskeleton to invade and create an intracellular niche supportive of bacterial replication. The cellular reservoir(s) supporting bacterial replication and persistence in gonorrhea infections are poorly defined. The manner in which gonococci colonize macrophages points to this innate immune phagocyte as a strong candidate for a cellular niche during natural infection. Here we investigate whether nutrients availability and immunological polarization alter macrophage colonization by Ng. Differentiation of macrophages in pro-inflammatory (M1-like) and tolerogenic (M2-like) phenotypes prior to infection reveals that Ng can invade macrophages in all activation states, albeit with lower efficiency in M1-like macrophages. These results suggest that during natural infection, bacteria could invade and grow within macrophages regardless of the nutrients availability and the macrophage immune activation status.

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.

Gonococcal OMV-delivered PorB induces epithelial cell mitophagy.

The bacterial pathogen Neisseria gonorrhoeae is able to invade epithelial cells and survive intracellularly. During this process, it secretes outer membrane vesicles (OMVs), however, the mechanistic details for interactions between gonococcal OMVs and epithelial cells and their impact on intracellular survival are currently not established. Here, we show that gonococcal OMVs induce epithelial cell mitophagy to reduce mitochondrial secretion of reactive oxygen species (ROS) and enhance intracellular survival. We demonstrate that OMVs deliver PorB to mitochondria to dissipate the mitochondrial membrane potential, resulting in mitophagy induction through a conventional PINK1 and OPTN/NDP52 mechanism. Furthermore, PorB directly recruits the E3 ubiquitin ligase RNF213, which decorates PorB lysine residue 171 with K63-linked polyubiquitin to induce mitophagy in a p62-dependent manner. These results demonstrate a mechanism in which polyubiquitination of a bacterial virulence factor that targets mitochondria directs mitophagy processes to this organelle to prevent its secretion of deleterious ROS.

Epidemiological profile and genetic resistance of Neisseria gonorrhoeae infection in women in a poor region of São Paulo, Brazil.

BACKGROUND AND AIM: Gonorrhea is a bacterial infection in the urogenital tract, transmitted by sexual or perinatal contact, caused by Neisseria gonorrhoeae, a gram-negative diplococcus. The present study evaluates the frequency of N. gonorrhoeae in women treated at Hospital Wladimir Arruda in poor area of São Paulo and also verifies the presence of genetic resistance against three antimicrobials of different classes: Tetracycline, Azithromycin and Ciprofloxacin. METHODS: This is an observational and descriptive study with a quantitative approach. Samples were collected at Hospital Escola Wladimir Arruda. The volunteers are women from 16 to 65 years of age. Sociodemographic, gynecological, sexual and health data are collected through a questionnaire, their symptoms/clinical manifestation were requested by the medical records, and then the participant is referred for collection of samples of cervical vaginal smear. The samples were screened for N. gonorrhoeae (dcmH gene) and tested for resistance genes to Tetracycline, Azithromycin and Ciprofloxacin through PCR. RESULTS: In the total of 127 samples analyzed by Real-Time PCR, 23 were positive and correspond to a general prevalence of a gonococcal infection in the studied population of 17% (CI:95%), and the participants were married (43.4%), had active sexual life (56.5%) and did not use any type of condom during sexual intercourse (52.1%). The resistance to the tetM ribosomal gene was found in 14 samples, prevalence of 60% (CI= 95%). CONCLUSIONS: We have described a concerning frequency of N. gonorrhoeae infection in females attended in an outcare patient. Also, most of the strains detected presented resistance to one or more antimicrobials.

Polyphenylene carboxymethylene (PPCM), the active component of the topical contraceptive Yaso-GEL, exhibits potent antimicrobial activity against Neisseria gonorrhoeae in preclinical studies.

INTRODUCTION: Polyphenylene carboxymethylene (PPCM) is a condensation polymer that has both contraceptive and antimicrobial activity against several sexually transmitted viruses including HIV, herpes simplex virus, Ebola virus and SARS-CoV-2 in preclinical studies. PPCM, both as an active pharmaceutical ingredient (API) and in a vaginal gel formulation (Yaso-GEL), has an excellent safety profile. Here, we evaluated the efficacy of PPCM against Neisseria gonorrhoeae in vitro and in a gonorrhoea mouse model. METHODS: The minimal inhibitory concentration (MIC) of PPCM was determined against 11 N. gonorrhoeae strains by agar dilution and a microtitre plate-based method. In vivo efficacy was tested in a murine model of N. gonorrhoeae genital tract infection by applying Yaso-GEL, PPCM incorporated in 2.7% hydroxyethylcellulose (HEC), or the HEC vehicle vaginally prior to challenge with N. gonorrhoeae. Vaginal swabs were quantitatively cultured over 5 days to assess efficacy. RESULTS: PPCM MIC against N. gonorrhoeae ranged between 5-100 µg/mL (agar dilution) and 50-200 µg/mL (microtitre plate method). PPCM/HEC gel applied vaginally prior to bacterial challenge resulted in a concentration-dependent inhibition of infection. Yaso-GEL containing 4% PPCM prevented infection in 100% of mice. Incubation of N. gonorrhoeae with PPCM increased membrane permeability, suggesting PPCM directly compromises N. gonorrhoeae viability, which may be a mechanism by which PPCM inhibits N. gonorrhoeae infection. CONCLUSIONS: Yaso-GEL containing the API PPCM showed significant activity against N. gonorrhoeae in vitro and in vivo in a female mouse model. These data support further development of Yaso-GEL as an inexpensive, non-hormonal and non-systemic product with both contraceptive and antimicrobial activity against gonorrhea and other common sexually transmitted infections (STIs). Such multipurpose prevention technology products are needed by women in all economic, social and cultural circumstances to prevent unintended pregnancy and STIs.

Functional characterization of the gonococcal polyphosphate pseudo-capsule.

Neisseria gonorrhoeae is an exclusively human pathogen able to evade the host immune system through multiple mechanisms. Gonococci accumulate a large portion of phosphate moieties as polyphosphate (polyP) on the exterior of the cell. Although its polyanionic nature has suggested that it may form a protective shield on the cell surface, its role remains controversial. Taking advantage of a recombinant His-tagged polyP-binding protein, the presence of a polyP pseudo-capsule in gonococcus was demonstrated. Interestingly, the polyP pseudo-capsule was found to be present in specific strains only. To investigate its putative role in host immune evasion mechanisms, such as resistance to serum bactericidal activity, antimicrobial peptides and phagocytosis, the enzymes involved in polyP metabolism were genetically deleted, generating mutants with altered polyP external content. The mutants with lower polyP content on their surface compared to the wild-type strains, became sensitive to complement-mediated killing in presence of normal human serum. Conversely, naturally serum sensitive strains that did not display a significant polyP pseudo-capsule became resistant to complement in the presence of exogenous polyP. The presence of polyP pseudo-capsule was also critical in the protection from antibacterial activity of cationic antimicrobial peptide, such as cathelicidin LL-37. Results showed that the minimum bactericidal concentration was lower in strains lacking polyP than in those harboring the pseudo-capsule. Data referring to phagocytic killing resistance, assessed by using neutrophil-like cells, showed a significant decrease in viability of mutants lacking polyP on their cell surface in comparison to the wild-type strain. The addition of exogenous polyP overturned the killing phenotype of sensitive strains suggesting that gonococcus could exploit environmental polyP to survive to complement-mediated, cathelicidin and intracellular killing. Taken together, data presented here indicate an essential role of the polyP pseudo-capsule in the gonococcal pathogenesis, opening new perspective on gonococcal biology and more effective treatments.

Stealthy microbes: How Neisseria gonorrhoeae hijacks bulwarked iron during infection.

Transition metals are essential for metalloprotein function among all domains of life. Humans utilize nutritional immunity to limit bacterial infections, employing metalloproteins such as hemoglobin, transferrin, and lactoferrin across a variety of physiological niches to sequester iron from invading bacteria. Consequently, some bacteria have evolved mechanisms to pirate the sequestered metals and thrive in these metal-restricted environments. Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, causes devastating disease worldwide and is an example of a bacterium capable of circumventing human nutritional immunity. Via production of specific outer-membrane metallotransporters, N. gonorrhoeae is capable of extracting iron directly from human innate immunity metalloproteins. This review focuses on the function and expression of each metalloprotein at gonococcal infection sites, as well as what is known about how the gonococcus accesses bound iron.

Pelviperitonitis gonocócica. A propósito de un caso / Gonococcal pelviperitonitis. A case report

Neisseria gonorrhoeae se considera uno de los agentes causales más importantes de la enfermedad pélvica inflamatoria (EPI) produciendo síntomas leves e inespecíficos, lo cual la convierte en un desafío diagnóstico. Se presenta un caso de una pelviperitonitis gonocócica aguda con dolor difuso, distensión abdominal, fiebre. El único hallazgo destacable fue un líquido peritoneal y endocervical purulento con reactantes de fase aguda elevados. El cultivo del líquido endocervical y peritoneal fue positivo para N. gonorrhoeae. En mujeres sexualmente activas y con sospecha de EPI es importante descartar enfermedades de transmisión sexual.

Neisseria gonorrhoeae is considered one of the most important causal agents of pelvic inflammatory disease, producing mild and nonspecific symptoms, which makes it a diagnostic challenge. A case of acute gonococcal pelviperitonitis with abdominal distension, fever and diffuse pain is presented. The only noteworthy finding was purulent peritoneal and endocervical fluid with elevated acute-phase reactants. Endocervical and peritoneal fluid culture showed infection with N. gonorrhoeae. Therefore, in sexually active women with suspected PID, it is important to rule out sexually transmitted diseases.

Atypical ocular Chlamydia trachomatis infections in two patients attending in a second-level hospital: a case report.

AIM: To report two atypical inclusion conjunctivitis cases due to Chlamydia trachomatis in young adults. METHOD: Transcription mediated amplification for C. trachomatis was performed using Aptima Combo 2 Assay (Hologic, Spain). RESULTS: The first patient was managed as an orbital disorder because he had unilateral location, and ptosis was observed. Orbital nuclear magnetic resonance revealed normal results, and conjunctival biopsy did not indicate significant results. For the second patient, thyroid eye disease was suspected, but the orbital nuclear magnetic resonance revealed normal results. Conjunctival exudate samples were collected and sent to the Microbiology Laboratory where C. trachomatis was confirmed. Both patients demonstrated a great improvement with oral azithromycin 1 g. CONCLUSION: Inclusion conjunctivitis could present as unspecified unilateral or bilateral chronic conjunctivitis. Thus, suspecting it would be important in order to prevent spread and wasting diagnostic resources.

Variable Expression of Opa Proteins by Neisseria gonorrhoeae Influences Bacterial Association and Phagocytic Killing by Human Neutrophils.

Neisseria gonorrhoeae infection is characterized by local and abundant recruitment of neutrophils. Despite neutrophils' antimicrobial activities, viable N. gonorrhoeae is recovered from infected individuals, leading to the question of how N. gonorrhoeae survives neutrophil attack. One feature impacting N. gonorrhoeae-neutrophil interactions is the phase-variable opacity-associated (Opa) proteins. Most Opa proteins engage human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to facilitate bacterial binding and invasion. Neutrophils express two transmembrane CEACAMs, CEACAM1 and the granulocyte-specific CEACAM3. While N. gonorrhoeae isolated from infected individuals is frequently Opa+, expression of OpaD from strain FA1090, which interacts with CEACAMs 1 and 3, is associated with reduced N. gonorrhoeae survival after exposure to human neutrophils. In this study, we hypothesized that the receptor-binding capability of individual Opa proteins impacts bacterial survival in the presence of neutrophils. To test this hypothesis, we introduced opa genes that are constitutively expressed into a derivative of strain FA1090 with all 11 opa genes deleted. The engineered genes encode Opa proteins that bind CEACAM1 and -3, CEACAM1 but not CEACAM3, or neither CEACAM1 nor -3. N. gonorrhoeae expressing CEACAM3-binding Opa proteins survived significantly less well than bacteria expressing other Opa proteins when exposed to primary human neutrophils. The CEACAM3-binding N. gonorrhoeae had significantly greater association with and internalization by neutrophils. However, once internalized, bacteria were similarly killed inside neutrophils, regardless of Opa expression. Furthermore, Opa expression did not significantly impact neutrophil granule mobilization. Our findings indicate that the extent to which Opa proteins mediate nonopsonic binding is the predominant determinant of bacterial survival from neutrophils. IMPORTANCE Neisseria gonorrhoeae, the cause of gonorrhea, is an urgent-threat pathogen due to increasing numbers of infections and increased antibiotic resistance. Many surface components of N. gonorrhoeae are phase variable, including the Opa protein family of adhesins and invasins. While Opa protein expression is selected for in vivo, bacteria expressing some Opa proteins are readily killed by neutrophils, which are recruited to sites of infection. The reason for this discrepancy has remained unresolved. Our work shows that Opa-dependent differences in bacterial survival after exposure to primary human neutrophils correlates with Opa-dependent bacterial binding and phagocytosis. These findings underscore how the ability of N. gonorrhoeae to change Opa expression through phase variation contributes to bacterial resistance to neutrophil clearance.

Self-Inhibitory Peptides Targeting the Neisseria gonorrhoeae MtrCDE Efflux Pump Increase Antibiotic Susceptibility.

Neisseria gonorrhoeae is an increasing public health threat due to its rapidly rising incidence and antibiotic resistance. There are an estimated 106 million cases per year worldwide, there is no vaccine available to prevent infection, and N. gonorrhoeae strains that are resistant to all antibiotics routinely used to treat the infection have emerged. In many strains, antibiotic resistance is mediated by overexpression of the MtrCDE efflux pump, which enables the bacteria to transport toxic antibiotics out of the cell. Genetic mutations that inactivate MtrCDE have previously been shown to render resistant strains susceptible to certain antibiotics. Here, we show that peptides rationally designed to target and disrupt the activity of each of the three protein components of MtrCDE were able to increase the susceptibility of N. gonorrhoeae strains to antibiotics in a dose-dependent manner and with no toxicity to human cells. Cotreatment of bacteria with subinhibitory concentrations of the peptide led to 2- to 64-fold increases in susceptibility to erythromycin, azithromycin, ciprofloxacin, and/or ceftriaxone in N. gonorrhoeae strains FA1090, WHO K, WHO P, and WHO X. The cotreatment experiments with peptides P-MtrC1 and P-MtrE1 resulted in increased susceptibilities of WHO P and WHO X to azithromycin, ciprofloxacin, and ceftriaxone that were of the same magnitude seen in MtrCDE mutants. P-MtrE1 was able to change the azithromycin resistance profile of WHO P from resistant to susceptible. Data presented here demonstrate that these peptides may be developed for use as a dual treatment with existing antibiotics to treat multidrug-resistant gonococcal infections.

Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages.

Dynamic reorganization of the actin cytoskeleton dictates plasma membrane morphogenesis and is frequently subverted by bacterial pathogens for entry and colonization of host cells. The human-adapted bacterial pathogen Neisseria gonorrhoeae can colonize and replicate when cultured with human macrophages, however the basic understanding of how this process occurs is incomplete. N. gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhea and tissue resident macrophages are present in the urogenital mucosa, which is colonized by the bacteria. We uncovered that when gonococci colonize macrophages, they can establish an intracellular or a cell surface-associated niche that support bacterial replication independently. Unlike other intracellular bacterial pathogens, which enter host cells as single bacterium, establish an intracellular niche and then replicate, gonococci invade human macrophages as a colony. Individual diplococci are rapidly phagocytosed by macrophages and transported to lysosomes for degradation. However, we found that surface-associated gonococcal colonies of various sizes can invade macrophages by triggering actin skeleton rearrangement resulting in plasma membrane invaginations that slowly engulf the colony. The resulting intracellular membrane-bound organelle supports robust bacterial replication. The gonococci-occupied vacuoles evaded fusion with the endosomal compartment and were enveloped by a network of actin filaments. We demonstrate that gonococcal colonies invade macrophages via a process mechanistically distinct from phagocytosis that is regulated by the actin nucleating factor FMNL3 and is independent of the Arp2/3 complex. Our work provides insights into the gonococci life-cycle in association with human macrophages and defines key host determinants for macrophage colonization.

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin.

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

Trends in asymptomatic STI among HIV-positive MSM and lessons for systematic screening.

The burden of STIs is particularly high in HIV-infected MSM patients. A recent increase in STIs prevalence has been noticed in the US and western European countries. We aim to assess trends in asymptomatic STIs following the publication of recommendations for STIs screening, i.e. Chlamydia (CT) and gonorrhea (NG). Seventeen centers located in the Paris area participated in the study. All asymptomatic HIV-infected MSM patients attending a follow up consultation were proposed to participated in the study. Asymptomatic patients were included over 2 periods: period 1 from April to December 2015 and period 2 from September to December 2017. Etiologic diagnosis of STIs including hepatitis B, C, syphilis, was performed using a serological test, including a non-treponemal titer with a confirmatory treponemal assay for syphilis. CT and NG were screened using a nucleic acid amplification test (NAATs) on 3 anatomical sites, i.e. urine, rectal and pharyngeal. Overall, 781 patients were included: 490 and 291 in periods 1 and 2 respectively. Asymptomatic CT, NG, and syphilis were diagnosed in 7.5%, 4.8% and, 4.2% respectively. The rate of patients having a multisite asymptomatic infection was 10.2% and 21.1% for CT and NG respectively. The most frequently involved anatomical sites for CT and NG asymptomatic infections were anorectal (66.1% and 55.2% respectively) and pharyngeal (47.4% and 60.5% respectively). CT and NG asymptomatic infection increased by 1.3- and 2-fold respectively between the two periods while syphilis decreased by 3 folds. Our results encourage to reconsider multisite screening for CT and NG in asymptomatic HIV positive MSM as the yield of screening urinary samples only might be low. Despite the more systematic STI screening of asymptomatic HIV positive MSM the prevalence of STI is increasing in MSM in France. Therefore, this strategy has not led to alter CT and NG transmission. The decrease of syphilis might involve self-medication by doxycycline, and the intensification of syphilis screening.

Incidence and risk factors of C. trachomatis and N. gonorrhoeae among young women from the Western Cape, South Africa: The EVRI study.

OBJECTIVE: Young women in South Africa are highly affected by sexually transmitted infections (STI), like C. trachomatis (CT) and N. gonorrhoeae (NG). We aimed to estimate the incidence of CT and NG, and its determinants, among young women from the Western Cape, South Africa, participating in an HPV vaccine trial (the EVRI study). METHODS: HIV-negative women aged 16-24 years were enrolled between October 2012 and July 2013. At enrolment and month 6 participants were screened for CT and NG (Anyplex CT/NG real-time detection method). A questionnaire on demographic and sexual history characteristics was completed at enrolment and month 7. Treatment for CT and/or NG was offered to infected participants. Incidence rates (IR) of CT and NG were estimated. Determinants of incident CT and NG infections were assessed using Poisson regression. RESULTS: 365 women were tested for CT and/or NG at least twice. Prevalence of CT and NG at baseline was 33.7% and 10.4%, respectively. Prevalence of co-infection with CT and NG was 7.1%. During 113.3 person-years (py), 48 incident CT infections were diagnosed (IR = 42.4 per 100 py, 95% confidence interval (CI) 31.9-56.2). Twenty-nine incident NG were diagnosed during 139.3 py (IR = 20.8 per 100 py, 95%CI 14.5-29.9). Prevalent CT infection at baseline was associated with incident CT (adjusted incidence rate ratio (aIRR) 5.8, 95%CI 3.0-11.23. More than three lifetime sex partners increased the risk for incident NG (3-4 partners aIRR = 7.3, 95%CI 2.1-26.0; ≥5 partners aIRR = 4.3, 95%CI 1.1-17.5). CONCLUSIONS: The IR of bacterial STIs among young women in the Western Cape is very high. Besides being previously infected and a higher lifetime number of sex partners, no other risk factors were found for CT and NG, suggesting that the majority of these women were at risk. This indicates the need for intensified prevention of STIs as well as screening and treatment programs to increase sexual health in this region.

Developing and validating a risk algorithm to diagnose Neisseria gonorrhoeae and Chlamydia trachomatis in symptomatic Rwandan women.

BACKGROUND: Algorithms that bridge the gap between syndromic sexually transmitted infection (STI) management and treatment based in realistic diagnostic options and local epidemiology are urgently needed across Africa. Our objective was to develop and validate a risk algorithm for Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) diagnosis among symptomatic Rwandan women and to compare risk algorithm performance to the current Rwandan National Criteria for NG/CT diagnosis. METHODS: The risk algorithm was derived in a cohort (n = 468) comprised of symptomatic women in Kigali who sought free screening and treatment for sexually transmitted infections and vaginal dysbioses at our research site. We used logistic regression to derive a risk algorithm for prediction of NG/CT infection. Ten-fold cross-validation internally validated the risk algorithm. We applied the risk algorithm to an external validation cohort also comprised of symptomatic Rwandan women (n = 305). Measures of calibration, discrimination, and screening performance of our risk algorithm compared to the current Rwandan National Criteria are presented. RESULTS: The prevalence of NG/CT in the derivation cohort was 34.6%. The risk algorithm included: age < =25, having no/primary education, not having full-time employment, using condoms only sometimes, not reporting genital itching, testing negative for vaginal candida, and testing positive for bacterial vaginosis. The model was well calibrated (Hosmer-Lemeshow p = 0.831). Higher risk scores were significantly associated with increased prevalence of NG/CT infection (p < 0.001). Using a cut-point score of > = 5, the risk algorithm had a sensitivity of 81%, specificity of 54%, positive predictive value (PPV) of 48%, and negative predictive value (NPV) of 85%. Internal and external validation showed similar predictive ability of the risk algorithm, which outperformed the Rwandan National Criteria. Applying the Rwandan National Criteria cutoff of > = 2 (the current cutoff) to our derivation cohort had a sensitivity of 26%, specificity of 89%, PPV of 55%, and NPV of 69%. CONCLUSIONS: These data support use of a locally relevant, evidence-based risk algorithm to significantly reduce the number of untreated NG/CT cases in symptomatic Rwandan women. The risk algorithm could be a cost-effective way to target treatment to those at highest NG/CT risk. The algorithm could also aid in sexually transmitted infection risk and prevention communication between providers and clients.