Refinement and optimisation of Neisseria gonorrhoeae NHBA and MetQ vaccine candidates.

Neisseria gonorrhoeae has a significant impact on reproductive health with an estimated 82 million new cases of infection per year worldwide. Due to the ongoing emergence of multidrug-resistant N. gonorrhoeae strains, the high number of asymptomatic cases, and the risk of disease sequelae, the development of a gonococcal vaccine is urgently needed. We have previously described two potential gonococcal vaccine antigens, cNHBA (C-terminal fragment of the Neisseria Heparin Binding Antigen) and MetQ (methionine-binding protein). This study aimed to optimise these antigens for improved immune responses and to facilitate vaccine production, by investigating cNHBA fusions with the full-length MetQ protein or N-terminal and C-terminal MetQ fragments (Met1 and Met2, respectively) adjuvanted with aluminium hydroxide. The cNHBA and MetQ fragments and fusion antigens were all immunogenic in mice, generating a predominantly IgG1 response. Antibodies mediated bacterial killing via both serum bactericidal activity (SBA) and opsonophagocytic activity (OPA), and reduced adherence to cervical and urethral epithelial cells. Among the antigen fusions tested, MetQ-cNHBA and cNHBA-Met2 generated the highest SBA, OPA and adherence blocking titres and are proposed as promising optimised antigens for N. gonorrhoeae vaccine development.

Immune responses to Neisseria gonorrhoeae and implications for vaccine development.

Neisseria gonorrheoae is the causative agent of gonorrhea, a sexually transmitted infection responsible for a major burden of disease with a high global prevalence. Protective immunity to infection is often not observed in humans, possible due to high variability of key antigens, induction of blocking antibodies, or a large number of infections being relatively superficial and not inducing a strong immune response. N. gonorrhoeae is a strictly human pathogen, however, studies using mouse models provide useful insights into the immune response to gonorrhea. In mice, N. gonorrhoea appears to avoid a protective Th1 response by inducing a less protective Th17 response. In mouse models, candidate vaccines which provoke a Th1 response can accelerate the clearance of gonococcus from the mouse female genital tract. Human studies indicate that natural infection often induces a limited immune response, with modest antibody responses, which may correlate with the clinical severity of gonococcal disease. Studies of cytokine responses to gonococcal infection in humans provide conflicting evidence as to whether infection induces an IL-17 response. However, there is evidence for limited induction of protective immunity from a study of female sex workers in Kenya. A controlled human infection model (CHIM) has been used to examine the immune response to gonococcal infection in male volunteers, but has not to date demonstrated protection against re-infection. Correlates of protection for gonorrhea are lacking, which has hampered the progress towards developing a successful vaccine. However, the finding that the Neisseria meningitidis serogroup B vaccines, elicit cross-protection against gonorrhea has invigorated the gonococcal vaccine field. More studies of infection in humans, either natural infection or CHIM studies, are needed to understand better gonococcal protective immunity.

Disseminated Gonococcal Infection Caused by a Multidrug-Resistant Pathogen Presenting with Hemorrhagic Manifestation: A Case Report.

The incidence of disseminated gonococcal infection (DGI) is rising in some parts of the world, but there is paucity of data on its true incidence from sub-Saharan Africa. DGI has varied manifestations in different population group. We report a case of a 30-year-old sexually active woman presenting with hemorrhagic symptoms 2 weeks after a surgery on account of diagnosis of uterine fibroid made at a peripheral hospital. A multidrug-resistant Neisseria gonorrhoeae was isolated from the wound on her surgical site and blood sample. She was managed with intravenous meropenem, pressure dressing, and blood products, with the patient making a full recovery after a week. This case is presented because it is a rare one. Moreover, there is the need to revive the awareness of clinicians on the existence of multidrug-resistant gonococcus in our environment. We herein report a case of DGI from Nigeria.

Penicillin-Binding Protein Occupancy Dataset for 18 ß-Lactams and 4 ß-Lactamase Inhibitors in Neisseria gonorrhoeae.

The lack of effective first-line antibiotic treatments against Neisseria gonorrhoeae, and the worldwide dissemination of resistant strains, are the main drivers of a worsening global health crisis. ß-lactam antibiotics have been the backbone of therapeutic armamentarium against gonococci. However, we are lacking critical insights to design rationally optimized therapies. In the present work, we generated the first PBP-binding data set on 18 currently available and clinically relevant ß-lactams and 4 ß-lactamase inhibitors in two N. gonorrhoeae ATCC type collection strains, 19424 and 49226 (PBP2 type XXII and A39T change in mtrR). PBP binding (IC50) was determined via the Bocillin FL binding assay in isolated membrane preparations. Three clusters of differential PBP IC50s were identified and were mostly consistent across both strains, but with quantitative differences. Carbapenems were coselective for PBP2 and PBP3 (0.01 to 0.03 mg/L). Third- and fourth-generation cephalosporins cefixime, cefotaxime, ceftazidime, cefepime, and ceftriaxone showed the lowest IC50 values for PBP2 (0.01 mg/L), whereas cefoxitin, ceftaroline, and ceftolozane required higher concentrations (0.04 to >2 mg/L). Aztreonam was selective for PBP2 in both strains (0.03 to 0.07 mg/L); amdinocillin bound this PBP at higher concentrations (1.33 to 2.94 mg/L). Penicillins specifically targeted PBP2 in strain ATCC 19424 (0.02 to 0.19 mg/L) and showed limited inhibition in strain ATCC 49226 (0.01 to >2 mg/L). Preferential PBP2 binding was observed by ß-lactam-based ß-lactamase inhibitors sulbactam and tazobactam (1.07 to 6.02 mg/L); meanwhile, diazabicyclooctane inhibitors relebactam and avibactam were selective for PBP3 (1.27 to 5.40 mg/L). This data set will set the bar for future studies that will help the rational use and translational development of antibiotics against multidrug-resistant (MDR) N. gonorrhoeae. IMPORTANCE The manuscript represents the first N. gonorrhoeae PBP-binding data set for 22 chemically different drugs in two type strains with different genetic background. We have identified three clusters of drugs according to their PBP binding IC50s and highlighted the binding differences across the two strains studied. With the currently available genomic information and the PBP-binding data, we have been able to correlate the target attainment differences and the mutations that affect the drug uptake with the MIC changes. The results of the current work will allow us to develop molecular tools of great practical use for the study and the design of new rationally designed therapies capable of combating the growing MDR gonococci threat.

Thiostrepton, a resurging drug inhibiting the stringent response to counteract antibiotic-resistance and expression of virulence determinants in Neisseria gonorrhoeae.

Due to the increased resistance to all available antibiotics and the lack of vaccines, Neisseria gonorrhoeae (the gonococcus) poses an urgent threat. Although the mechanisms of virulence and antibiotic resistance have been largely investigated in this bacterium, very few studies have addressed the stringent response (SR) that in pathogenic bacteria controls the expression of genes involved in host-pathogen interaction and tolerance and persistence toward antibiotics. In this study, the results of the transcriptome analysis of a clinical isolate of N. gonorrhoeae, after induction of the SR by serine hydroxamate, provided us with an accurate list of genes that are transcriptionally modulated during the SR. The list includes genes associated with metabolism, cellular machine functions, host-pathogen interaction, genome plasticity, and antibiotic tolerance and persistence. Moreover, we found that the artificial induction of the SR in N. gonorrhoeae by serine hydroxamate is prevented by thiostrepton, a thiopeptide antibiotic that is known to interact with ribosomal protein L11, thereby inhibiting functions of EF-Tu and EF-G, and binding of pppGpp synthase I (RelA) to ribosome upon entry of uncharged tRNA. We found that N. gonorrhoeae is highly sensitive to thiostrepton under in vitro conditions, and that thiostrepton, in contrast to other antibiotics, does not induce tolerance or persistence. Finally, we observed that thiostrepton attenuated the expression of key genes involved in the host-pathogen interaction. These properties make thiostrepton a good drug candidate for dampening bacterial virulence and preventing antibiotic tolerance and persistence. The ongoing challenge is to increase the bioavailability of thiostrepton through the use of chemistry and nanotechnology.

Insight into Prevention of Neisseria Gonorrhoeae: A Short Review.

Neisseria gonorrhoeae (gonococcus) and Neisseria meningitidis (meningococcus) are important global pathogens which cause the sexually transmitted diseases gonorrhea and meningitis, respectively, as well as sepsis. We prepared a review according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA), with the aims of (a) evaluating the data on the MenB vaccination as protection against sexually transmitted infections by N. gonorrhoeae and (b) to briefly comment on the data of ongoing studies of new vaccines. We evaluated existing evidence on the effect of 4CMenB, a multi-component vaccine, on invasive diseases caused by different meningococcal serogroups and on gonorrhea. Non-B meningococcal serogroups showed that the 4CMenB vaccine could potentially offer some level of protection against non-B meningococcal serogroups and N. gonorrhoeae. The assessment of the potential protection conferred by 4CMenB is further challenged by the fact that further studies are still needed to fully understand natural immune responses against gonococcal infections. A further limitation could be the potential differences between the protection mechanisms against N. gonorrhoeae, which causes local infections, and the protection mechanisms against N. meningitidis, which causes systemic infections.

Gonococcal arthritis: case series of 58 hospital cases.

OBJECTIVES: Extra-genital manifestations of gonococcal infection are rare (0.5-3%). Among them, gonococcal arthritis (GA) is the most frequent, accounting for 30-90% of disseminated infections. Our study aimed to describe all hospital cases of GA in Reunion Island, a French overseas territory. METHODS: We conducted a retrospective, multicentric, observational study of all cases of certain, probable or possible GA from 2008 to 2020. RESULTS: We identified 58 cases of GA, mostly certain cases (n = 48). Sex ratio was balanced, but men were older than women (51 vs 27 years, p < 0.001). A total of 41% had travelled abroad during the previous 3 months, mostly in Madagascar or South-East Asia. The most frequently infected joint was the knee, followed by ankle, wrist and fingers or carpal joints. Only 16% of cases had genital symptoms, but 50% had another extra-genital manifestation, mainly skin lesions (40%). Positivity rate of joint puncture was 91%, with a purulent liquid. Only 58% had a positive culture, and 33% had only a positive PCR. There was no 3GC-resistant strain. In comparison with gonococcal infection without arthritis, patients were older and had fewer genital but more extra-genital symptoms. On discharge 60% had persistent articular symptoms. GA represented 18% of all hospitalised septic arthritis cases with microbial identification in 2019. CONCLUSIONS: GA is rare but it is important to make an early diagnosis and treat promptly, as joint destruction may be important, leading to persistent symptoms after discharge. PCR use in joint puncture is useful in cases with negative culture.

Meningococcal Detoxified Outer Membrane Vesicle Vaccines Enhance Gonococcal Clearance in a Murine Infection Model.

BACKGROUND: Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea. METHODS: We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets. RESULTS: Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB. CONCLUSIONS: Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine.

Gonococcal bacterial load in PrEP users with Mycoplasma genitalium coinfection.

OBJECTIVES: Gonococcal infections with a higher bacterial load may pose a higher risk of transmission. We assessed the association between gonococcal bacterial load and coinfection with Mycoplasma genitalium. METHODS: From September 2015 until May 2018, 200 men and transgender women who have sex with men participated in an HIV pre-exposure prophylaxis (PrEP) demonstration trial in Antwerp, Belgium. They underwent 3-monthly 3-site (anus, urine, and pharynx) molecular testing for N. gonorrhoeae and C. trachomatis and M. genitalium, irrespective of symptoms. Gonococcal bacterial load was determined on remnant DNA extracts using an in-house quantitative PCR. Results were expressed as log10 transformed copies/mL and analyzed with a linear regression model. RESULTS: Gonococcal bacterial load could be determined for 82 (80.4%) of 102 anal, 17 (73.9%) of 23 urine, and 64 (90.1%) of 71 pharyngeal samples. M. genitalium was detected in five of these anal, two urine, and two pharyngeal samples and C. trachomatis was detected in 16 anal, one urine, and two pharyngeal samples. Gonococcal bacterial load was significantly higher in the presence of M. genitalium (difference 0.92 log copies/mL, 95% CI 0.16-1.67). CONCLUSIONS: Gonococcal bacterial load was higher with M. genitalium coinfection. M. genitalium may thus be a cofactor in gonococcal transmission.

[Infectious endocarditis due to Neisseria gonorrhoeae]. / Endocarditis infecciosa por Neisseria gonorroheae.

We report a case of infectious endocarditis due to Neisseria gonorrhoeae in a 38-year-old male patient with no cardiovascular risk factors or past medical history who presented with prolonged febrile illness, asthenia and weight loss. The blood cultures were positive for gonococcus. He received antibiotic treatment with ceftriaxone for 29 days. The patient developed severe aortic regurgitation and underwent surgical aortic valve replacement with a bileaflet mechanical prosthesis, with favorable outcome.

Se presenta un caso de endocarditis infecciosa por Neisseria gonorrhoeae, en un paciente masculino de 38 años, sin factores de riesgo cardiovascular ni otros antecedentes previos. La sospecha diagnóstica comienza por síndrome febril prolongado, astenia y pérdida de peso, confirmada con rescate de gonococo en los hemocultivos. Cumplió tratamiento antibiótico con ceftriaxona por 29 días. Evoluciona con insuficiencia aórtica grave por lo cual se realiza cirugía de reemplazo valvular por prótesis mecánica bidisco exitosa, con una evolución favorable.

Endocarditis infecciosa por Neisseria gonorroheae / Infectious endocarditis due to Neisseria gonorrhoeae

Resumen Se presenta un caso de endocarditis infecciosa por Neisseria gonorrhoeae, en un paciente masculino de 38 años, sin factores de riesgo cardiovascular ni otros antecedentes previos. La sospecha diagnóstica comienza por síndrome febril prolongado, astenia y pérdida de peso, confirmada con rescate de gonococo en los hemocultivos. Cumplió tratamiento antibiótico con ceftriaxona por 29 días. Evoluciona con insu ficiencia aórtica grave por lo cual se realiza cirugía de reemplazo valvular por prótesis mecánica bidisco exitosa, con una evolución favorable.

Abstract We report a case of infectious endocarditis due to Neisseria gonorrhoeae in a 38-year-old male patient with no cardiovascular risk factors or past medical history who presented with prolonged febrile illness, asthenia and weight loss. The blood cultures were positive for gonococcus. He received antibiotic treatment with ceftriaxone for 29 days. The patient developed severe aortic regurgitation and underwent surgical aortic valve replacement with a bileaflet mechanical prosthesis, with favorable outcome.

Neisseria gonorrhoeae: un patógeno díscolo: conceptos microbiológicos, resistencia a antimicrobianos y su vigilancia epidemiológica en Chile / Neisseria gonorrhoeae: a wayward pathogen: microbiological concepts, antimicrobial resistance and its epidemiological surveillance in Chile

Resumen Neisseria gonorrhoeae es un diplococo gramnegativo, no móvil, esporulado, aerobio o anaerobio facultativo, catalasa y oxidasa positivas. Las infecciones de transmisión sexual causadas por este microorganismo son un problema de salud pública definido como tal desde el siglo XIX, representando una gran amenaza para la salud humana debido a la su alta prevalencia y multirresistencia a antimicrobianos. En las últimas décadas han aumentado los reportes de cepas resistentes a penicilina, fluoroquinolonas, sulfonamidas, tetraciclina, macrólidos, y más recientemente a cefalosporinas y azitromicina. Tal panorama ha generado preocupación a nivel mundial, debido al aumento de casos de gonorrea asociados a cepas multirresistentes. En Chile se desarrolló desde el 2010 hasta el 2018 el Programa de Vigilancia de N. gonorrhoeae a nivel nacional con el objeto de caracterizar esta infección en las regiones y registrar la resistencia a los antimicrobianos. Esta revisión presenta un análisis sistemático bibliográfico, actualizado, de los principales aspectos de este microorganismo, su respuesta a antimicrobianos, y entrega pautas de diagnóstico y tratamiento, a la espera de avanzar en la comprensión del mecanismo molecular y las interacciones metabólicas e inmunológicas que determinan la infección, con miras a diseñar una vacuna efectiva.

Abstract Neisseria gonorrhoeae is a nonmotile, sporulated, aerobic or facultative anaerobic gram-negative diplococcus, catalase and oxidase positive. Sexually transmitted infections caused by this microorganism were established as public health problem since the 19th century, representing a great threat to human health due to its high prevalence and multi-resistance to antimicrobials. In recent decades, reports of strains resistant to penicillin, fluoroquinolones, sulfonamides, tetracycline, macrolides, and more recently to cephalosporins and azithromycin have increased. Such a panorama has generated concern worldwide, due to the increase in cases of gonorrhea associated with multi-resistant strains. In Chile, from 2010 to 2018, the National Surveillance Program for N. gonorrhoeae was developed in order to characterize this infection in the regions and record antimicrobial resistance. This review presents an updated, systematic bibliographic analysis of the main aspects of this microorganism, its response to antimicrobials, and provides diagnostic and treatment guidelines, while waiting to advance in the understanding of the molecular mechanism and the metabolic and immunological interactions that determine infection, with a view to designing an effective vaccine.

Biochemical characterization and redesign of the coenzyme specificity of a novel monofunctional NAD+-dependent homoserine dehydrogenase from the human pathogen Neisseria gonorrhoeae.

Gonorrhoea, caused by Neisseria gonorrhoeae, is a major global public health concern. Homoserine dehydrogenase (HSD), a key enzyme in the aspartate pathway, is a promising metabolic target against pathogenic infections. In this study, a monofunctional HSD from N. gonorrhoeae (NgHSD) was overexpressed in Escherichia coli and purified to >95% homogeneity for biochemical characterization. Unlike the classic dimeric structure, the purified recombinant NgHSD exists as a tetramer in solution. We determined the enzymatic activity of recombinant NgHSD for l-homoserine oxidation, which revealed that this enzyme was NAD+ dependent, with an approximate 479-fold (kcat/Km) preference for NAD+ over NADP+, and that optimal activity for l-homoserine oxidation occurred at pH 10.5 and 40 °C. At 800 mM, neither NaCl nor KCl increased the activity of NgHSD, in contrast to the behavior of several reported NAD+-independent homologs. Moreover, threonine did not markedly inhibit the oxidation activity of NgHSD. To gain insight into the cofactor specificity, site-directed mutagenesis was used to alter coenzyme specificity. The double mutant L45R/S46R, showing the highest affinity for NADP+, caused a shift in coenzyme preference from NAD+ to NADP+ by a factor of ~974, with a catalytic efficiency comparable with naturally occurring NAD+-independent homologs. Collectively, our results should allow the exploration of drugs targeting NgHSD to treat gonococcal infections and contribute to the prediction of the coenzyme specificity of novel HSDs.

Multidrug-Resistant Bacteria in the Community: An Update.

Multidrug-resistant bacteria are among the most important current threats to public health. Typically, they are associated with nosocomial infections. However, some have become prevalent causes of community-acquired infections, such as Neisseria gonorrhoeae, Shigella, Salmonella, and Streptococcus pneumoniae. The community spread of multidrug-resistant bacteria is also a crucial development. An important global threat on the horizon is represented by production of carbapenemases by community-acquired hypervirulent Klebsiella pneumoniae. Such strains have already been found in Asia, Europe, and North America. Prevention of further community spread of multidrug-resistant bacteria is of the utmost importance, and will require a multidisciplinary approach involving all stakeholders.

Azithromycin resistant gonococci: a literature review.

OBJECTIVE: Gonorrhea is the second most common sexually transmitted bacterial infection (STI) next to Chlamydia. Untreated cases could results in major complications like pelvic inflammatory disease (PID), ectopic pregnancy, infertility, miscarriage, fetal death and congenital infections. Gonorrhea has been treated with antibiotics for more than eight decades. However, the emergence and spread of antimicrobial resistance (AMR) in gonococcus seriously compromises the management of the disease. The aim of this review was to describe the current developments in the field of azithromycin resistant gonococci. METHODS: Literatures published in English in the last 10 years were retrieved from PubMed, SCOPUS, Google scholar, Cochrane library and the Google databases using relevant searching terms. RESULTS: Gonococcus is capable of using a number of strategies to confer resistance as the bacterium has an extraordinary capacity to alter its genome. So far the accumulated data on the field showed that the world is heading towards a pandemic of extensively drug-resistant (XDR) gonococcus which is now seems to be evolving into a true "superbug". Hence, in the near future gonorrhea may become untreatable on the international basis unless new drugs become available. An antibiotic resistance in gonococcus has been noted beginning in 1940s against sulfonamides. Since then, resistance has rapidly emerged to penicillins, tetracyclines, macrolides, fluoroquinolones, and cephalosporins. Currently, in most nations, the injectable extended-spectrum cephalosporin (ESC), i.e. ceftriaxone based therapy is the only remaining option for gonorrhea. Based on the WHO and the US-CDC recommendations, countries are increasingly using a combination of cephalosporin and azithromycin for the treatment of gonorrhoea. Azithromycin revolutionized gonoccocal therapy as it shortened treatment time by more than half from 7 to 14 days and improved patient compliance due to high tissue levels and long half-life. However, constantly emerging reports from different parts of the globe showed that N. gonorrhoeae is developing significant level of resistance against azithromycin, and so far more than 33% level of resistance was reported. Two strategies have been commonly implicated in gonococcal resistance against azithromycin: over expression of an efflux pump (due to mutations at mtrR coding region) and decreased antimicrobial affinity (due to mutations in genes encoding the 23S ribosomal subunit). CONCLUSIONS: With no alternative antimicrobial treatment options for gonorrhoea and only a few new drugs in the development pipeline, it is necessary to monitor drug resistance and optimize treatment regimens regularly. Moreover, investigations for novel drugs should be wired.

The Neisseria gonorrhoeae Vaccine Candidate NHBA Elicits Antibodies That Are Bactericidal, Opsonophagocytic and That Reduce Gonococcal Adherence to Epithelial Cells.

Due to the continuing emergence of multidrug resistant strains of Neisseria gonorrhoeae there is an urgent need for the development of a gonococcal vaccine. We evaluated the gonococcal Neisseria heparin binding antigen (NHBA) as a potential vaccine candidate, in terms of its sequence conservation and expression in a range of N. gonorrhoeae strains, as well as its immunogenicity and the functional activity of antibodies raised to either the full length NHBA or a C-terminal fragment of NHBA (NHBA-c). The gene encoding NHBA is highly conserved and expressed in all N. gonorrhoeae strains investigated. Recombinant NHBA is immunogenic, and mice immunized with either NHBA or NHBA-c adjuvanted with either Freund's or aluminium hydroxide (alum) generated a humoral immune response, with predominantly IgG1 antibodies. Antibodies generated by both NHBA and NHBA-c antigens promoted complement activation and mediated bacterial killing via both serum bactericidal activity and opsonophagocytic activity, with slightly higher titers seen for the NHBA-c antigen. Anti-NHBA was also able to block the functional activity of NHBA by reducing binding to heparin and adherence to cervical and urethral epithelial cells. These data suggest that the gonococcal NHBA is a promising vaccine antigen to include in a vaccine to control N. gonorrhoeae.

Role of the Gonococcal Neisserial Heparin Binding Antigen in Microcolony Formation, and Serum Resistance and Adherence to Epithelial Cells.

The sexually transmitted infection gonorrhoea is on the rise worldwide and an increased understanding of the mechanisms of colonization and pathogenesis of Neisseria gonorrhoeae is required to aid development of new treatment and prevention strategies. In the current study, we investigate the neisserial heparin-binding antigen (NHBA) of N. gonorrhoeae and confirm its role in binding to several glycans, including heparin, and identify interactions of NHBA with both gonococcal and host cells. Furthermore, we report that a gonococcal nhba mutant displays decreased cell aggregation and microcolony formation, as well as reduced survival in human serum and reduced adherence to human cervical and urethral epithelial cells, relative to the wild-type strain. These data indicate that the gonococcal NHBA contributes to several aspects of the colonization and survival of N. gonorrhoeae and may be a target for new antimicrobial or vaccines.

Two Distinct L-Lactate Dehydrogenases Play a Role in the Survival of Neisseria gonorrhoeae in Cervical Epithelial Cells.

L-lactate is an abundant metabolite in a number of niches in host organisms and represents an important carbon source for bacterial pathogens such as Neisseria gonorrhoeae. In this study, we describe an alternative, iron-sulfur cluster-containing L-lactate dehydrogenase (LutACB), that is distinct from the flavoprotein L-lactate dehydrogenase (LldD). Expression of lutACB was found to be positively regulated by iron, whereas lldD was more highly expressed under conditions of iron-limitation. The functional role of LutACB and LldD was reflected in in vitro studies of growth and in the survival of N gonorrhoeae in primary cervical epithelial cells.

Where have all the susceptible gonococci gone? A historical review of changes in MIC distribution over the past 75 years.

BACKGROUND: Does the emergence of antimicrobial resistance in Neisseria gonorrhoeae include the erasure of highly susceptible strains or does it merely involve a stretching of the MIC distribution? If it was the former this would be important to know as it would increase the probability that the loss of susceptibility is irreversible. METHODS: We conducted a historical analysis based on a literature review of changes of N. gonorrhoeae MIC distribution over the past 75 years for 3 antimicrobials (benzylpenicillin, ceftriaxone and azithromycin) in five countries (Denmark, Japan, South Africa, the United Kingdom and the United States). RESULTS: Changes in MIC distribution were most marked for benzylpenicillin and showed evidence of a right shifting of MIC distribution that was associated with a reduction/elimination of susceptible strains in all countries. In the case of ceftriaxone and azithromycin, where only more recent data was available, right shifting was also found in all countries but the extent of right shifting varied and the evidence for the elimination of susceptible strains was more mixed. CONCLUSIONS: The finding of right shifting of MIC distribution combined with reduction/elimination of susceptible strains is of concern since it suggests that this shifting may not be reversible. Since excess antimicrobial consumption is likely to be responsible for this right shifting, this insight provides additional impetus to promote antimicrobial stewardship.

High prevalence of TEM-135 expression from the Asian plasmid in penicillinase-producing Neisseria gonorrhoeae from Hangzhou, China.

Penicillinase-producing Neisseria gonorrhoeae (PPNG) expressing the TEM ß-lactamase variant TEM-135 are a global public-health concern as this variant requires only a single amino acid substitution to develop into an extended-spectrum ß-lactamase. The aim of this study was to investigate the epidemiology of blaTEM-135 in 505 N. gonorrhoeae isolates from Hangzhou, China, during the periods 2011-2012 and 2015-2017. Investigation by nitrocefin test and mismatch amplification PCR showed that 41.0% (207/505) of the isolates were PPNG, of which 37.2% (77/207) contained the blaTEM-135 gene. Further PCR-based plasmid typing showed that blaTEM-135 was predominantly expressed from the Asian plasmid (94%). PPNG isolates consisted of three major clusters, namely Asian plasmid/blaTEM-135 (34.8%), Asian plasmid/blaTEM-1 (32.4%) and African plasmid/blaTEM-1 (28.0%), which showed significant differences in penicillin minimum inhibitory concentrations (MICs) determined by the agar dilution method. Representative isolates were investigated by quantitative real-time PCR (plasmid copy number and blaTEM gene expression), western blot analysis (TEM levels and TEM stability) and in vivo ß-lactamase activity assays to elucidate the cause of the observed differences in penicillin MIC. Overall, isolates of the Asian plasmid/blaTEM-135 cluster showed the highest ß-lactamase activity, which was explained by higher blaTEM gene expression (Asian versus African plasmid) and higher TEM stability (TEM-135 versus TEM-1). In conclusion, the blaTEM-135 gene is commonly present on the Asian plasmid in PPNG isolates from Hangzhou. The PPNG isolate cluster containing the Asian plasmid and blaTEM-135 showed the highest penicillin MICs, which might explain its abundance in the Hangzhou population.