RESUMO
There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.
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There is an unmet need for developing drugs for the treatment of gonorrhea, due to rapidly evolving resistance of Neisseria gonorrhoeae against antimicrobial drugs used for empiric therapy, an increase in globally reported multidrug resistant cases, and the limited available therapeutic options. Furthermore, few drugs are under development. Development of antimicrobials is hampered by challenges in clinical trial design, limitations of available diagnostics, changes in and varying standards of care, lack of robust animal models, and clinically relevant pharmacodynamic targets. On April 23, 2021, the U.S. Food and Drug Administration; Centers for Disease Control and Prevention; and National Institute of Allergy and Infectious Diseases, National Institutes of Health co-sponsored a workshop with stakeholders from academia, industry, and regulatory agencies to discuss the challenges and strategies, including potential collaborations and incentives, to facilitate the development of drugs for the treatment of gonorrhea. This article provides a summary of the workshop.
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The bacterial pathogen Neisseria gonorrhoeae is an urgent global health problem due to increasing numbers of infections, coupled with rampant antibiotic resistance. Vaccines against gonorrhea are being prioritized to combat drug-resistant N. gonorrhoeae. Meningococcal serogroup B vaccines such as four-component meningococcal B vaccine (4CMenB) are predicted by epidemiology studies to cross-protect individuals from natural infection with N. gonorrhoeae and elicit antibodies that cross-react with N. gonorrhoeae. Evaluation of vaccine candidates for gonorrhea requires a suite of assays for predicting efficacy in vitro and in animal models of infection, including the role of antibodies elicited by immunization. Here, we present the development and optimization of assays to evaluate antibody functionality after immunization of mice: antibody binding to intact N. gonorrhoeae, serum bactericidal activity, and opsonophagocytic killing activity using primary human neutrophils [polymorphonuclear leukocytes (PMNs)]. These assays were developed with purified antibodies against N. gonorrhoeae and used to evaluate serum from mice that were vaccinated with 4CMenB or given alum as a negative control. Results from these assays will help prioritize gonorrhea vaccine candidates for advanced preclinical to early clinical studies and will contribute to identifying correlates and mechanisms of immune protection against N. gonorrhoeae.
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Gonorreia , Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis Sorogrupo B , Neisseria meningitidis , Humanos , Camundongos , Animais , Neisseria gonorrhoeae , Gonorreia/microbiologia , Infecções Meningocócicas/microbiologia , Vacinas Bacterianas , Anticorpos , Vacinas Combinadas , Anticorpos Antibacterianos , Antígenos de BactériasRESUMO
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.
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Gonorreia , Infecções Sexualmente Transmissíveis , Feminino , Humanos , Animais , Camundongos , Neisseria gonorrhoeae , Antibacterianos/uso terapêutico , Anticoncepcionais/uso terapêutico , Ágar/uso terapêutico , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Polímeros/farmacologia , Polímeros/uso terapêutico , Testes de Sensibilidade MicrobianaRESUMO
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.
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Gonorreia , Vacinas Meningocócicas , Neisseria meningitidis , Animais , Anticorpos Antibacterianos , Antígenos de Bactérias , Proteínas da Membrana Bacteriana Externa , Vacinas Bacterianas , Feminino , Gonorreia/prevenção & controle , Imunoglobulina G , Camundongos , Neisseria gonorrhoeaeRESUMO
Drug-resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance and limit the development of new resistances. Here, we describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6-fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2-oxa-8-azaspiro[4.5]decan-3-yl)methanol], which was developed to target both ATP-binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae, including drug-resistant strains. A phosphate pro-drug, JSF-2659, was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram-positive organisms, and JSF-2659 was shown in a deep tissue model of vancomycin-resistant Staphylococcus aureus (VRSA) and a model of Clostridioides difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel preclinical drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.
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Gonorreia , Staphylococcus aureus Resistente à Meticilina , Pró-Fármacos , Trifosfato de Adenosina , Animais , Antibacterianos/química , Antibacterianos/farmacologia , DNA Girase/genética , Farmacorresistência Bacteriana , Feminino , Gonorreia/tratamento farmacológico , Metanol/farmacologia , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae , Fosfatos/farmacologia , Pró-Fármacos/farmacologia , Inibidores da Topoisomerase II/farmacologiaRESUMO
The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.
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Gonorreia , Neisseria gonorrhoeae , Substituição de Aminoácidos , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Feminino , Gentamicinas/farmacologia , Gentamicinas/uso terapêutico , Gonorreia/tratamento farmacológico , Camundongos , Testes de Sensibilidade Microbiana , Fator G para Elongação de PeptídeosRESUMO
There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted with Ng OMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized several Ng surface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognized Ng PilQ and several proteins of similar apparent molecular weight, but MtrE was only recognized by mouse serum. Pooled sera from 4CMenB-immunized mice showed a 4-fold increase in serum bactericidal50 titers against the challenge strain; in contrast, no significant difference in bactericidal activity was detected when sera from 4CMenB-immunized and unimmunized subjects were compared. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against gonorrhea, and implicate several Ng surface antigens as potentially protective targets. Additionally, this study further defines the usefulness of murine infection model as a relevant experimental system for gonorrhea vaccine development.
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Proteção Cruzada/imunologia , Vacinas Meningocócicas/farmacologia , Neisseria gonorrhoeae/imunologia , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Estudos de Casos e Controles , Reações Cruzadas/imunologia , Feminino , Gonorreia/imunologia , Humanos , Soros Imunes/imunologia , Imunização/métodos , Masculino , Infecções Meningocócicas/microbiologia , Vacinas Meningocócicas/imunologia , Vacinas Meningocócicas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Estudos Retrospectivos , Sorogrupo , Vacinação/métodosRESUMO
Gonorrhea, an obligate human infection, is on the rise worldwide and gonococcal strains resistant to many antibiotics are emerging. Appropriate antimicrobial treatment and prevention, including effective vaccines, are urgently needed. To guide investigation, an experimental model of genital tract infection has been developed in female mice to study mechanisms by which Neisseria gonorrhoeae evades host-derived antimicrobial factors and to identify protective and immunosuppressive pathways. Refinements of the animal model have also improved its use as a surrogate host of human infection and accelerated the testing of novel therapeutic and prophylactic compounds against gonococcal infection. Reviewed herein are the (a) history of antibiotic usage and resistance against gonorrhea and the consequences of resistance mechanisms that may increase gonococcal fitness and therefore the potential for spread, (b) use of gonococcal infection in the animal model system to study mechanisms of pathogenesis and host defenses, and
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Antibacterianos/uso terapêutico , Vacinas Bacterianas/imunologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Gonorreia/tratamento farmacológico , Gonorreia/prevenção & controle , Neisseria gonorrhoeae/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Vacinas Bacterianas/isolamento & purificação , Descoberta de Drogas , Feminino , Gonorreia/microbiologia , Camundongos , Neisseria gonorrhoeae/imunologiaRESUMO
Murine models of Neisseria gonorrhoeae lower reproductive tract infection are valuable systems for studying N. gonorrhoeae adaptation to the female host and immune responses to infection. These models have also accelerated preclinical testing of candidate therapeutic and prophylactic products against gonorrhea. However, because N. gonorrhoeae infection is restricted to the murine cervicovaginal region, there is a need for an in vivo system for translational work on N. gonorrhoeae pelvic inflammatory disease (PID). Here we discuss the need for well-characterized preclinical upper reproductive tract infection models for developing candidate products against N. gonorrhoeae PID, and report a refinement of the gonorrhea mouse model that supports sustained upper reproductive tract infection. To establish this new model for vaccine testing, we also tested the licensed meningococcal 4CMenB vaccine, which cross-protects against murine N. gonorrhoeae lower reproductive tract infection, for efficacy against N. gonorrhoeae in the endometrium and oviducts following transcervical or vaginal challenge.
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Anti-Infecciosos/administração & dosagem , Gonorreia/prevenção & controle , Doença Inflamatória Pélvica/prevenção & controle , Infecções do Sistema Genital/microbiologia , Animais , Modelos Animais de Doenças , Feminino , Gonorreia/tratamento farmacológico , Camundongos , Neisseria gonorrhoeae/imunologia , Doença Inflamatória Pélvica/microbiologiaRESUMO
The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane, MlaA (VacJ) acts as a channel to shuttle phospholipids from the outer leaflet. We identified Neisseria gonorrhoeae MlaA (ngo2121) during high-throughput proteomic mining for potential therapeutic targets against this medically important human pathogen. Our follow-up phenotypic microarrays revealed that lack of MlaA results in a complex sensitivity phenome. Herein we focused on MlaA function in cell envelope biogenesis and pathogenesis. We demonstrate the existence of two MlaA classes among 21 bacterial species, characterized by the presence or lack of a lipoprotein signal peptide. Purified truncated N. gonorrhoeae MlaA elicited antibodies that cross-reacted with a panel of different Neisseria. Little is known about MlaA expression; we provide the first evidence that MlaA levels increase in stationary phase and under anaerobiosis but decrease during iron starvation. Lack of MlaA resulted in higher cell counts during conditions mimicking different host niches; however, it also significantly decreased colony size. Antimicrobial peptides such as polymyxin B exacerbated the size difference while human defensin was detrimental to mutant viability. Consistent with the proposed role of MlaA in vesicle biogenesis, the ΔmlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from ΔmlaA and wild type bacteria revealed enrichment of TadA-which recodes proteins through mRNA editing-as well as increased levels of adhesins and virulence factors. MlaA-deficient gonococci significantly outcompeted (up to 16-fold) wild-type bacteria in the murine lower genital tract, suggesting the growth advantage or increased expression of virulence factors afforded by inactivation of mlaA is advantageous in vivo. Based on these results, we propose N. gonorrhoeae restricts MlaA levels to modulate cell envelope homeostasis and fine-tune virulence.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Neisseria gonorrhoeae/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bactérias , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias , Transporte Biológico , Membrana Celular , Parede Celular , Proteínas de Escherichia coli , Gonorreia , Bactérias Gram-Negativas/metabolismo , Humanos , Neisseria gonorrhoeae/fisiologia , Fosfolipídeos/metabolismo , Fosfolipídeos/fisiologia , Filogenia , Proteômica , Virulência , Fatores de VirulênciaRESUMO
ABSTRACT: Declining gonococcal susceptibility to ceftriaxone and azithromycin has raised the possibility of untreatable gonorrhea in the future and reignited interest in gonococcal vaccine development. Despite decades of research, previous gonococcal vaccine candidates have been ineffective. A growing body of data suggests that meningococcal group B outer-membrane vaccines may be cross-protective against Neisseria gonorrhoeae. Clinical trials of a licensed vaccine against Neisseria meningitidis serogroup B containing an outer-membrane vaccine component are underway to determine its efficacy against N. gonorrhoeae. Other experimental gonococcal vaccine candidates are in the preclinical phases. Population impact of future gonococcal vaccines with different levels of efficacy and duration of protection in various populations is being evaluated using modeling studies. Despite recent progress, gaps in gonococcal vaccine research remain. Research is needed to evaluate vaccine efficacy in preventing gonococcal infections acquired via various anatomic routes and among patients coinfected with other sexually transmitted infections. Studies that model the impact of a future vaccine on high-burden populations such as men who have sex with men and estimate both vaccine cost-effectiveness and the incremental cost-effectiveness ratio of vaccination to antimicrobial resistance and treatment costs are warranted. This narrative review examines the current state of gonococcal vaccine research, the possible impact of a gonococcal vaccine on gonorrhea rates based on modeling studies, gaps in the gonococcal vaccine literature, and public health implications of a future gonococcal vaccine on reducing the gonorrhea burden in the United States.
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Gonorreia , Minorias Sexuais e de Gênero , Gonorreia/epidemiologia , Gonorreia/prevenção & controle , Homossexualidade Masculina , Humanos , Masculino , Neisseria gonorrhoeae , Saúde Pública , Estados UnidosRESUMO
Gonorrhea remains a major public health challenge, and current recommendations for gonorrhea treatment are threatened by evolving antimicrobial resistance and a diminished pipeline for new antibiotics. Evaluations of potential new treatments for gonorrhea currently make limited use of new understanding of the pharmacokinetic and pharmacodynamic contributors to effective therapy, the prevention of antimicrobial resistance, and newer designs for clinical trials. They are hampered by the requirement to utilize combination ceftriaxone/azithromycin therapy as the comparator regimen in noninferiority trials designed to seek an indication for gonorrhea therapy. Evolving gonococcal epidemiology and clinical trial design constraints hinder the enrollment of those populations at the greatest risk for gonorrhea (adolescents, women, and persons infected with antibiotic-resistant Neisseria gonorrhoeae). This article summarizes a recent meeting on the evaluation process for antimicrobials for urogenital gonorrhea treatment and encourages the consideration of new designs for the evaluation of gonorrhea therapy.
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Anti-Infecciosos , Gonorreia , Adolescente , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Infecciosos/farmacologia , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Ensaios Clínicos como Assunto , Farmacorresistência Bacteriana , Feminino , Gonorreia/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeaeRESUMO
Lysozymes are nearly omnipresent as the first line of immune defense against microbes in animals. They exert bactericidal action through antimicrobial peptide activity and peptidoglycan hydrolysis. Gram-negative bacteria developed several weapons to battle lysozymes, including inhibitors of c-type lysozymes in the MliC/PliC family and the Neisseria adhesin complex protein (ACP). Until the recent discovery of ACP, no proteinaceous lysozyme inhibitors were reported for the genus Neisseria, including the important human pathogen N. gonorrhoeae. Here, we describe a previously unrecognized gonococcal virulence mechanism involving a protein encoded by the open reading frame ngo1063 that acts to counteract c-type Iysozyme and provides a competitive advantage in the murine model of gonorrhea. We named this protein SliC as a surface-exposed lysozyme inhibitor of c-type lysozyme. SliC displays low overall primary sequence similarity to the MliC/PliC inhibitors, but we demonstrate that it has a parallel inhibitory mechanism. Our studies provide the first evidence that bacterial proteinaceous lysozyme inhibitors protect against host lysozyme during infection based on lack of attenuation of the ΔsliC mutant in lysozyme knock-out mice, and that the conserved residues involved in lysozyme inhibition, S83 and K103, are functionally indispensable during infection in wild type mice. Recombinant SliC completely abrogated the lytic activity of human and chicken c-type lysozymes, showing a preference towards human lysozyme with an IC50 of 1.85 µM and calculated KD value of 9.2 ± 1.9 µM. In contrast, mutated SliC bearing S83A and K103A substitutions failed to protect fluorescein-labeled cell-wall from lysozyme-mediated hydrolysis. Further, we present data revealing that SliC is a surface-displayed lipoprotein released in membrane vesicles that is expressed throughout all phases of growth, in conditions relevant to different niches of the human host, and during experimental infection of the murine genital tract. SliC is also highly conserved and expressed by diverse gonococcal isolates as well as N. meningitidis, N. lactamica, and N. weaveri. This study is the first to highlight the importance of an anti-lysozyme strategy to escape the innate immune response during N. gonorrhoeae infection.
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Proteínas de Bactérias/metabolismo , Gonorreia/metabolismo , Muramidase/metabolismo , Neisseria gonorrhoeae/metabolismo , Fatores de Virulência/metabolismo , Virulência/fisiologia , Animais , Galinhas , Humanos , Camundongos , Neisseria gonorrhoeae/patogenicidadeRESUMO
There is a pressing need for drug development for gonorrhea. Here we describe a pharmacokinetic (PK)/pharmacodynamic (PD) analysis of extended-spectrum cephalosporins (ESC) against drug-susceptible and drug-resistant gonococcal strains in a murine genital tract infection model. The PK determined in uninfected mice displayed a clear dose-response in plasma levels following single doses of ceftriaxone (CRO) (intraperitoneal) or cefixime (CFM) (oral). The observed doses required for efficacy against ESC-susceptible (ESCs) strain FA1090 were 5 mg/kg of body weight (CRO) and 12 mg/kg (CFM); these doses had estimated therapeutic times (the time that the free drug concentration remains above the MIC [fTMIC]) of 24 h and 37 h, respectively. No single dose of CRO or CFM was effective against ESC-resistant (ESCr) strain H041. However, fractionation (three times a day every 8 h [TIDq8h]) of a 120-mg/kg dose of CRO resulted in estimated therapeutic times in the range of 23 h and cleared H041 infection in a majority (90%) of mice, comparable to the findings for gentamicin. In contrast, multiple CFM doses of 120 or 300 mg/kg administered TIDq8h cleared infection in ≤50% of mice, with the therapeutic times estimated from single-dose PK data being 13 and 27 h, respectively. This study reveals a clear relationship between plasma ESC levels and bacterial clearance rates in the gonorrhea mouse model. The PK/PD relationships observed in mice reflected those observed in humans, with in vivo efficacy against an ESCs strain requiring doses that yielded an fTMIC in excess of 20 to 24 h. PK data also accurately predicted the failure of single doses of ESCs against an ESCr strain and were useful in designing effective dosing regimens.
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Antibacterianos/sangue , Cefixima/sangue , Ceftriaxona/sangue , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Cefixima/farmacologia , Ceftriaxona/farmacologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade MicrobianaRESUMO
Efforts to develop vaccines against Neisseria gonorrhoeae have become increasingly important, given the rising threat of gonococcal antimicrobial resistance (AMR). Recent data suggest vaccines for gonorrhoea are biologically feasible; in particular, epidemiological evidence shows that vaccines against a closely related pathogen, serogroup B Neisseria meningitidis outer membrane vesicle (OMV) vaccines, may reduce gonorrhoea incidence. Vaccine candidates using several approaches are currently in preclinical development, including meningococcal and gonococcal OMV vaccines, a lipooligosaccharide epitope and purified protein subunit vaccines. The Global STI Vaccine Roadmap provides action steps to build on this technical momentum and advance gonococcal vaccine development. Better quantifying the magnitude of gonorrhoea-associated disease burden, for outcomes like infertility, and modelling the predicted role of gonococcal vaccines in addressing AMR will be essential for building a full public health value proposition, which can justify investment and help with decision making about future vaccine policy and programs. Efforts are underway to gain consensus on gonorrhoea vaccine target populations, implementation strategies and other preferred product characteristics that would make these vaccines suitable for use in low- and middle-income, as well as high-income, contexts. Addressing these epidemiological, programmatic and policy considerations in parallel to advancing research and development, including direct assessment of the ability of meningococcal B OMV vaccines to prevent gonorrhoea, can help bring about the development of viable gonococcal vaccines.
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Vacinas Bacterianas/uso terapêutico , Gonorreia/prevenção & controle , Neisseria gonorrhoeae/imunologia , Vacinas Bacterianas/imunologia , Pesquisa Biomédica , Gonorreia/imunologia , HumanosRESUMO
Neisseria gonorrhoeae is an urgent antibiotic-resistant threat. This study determined the MICs of mupirocin to be 0.0039 to 0.0625 µg/ml for 94 N. gonorrhoeae strains. Cross-resistance with other antibiotics was not detected. Mupirocin, which is currently limited to topical administration, demonstrated activity by injection when delivered in nanoliposomes. The nanoliposomal formulation of mupirocin is a potential treatment for drug-resistant N. gonorrhoeae.
Assuntos
Antibacterianos/farmacologia , Lipossomos/química , Mupirocina/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Antibacterianos/química , Testes de Sensibilidade Microbiana , Mupirocina/químicaRESUMO
The gonococcal NorM efflux pump exports substrates with a cationic moiety, including quaternary ammonium compounds such as berberine (BE) and ethidium bromide (EB) as well as antibiotics such as ciprofloxacin and solithromycin. The norM gene is part of a four-gene operon that is transcribed from a promoter containing a polynucleotide tract of 6 or 7 thymidines (T's) between the -10 and -35 hexamers; the majority of gonococcal strains analyzed in this study contained a T-6 sequence. Primer extension analysis showed that regardless of the length of the poly(T) tract, the same transcriptional start site (TSS) was used for expression of norM Interestingly, the T-6 tract correlated with a higher level of both norM expression and gonococcal resistance to NorM substrates BE and EB. Analysis of expression of genes downstream of norM showed that the product of the tetR-like gene has the capacity to activate expression of norM as well as murB, which encodes an acetylenolpyroylglucosamine reductase predicted to be involved in the early steps of peptidoglycan synthesis. Moreover, loss of the TetR-like transcriptional regulator modestly increased gonococcal susceptibility to NorM substrates EB and BE. We conclude that both cis- and trans-acting regulatory systems can regulate expression of the norM operon and influence levels of gonococcal susceptibility to antimicrobials exported by NorM.
Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/metabolismo , Gonorreia/metabolismo , Gonorreia/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Neisseria gonorrhoeae/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Bacterial sexually transmitted infections are widespread and common, with Neisseria gonorrhoeae (gonorrhea) and Chlamydia trachomatis (chlamydia) being the two most frequent causes. If left untreated, both infections can cause pelvic inflammatory disease, infertility, ectopic pregnancy, and other sequelae. The recommended treatment for gonorrhea is ceftriaxone plus azithromycin (to empirically treat chlamydial coinfections). Antibiotic resistance to all existing therapies has developed in gonorrheal infections. The need for new antibiotics is great, but the pipeline for new drugs is alarmingly small. The aminomethyl spectinomycins, a new class of semisynthetic analogs of the antibiotic spectinomycin, were developed on the basis of a computational analysis of the spectinomycin binding site of the bacterial 30S ribosome and structure-guided synthesis. The compounds display particular potency against common respiratory tract pathogens as well as the sexually transmitted pathogens that cause gonorrhea and chlamydia. Here, we demonstrate the in vitro potencies of several compounds of this class against both bacterial species; the compounds displayed increased potencies against N. gonorrhoeae compared to that of spectinomycin and, significantly, demonstrated activity against C. trachomatis that is not observed with spectinomycin. Efficacies of the compounds were compared to those of spectinomycin and gentamicin in a murine model of infection caused by ceftriaxone/azithromycin-resistant N. gonorrhoeae; the aminomethyl spectinomycins significantly reduced the colonization load and were as potent as the comparator compounds. In summary, data produced by this study support aminomethyl spectinomycins as a promising replacement for spectinomycin and antibiotics such as ceftriaxone for treating drug-resistant gonorrhea, with the added benefit of treating chlamydial coinfections.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Chlamydia/tratamento farmacológico , Chlamydia trachomatis/efeitos dos fármacos , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/efeitos dos fármacos , Doenças Bacterianas Sexualmente Transmissíveis/tratamento farmacológico , Espectinomicina/análogos & derivados , Espectinomicina/uso terapêutico , Animais , Azitromicina/farmacologia , Ceftriaxona/farmacologia , Infecções por Chlamydia/microbiologia , Coinfecção/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla , Feminino , Gentamicinas/uso terapêutico , Gonorreia/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Subunidades Ribossômicas Menores de Bactérias/efeitos dos fármacosRESUMO
Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform-isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry-to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens.