Whole-genome sequencing resolves biochemical misidentification of Neisseria species from urogenital specimens.

Neisseria meningitidis (Nm) and Neisseria gonorrhoeae (Ng) are human pathogens that sometimes occupy the same anatomical niche. Ng, the causative agent of gonorrhea, infects 87 million individuals annually worldwide and is an urgent threat due to increasing drug resistance. Ng is a pathogen of the urogenital tract and may infect the oropharyngeal or rectal site, often asymptomatically. Conversely, Nm is an opportunistic pathogen. While often a commensal in the oropharyngeal tract, it is also the leading cause of bacterial meningitis with 1.2 million cases globally, causing significant morbidity and mortality. Horizontal gene transfer (HGT) is likely to occur between Ng and Nm due to their shared anatomical niches and genetic similarity, which poses challenges for accurate detection and treatment. Routine surveillance through the Gonococcal Isolate Surveillance Project and Strengthening the U.S. Response to Resistant Gonorrhea detected six concerning urogenital Neisseria isolates with contradicting species identification in Milwaukee (MIL). While all six isolates were positive for Ng using nucleic acid amplification testing (NAAT) and matrix-assisted laser desorption/ionization time of flight identified the isolates as Ng, two biochemical tests, Gonochek-II and API NH, classified them as Nm. To address this discrepancy, we performed whole-genome sequencing (WGS) using Illumina MiSeq on all isolates and employed various bioinformatics tools. Species detection analysis using BMScan, which uses WGS data, identified all isolates as Ng. Furthermore, Kraken revealed over 98% of WGS reads mapped to the Ng genome and <1% to Nm. Recombination analysis identified putative HGT in all MIL isolates within the γ-glutamyl transpeptidase (ggt) gene, a key component in the biochemical tests used to differentiate between Nm and Ng. Further analysis identified Nm as the source of HGT event. Specifically, the active Nm ggt gene replaced the Ng pseudogenes, ggt1 and ggt2. Together, this study demonstrates that closely related Neisseria species sharing a niche underwent HGT, which led to the misidentification of species following biochemical testing. Importantly, NAAT accurately detected Ng. The misidentification highlights the importance of using WGS to continually evaluate diagnostic or bacterial identification tests.

CARD8 polymorphisms among bacterial meningitis patients in North-West Ethiopia.

BACKGROUND: The severity of infectious disease outcomes is dependent on the virulence factors of the pathogen and the host immune response. CARD8 is a major regulator of the innate immune proinflammatory response and has been suggested to modulate the host response to common inflammatory diseases. In the present study, the C10X genetic polymorphism in the CARD8 gene was investigated in relation to bacterial meningitis. METHODS: A total of 400 clinically suspected meningitis patients hospitalized at the University of Gondar Hospital were enrolled in the study. Cerebrospinal fluid (CSF) and blood samples were collected for laboratory investigations. The collected CSF was cultured, and all the results obtained from the culture were confirmed using direct RT‒PCR. Genotyping of whole-blood samples was performed using a TaqMan assay. The results were compared with apparently healthy controls and with PCR-negative meningitis suspected patients. RESULTS: Of the included patients, 57% were men and the most common clinical signs and symptoms were fever (81%), headache (80%), neck stiffness (76%), nausea (68%), and vomiting (67%). Microbiology culture identified 7 patients with bacterial meningitis caused by Neisseria meningitidis (n = 4) and Streptococcus pneumoniae (n = 3). The RT-PCR revealed 39 positive samples for N. meningitidis (n = 10) and S. pneumoniae (n = 29). A total of 332 whole-blood samples were genotyped with the following results: 151 (45.5%) C10X heterozygotes, 59 (17.7%) C10X homozygotes and 122 (36.7%) wild genotypes. The polymorphic gene carriers among laboratory confirmed, clinically diagnosed meningitis and healthy controls were 23(46%), 246(40%), and 1526(39%), respectively with OR = 1.27 (0.7-2.3) and OR = 1.34 (0.76-2.4). The presence of the C10X polymorphism in the CARD8 gene was more prevalent in suspected meningitis patients than in healthy controls (OR 1.2; 1.00-1.5). Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease. The presence of viable or active bacterial infection was found to be associated with the presence of heterozygous C10X carriers. CONCLUSIONS: A greater proportion of C10X in the CARD8 gene in confirmed bacterial meningitis patients and clinically diagnosed meningitis patients than in healthy controls. Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease than heterozygote gene carriers and healthy controls.

Characterisation and Immunogenicity of Neisseria cinerea outer membrane vesicles displaying NadA, NHBA and fHbp from Neisseria meningitidis serogroup B.

More affordable and effective vaccines against bacterial meningitis caused by Neisseria meningitidis serogroup B are still required for global prevention. We have previously shown that modified outer membrane vesicles (mOMVs) from commensal Neisseria cinerea can be used as a platform to induce immune responses against meningococcal antigens. The aim of the present study was to use a combination of two genetically engineered mOMVs to express multiple antigens from N. meningitidis known to be involved in protective immunity to meningococcal meningitis (different variants of factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisseria Adhesin A (NadA)). Antigen expression in the mOMVs was confirmed by Western blotting; detoxification of the lipooligosaccharide (LOS) was confirmed by measuring human Toll-like receptor 4 (hTLR4) activation using in vitro cell assays. Mice immunised with a combination of two mOMVs expressing fHbp, NHBA and NadA produced antibodies to all the antigens. Furthermore, serum bactericidal activity (SBA) was induced by the immunisation, with mOMVs expressing NadA displaying high SBA titres against a nadA+ MenB strain. The work highlights the potential of mOMVs from N. cinerea to induce functional immune responses against multiple antigens involved in the protective immune response to meningococcal disease.

Neisseria meningitidis activates pyroptotic pathways in a mouse model of meningitis: role of a two-partner secretion system.

There is evidence that in infected cells in vitro the meningococcal HrpA/HrpB two-partner secretion system (TPS) mediates the exit of bacteria from the internalization vacuole and the docking of bacteria to the dynein motor resulting in the induction of pyroptosis. In this study we set out to study the role of the HrpA/HrpB TPS in establishing meningitis and activating pyroptotic pathways in an animal model of meningitis using a reference serogroup C meningococcal strain, 93/4286, and an isogenic hrpB knockout mutant, 93/4286ΩhrpB. Survival experiments confirmed the role of HrpA/HrpB TPS in the invasive meningococcal disease. In fact, the ability of the hrpB mutant to replicate in brain and spread systemically was impaired in mice infected with hrpB mutant. Furthermore, western blot analysis of brain samples during the infection demonstrated that: i. N. meningitidis activated canonical and non-canonical inflammasome pyroptosis pathways in the mouse brain; ii. the activation of caspase-11, caspase-1, and gasdermin-D was markedly reduced in the hrpB mutant; iii. the increase in the amount of IL-1ß and IL-18, which are an important end point of pyroptosis, occurs in the brains of mice infected with the wild-type strain 93/4286 and is strongly reduced in those infected with 93/4286ΩhrpB. In particular, the activation of caspase 11, which is triggered by cytosolic lipopolysaccharide, indicates that during meningococcal infection pyroptosis is induced by intracellular infection after the exit of the bacteria from the internalizing vacuole, a process that is hindered in the hrpB mutant. Overall, these results confirm, in an animal model, that the HrpA/HrpB TPS plays a role in the induction of pyroptosis and suggest a pivotal involvement of pyroptosis in invasive meningococcal disease, paving the way for the use of pyroptosis inhibitors in the adjuvant therapy of the disease.

Meningococcal Pneumonia in a Patient With Pulmonary Amyloidosis and a Recent COVID-19 Infection.

A 64-year-old man visited our outpatient clinic with chief complaints of high fever and throat pain. His medical history was significant for pulmonary amyloidosis that was observed at our outpatient clinic, and his recent recovery from COVID-19. Findings from imaging studies included thickening of the bronchial walls, infiltrates of the left upper lobe, and pre-existing pulmonary nodules from amyloidosis. A peripheral blood examination revealed leukocytosis and elevated C-reactive protein levels. His signs and symptoms suggested bronchopneumonia and antimicrobial treatment was initiated. Sputum microscopic examination revealed gram-negative cocci and culture growth revealed to be Neisseria meningitidis, with follow-up bacterial specimens after treatment demonstrating diminished microbes. Despite a medical history of amyloidosis and COVID-19, the patient's clinical course resulted in favorable outcomes. The N. meningitidis infection is a rare condition in generally healthy individuals, and certain conditions may be related to the contraction of the pathogen, otherwise seen primarily in immunocompromised hosts. In our case, the medical history of amyloidosis and recent COVID-19 infection may have contributed to the development of meningococcal bronchopneumonia.

Factor H binding protein (FHbp): An evaluation of genotypic diversity across Neisseria meningitidis serogroups.

Neisseria meningitidis serogroups A, B, C, W, X, and Y cause invasive meningococcal disease (IMD) worldwide. Factor H binding protein (FHbp), a key meningococcal virulence factor, is an antigen included in both licensed meningococcal serogroup B (MenB) vaccines. This review examines the biology and epidemiology of FHbp and assesses the ability and potential of FHbp vaccine antigens to protect against IMD. Using evidence from the literature and the contemporary PubMLST database, we discuss analyses of MenB genotypes on the representation of the most prevalent multilocus sequence typing (MLST)/clonal complexes, FHbp subfamily distribution, and FHbp and porin A (PorA) variants. We further discuss that the similar genotypes, distribution, and diversity of FHbp variant types have remained stable over long time periods, supporting the potential for FHbp-containing, protein-based vaccines to protect against IMD, including MenB-FHbp (Trumenba®), which contains two lipidated FHbp antigens (one each from both FHbp subfamilies: A and B).

A Presentation of Neisseria meningitidis in a Patient Taking Adalimumab as Immunosuppressive Therapy for Hidradenitis Suppurativa.

Neisseria meningitidis is common within the human population. Most patients with N. meningitidis colonization are asymptomatic, but invasive disease can result in meningitis, fulminant septicemia, and disseminated intravascular coagulation. This case report describes a patient who presented with symptoms of sepsis and was later diagnosed with N. meningitidis. The cause of her infection was believed to be immunosuppression from adalimumab, which she was taking for systemic hidradenitis suppurativa.

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.

Exploring the prevalence of chlamydial and gonorrheal infections in pregnant women: a multicenter study in Egypt.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are widespread, treatable sexually transmitted infections (STIs) of global significance, affecting millions annually. Left untreated, they pose significant risks, including pelvic inflammatory disease (PID), infertility, and complications during pregnancy. The U.S. Centers for Disease Control recommends annual chlamydial screening for sexually active women to address these risks. Responding to this global challenge, the World Health Organization (WHO) has formulated a global health sector strategy on sexually transmitted infections, outlining priority actions to strengthen STI responses in countries. However, STI epidemiological studies encounter challenges in developing nations like Egypt due to socio-cultural factors, poverty, and limited diagnostic facilities. In Egypt, STI diagnosis primarily relies on clinical presentations, lacking structured screening programs for CT and NG. This study's main objective is to estimate the prevalence of Chlamydial and gonorrheal infections, advocating for supportive STI strategies in Egypt. Additionally, the study aims to provide a foundation for national prevalence estimates of CT and NG infections. METHODS: A cross-sectional study encompassed five antenatal clinics in different regions of Egypt. A total of 1040 pregnant women attending these clinics were consecutively sampled. Data collection involved structured questionnaires, and urine samples were subjected to the GeneXpert CT/NG qualitative real-time PCR test. RESULTS: The prevalence of CT infections was 0.29% (95% CI, 0.10-0.86%), with no detected NG infections. The three CT-positive cases were distributed across different recruitment centers, with no statistically significant differences observed between infected and non-infected participants. Notably, 40.3% of recruited women reported gynecological symptoms, primarily discharge. Additionally, 9.6% had undergone previous testing for sexually transmitted infections, with 8.2% receiving positive results. CONCLUSIONS: This study provides valuable data on the prevalence of CT and NG infections among pregnant women attending ANC clinics in Egypt. The findings underscore the importance of ongoing surveillance, routine screening, and targeted interventions to ensure the reproductive health and well-being of pregnant women and their infants. Further research is warranted to explore the broader implications of STIs in different populations and to inform evidence-based guidelines for screening and management in diverse settings. TRIAL REGISTRATION: IRB no.: 17,400,017; WHO ERC Protocol Id. A66005.

Emergence and evolution of mosaic penA-60 and penA-237 alleles in a Neisseria gonorrhoeae core genogroup that was historically susceptible to extended spectrum cephalosporins.

Introduction: Neisseria gonorrhoeae (Ng) has successively developed resistance to all previously recommended antimicrobial therapies, with ceftriaxone being the last option for monotherapy of gonorrhea. Global emergence and international spread of the FC428 clone derived mosaic penA-60 allele, associated with highlevel ceftriaxone minimum inhibitory concentrations (MICs) in non FC428 clone Ng lineages, has become an increasing concern. The penA-60 allele carrying Ng was first identified in the U.S. in Las Vegas, Nevada (2019; GCWGS-102723), with a multi-locus sequence type (MLST)-1901 strain, in a non FC428 clone Ng lineage, which is associated with a historically ceftriaxone susceptible core genogroup. Later in 2022, an allele genetically similar to penA-60, mosaic penA-237, was identified in the UK (H22-722) and France (F92) with high-level ceftriaxone MICs and both belonged to MLST-1901. Methods: In this study, we assessed phylogenomic relatedness and antimicrobial resistance (AMR) determinant profiles of these three isolates with high-level ceftriaxone MICs among a global collection of 2,104 genomes belonging to the MLST-1901 core genome cluster group 31, which includes strains separated by a locus threshold of 200 or fewer differences (Ng_cgc_200). Recombination events in and around the penA coding region were catalogued and potential sources of inter species recombinant DNA were also inferred. Results: The global population structure of MLST-1901 core genogroup falls into 4 major lineages. Isolates GCWGS-10723, F92, and H22-722 clustered within Lineage 1, which was dominated by non-mosaic penA-5 alleles. These three isolates formed a clade within Lineage 1 that consisted of isolates from North America and southeast Asia. Neisseria subflava and Neisseria sicca were identified as likely progenitors of two independent recombination events that may have led to the generation of mosaic penA-60 and penA-237, within a possible non-mosaic penA-5 background. Discussions: Our study suggests that there are multiple evolutionary pathways that could generate concerning mosaic penA alleles via homologous recombination of historically susceptible Ng lineages with Neisseria commensals. Enhanced surveillance of gonococcal strains and Neisseria commensals is crucial for understanding of the evolution of AMR, particularly in less-studied regions (e.g., Asia), where high-level ceftriaxone MICs and multi-drug resistance are more prevalent.

Neisseria gonorrhoeae carbonic anhydrase inhibition.

Carbonic anhydrases (CAs) are ubiquitous enzymes that are found in all kingdoms of life. Though different classes of CAs vary in their roles and structures, their primary function is to catalyze the reaction between carbon dioxide and water to produce bicarbonate and a proton. Neisseria gonorrhoeae encodes for three distinct CAs (NgCAs) from three different families: an α-, a ß-, and a γ-isoform. This chapter details the differences between the three NgCAs, summarizing their subcellular locations, roles, essentiality, structures, and enzyme kinetics. These bacterial enzymes have the potential to be drug targets; thus, previous studies have investigated the inhibition of NgCAs-primarily the α-isoform. Therefore, the classes of inhibitors that have been shown to bind to the NgCAs will be discussed as well. These classes include traditional CA inhibitors, such as sulfonamides, phenols, and coumarins, as well as non-traditional inhibitors including anions and thiocarbamates.

Genetic Manipulation of Neisseria gonorrhoeae and Commensal Neisseria Species.

The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture Basic Protocol 4: Electroporation of Neisseria gonorrhoeae Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5 Basic Protocol 7: Chemical mutagenesis Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome Alternate Protocol 1: Complementation with replicating plasmids Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies.

Alternative drugs for the treatment of gonococcal infections: old and new.

INTRODUCTION: The rise in antibiotic resistance to N. gonorrhoeae poses a substantial threat to effective gonorrhea treatment. Historical progression of resistance from sulfonamides to the more recent declines in efficacy of fluoroquinolones and susceptibilities of ceftriaxone highlight the urgent need for novel therapeutic approaches, necessitating the examination of alternative and new antibiotics. AREAS COVERED: This review examines the potential of repurposing older antibiotics for gonorrhea treatment with a focus on their efficacy and limitations. These include aztreonam, ertapenem, and fosfomycin. New oral drugs zoliflodacin and gepotidacin are in late clinical development, but there are concerns regarding their effectiveness for extragenital infections and the development of resistance. EXPERT OPINION: While ceftriaxone remains the best treatment for gonorrhea across all anatomic sites, resistance may eventually limit its use. Among older antibiotics, ertapenem shows the most potential as an alternative but shares the same administrative drawbacks as ceftriaxone. New oral drugs zoliflodacin and gepotidacin initially appeared promising, but their efficacy for pharyngeal infections and potential for resistance development are concerning. Phase 3 trial results have not been made available except through press releases, which perpetuates concerns. Understanding pharmacokinetic and pharmacodynamic profiles of antibiotics will be key in optimizing future treatment recommendations.

Invasive Neisseria meningitidis subtype C in gay, bisexual and other men who have sex with men: a systematic review.

INTRODUCTION: Outbreaks of invasive Neisseria meningitidis subtype C in networks of gay, bisexual and other men who have sex with men (MSM) have been reported. We aimed to explore any factors seen in MSM with invasive N.meningitidis subtype C. METHOD: We searched three bibliographical databases for manuscripts written in English exploring at least one factor seen in MSM with invasive N. meningitidis subtype C published up to May 2024. Following an initial search, removal of duplicates and abstract review, two authors independently reviewed full-text manuscripts and performed a risk of bias assessment using the Joanna Briggs Institute toolkit. Narrative data were synthesised to generate themes. RESULTS: 16 manuscripts were included in this review from the USA (n=10), Germany (n=2), France (n=2), Canada (n=1) and Italy (n=1) and consisted of nine case series, four cross-sectional studies, two case reports and one case-control study published between 2003 and 2024 involving 236 MSM with invasive N. meningitidis subtype C, of which at least 64 died. We have highlighted some demographic (African-American or Hispanic identity in the USA, living with HIV), behavioural (kissing, sharing drinks, visiting sex-on-premises venues, visiting gay-oriented venues, using websites/mobile phone apps to meet sexual partners, recreational drug use, multiple and non-regular sexual partners) and infection (previous Chlamydia trachomatis, Treponema pallidum, Neisseria gonorrhoeae, Mpox) factors in MSM with invasive N. meningitidis subtype C. CONCLUSION: These data serve as an important resource to inform and target future public health strategies and outbreak control measures for the prevention of invasive N. meningitidis subtype C in MSM. PROSPERO REGISTRATION NUMBER: CRD42024543551.

Disseminated Gonococcal Infection Presenting as Isolated Septic Arthritis: A Case Report and Review of Literature.

Neisseria gonorrhoeae is a gram-negative diplococcus that passes from one person to another through sexual contact. On rare occasions, Neisseria gonorrhoeae may spread from the primary mucosal site to distant parts of the body and present with signs of systemic illness; this is commonly known as disseminated gonococcal infection (DGI). We present a case report of an 18-year-old patient who was diagnosed with septic arthritis of the right third metacarpophalangeal (MCP) joint without mucosal involvement or systemic symptoms and who was found to have gonococcal bacteremia. This case highlights the importance of clinician awareness of the many extragenital manifestations of DGI and a high index of suspicion in the setting of septic arthritis and high-risk sexual practices. Diagnosing DGI early and providing prompt treatment may prevent complications of sepsis, joint destruction, and a prolonged hospital stay.

Antimicrobial activity of compounds identified by artificial intelligence discovery engine targeting enzymes involved in Neisseria gonorrhoeae peptidoglycan metabolism.

BACKGROUND: Neisseria gonorrhoeae (Ng) causes the sexually transmitted disease gonorrhoea. There are no vaccines and infections are treated principally with antibiotics. However, gonococci rapidly develop resistance to every antibiotic class used and there is a need for developing new antimicrobial treatments. In this study we focused on two gonococcal enzymes as potential antimicrobial targets, namely the serine protease L,D-carboxypeptidase LdcA (NgO1274/NEIS1546) and the lytic transglycosylase LtgD (NgO0626/NEIS1212). To identify compounds that could interact with these enzymes as potential antimicrobials, we used the AtomNet virtual high-throughput screening technology. We then did a computational modelling study to examine the interactions of the most bioactive compounds with their target enzymes. The identified compounds were tested against gonococci to determine minimum inhibitory and bactericidal concentrations (MIC/MBC), specificity, and compound toxicity in vitro. RESULTS: AtomNet identified 74 compounds that could potentially interact with Ng-LdcA and 84 compounds that could potentially interact with Ng-LtgD. Through MIC and MBC assays, we selected the three best performing compounds for both enzymes. Compound 16 was the most active against Ng-LdcA, with a MIC50 value < 1.56 µM and MBC50/90 values between 0.195 and 0.39 µM. In general, the Ng-LdcA compounds showed higher activity than the compounds directed against Ng-LtgD, of which compound 45 had MIC50 values of 1.56-3.125 µM and MBC50/90 values between 3.125 and 6.25 µM. The compounds were specific for gonococci and did not kill other bacteria. They were also non-toxic for human conjunctival epithelial cells as judged by a resazurin assay. To support our biological data, in-depth computational modelling study detailed the interactions of the compounds with their target enzymes. Protein models were generated in silico and validated, the active binding sites and amino acids involved elucidated, and the interactions of the compounds interacting with the enzymes visualised through molecular docking and Molecular Dynamics Simulations for 50 ns and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA). CONCLUSIONS: We have identified bioactive compounds that appear to target the N. gonorrhoeae LdcA and LtgD enzymes. By using a reductionist approach involving biological and computational data, we propose that compound Ng-LdcA-16 and Ng-LtgD-45 are promising anti-gonococcal compounds for further development.

Whole-genome sequencing of Neisseria meningitidis collected in Chile from pediatric patients during 2016-2019 and coverage vaccine prediction.

BACKGROUND: Over the past few years, whole-genome sequencing (WGS) has become a valuable tool for global meningococcal surveillance. The objective of this study was to genetically characterize Neisseria meningitidis strains isolated from children in Chile through WGS and predicting potential vaccine coverage using gMATS and MenDeVAR. METHODS: WGS of 42 N.meningitidis from pediatric patients were processed and assembled using different software. We analyzed genomes with BIGSdb platform hosted at PubMLST.org, and predicted vaccine coverage using MenDeVAR and gMATS tools. RESULTS: Among 42 strains, 25 were MenB, 16 MenW, and 1 MenC. The cc11 and cc 41/44 were the most frequents. The main frequent deduced peptide sequence for PorA was P1.5,2 (40 %), peptide P1.4 was present in one MenB strain; NHBA-29 (64 %), none having peptide 2; fHbp-2 (76 %), one strain had peptide-1, and two had peptide 45; NadA was detected in 52 %, peptide-6 was present in 84 %, none had peptide 8. The MenDeVAR index predicted a coverage in MenB strains for 4CMenB 8 % exact matches, 12 % cross-reactivity, 8 % not coverage and 64 % had insufficient data. gMATS predicted 16 % was covered, 8 % not covered and 76 % unpredictable, and overall coverage of 54 %. For rLP2086-fHbp, the MenDeVAR index predicted exact match in 8 %, cross-reactivity in 64 %, and insufficient data in 28 % and an overall coverage of 72 %. In non-MenB strains, the MenDeVAR index predicted for 4CMenB vaccine: cross-reactivity 88 %, 6 % for not covered and insufficient data. For rLP2086-fHbp, predicted cross-reactivity 12 % and insufficient data in 88 %. gMATS predicted an overall coverage of 50 % for Non-MenB. CONCLUSION: genetic variability of the Chilean strains that its different from other countries, and until now limit the coverage prediction of vaccine with the available tools like gMATS and MenDeVAR.

Assessing novel partner antimicrobials to protect ceftriaxone against gonococcal resistance: An in vitro evaluation.

BACKGROUND: The emergence of ceftriaxone-resistant Neisseria gonorrhoeae poses a significant threat to existing treatment regimens. Our study aimed to assess the efficacy of antimicrobials that could be combined with ceftriaxone to reduce the probability of ceftriaxone resistance emerging and spreading in N. gonorrhoeae. METHODS AND RESULTS: Broth microdilution was used to determine the minimal inhibitory concentrations (MICs) for a panel of ceftriaxone-resistant (WHO X, Y, Z) and ceftriaxone-susceptible (WHO L, N, P) N. gonorrhoeae WHO reference strains for the following antimicrobials: ceftriaxone, doxycycline, azithromycin, zoliflodacin, fosfomycin, pristinamycin, ramoplanin, gentamicin and NAI-107. The MICs for zoliflodacin and pristinamycin for all strains were lower than or equal to the available breakpoints. A checkerboard assay was used to determine the drug-drug combination effect, which showed either an indifferent or an additive effect for all the combinations tested with ceftriaxone. CONCLUSIONS: The low MICs of zoliflodacin and pristinamycin for the three ceftriaxone-resistant strains suggest that these antimicrobials could be used as partner drugs with ceftriaxone to reduce the probability of ceftriaxone resistance spreading in areas with a high prevalence of ceftriaxone resistance.

Optimal Protein Allocation Controls the Inhibition of GltA and AcnB in Neisseria gonorrhoeae.

Neisseria gonorrhea (Ngo) is a major concern for global public health due to its severe implications for reproductive health. Understanding its metabolic phenotype is crucial for comprehending its pathogenicity. Despite Ngo's ability to encode TCA cycle proteins, GltA and AcnB, their activities are notably restricted. To investigate this phenomenon, we used the iNgo_557 metabolic model and incorporated a constraint on total cellular protein content. Our results indicate that low cellular protein content severely limits GltA and AcnB activity, leading to a shift towards acetate overflow for ATP production, which is more efficient in terms of protein usage. Surprisingly, increasing cellular protein content alleviates this restriction on GltA and AcnB and delays the onset of acetate overflow, highlighting protein allocation as a critical determinant in understanding Ngo's metabolic phenotype. These findings underscore the significance of Ngo's metabolic adaptation in light of optimal protein allocation, providing a blueprint to understand Ngo's metabolic landscape.