Neisseria Gonorrhoeae Research Articles

Clinical Studies Are Needed to Determine the Efficacy of Ceftriaxone and Other Interventions in Addressing Resistant Neisseria gonorrhoeae Infection.

Clinical Studies Are Needed to Determine the Efficacy of Ceftriaxone and Other Interventions in Addressing Resistant Neisseria gonorrhoeae Infection.

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.

Prevalence of sexually transmitted infections (STIs) among first time visitors at STIs clinic in Hangzhou, China: Assessing the influence of the COVID-19 pandemic.

This study assesses the prevalence of sexually transmitted infections (STIs) in first time visitors to the STIs clinic in Hangzhou, China, considering different genders, ages and symptoms. And also explores howthe COVID-19 pandemic has affected on STIs. From 2019 to 2023, 27,283 first time visitors were tested for nine distinct STIs, including Human Papillomavirus (HPV), Human Immunodeficiency Virus (HIV), syphilis, Herpes Simplex Virus type 2 (HSV-2), Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), and vaginal Candida. Symptomatic male and female visitors showed overall STI-positive rates of 39.27% and 59.20%, respectively(p < .001). The top three pathogens in both genders were HPV (47.56% and 56.71%), UU (29.21% and 56.47%), and HSV-2 (22.41% and 52.94%). Among asymptomatic visitors, the total STI-positive rate was 36.63% in males and 52.03% in females. Age-stratified analysis revealed higher STI rates in visitors ≤ 20 or >50 years, regardless of gender and symptoms. During the COVID-19 pandemic, symptomatic visitors showed lower positive rates for HPV, HIV, syphilis, and HSV-2, while Candida, UU, CT, NG, and multiple infections increased. Among asymptomatic visitors, HPV had the lowest positive rate, while NG and multiple infections increased during the pandemic. STI prevalence is notably high, particularly in those aged ≤ 20 and >50 years. It emphasizes the need for enhanced health education, condom use, and vaccination. The COVID-19 pandemic impacting STIs through varied factors, such as reduced sexual activity and clinical service interruption.

Evaluation of antibacterial activity of ozonated oil and ozonated water against Neisseria gonorrhoeae and Neisseria meningitidis by broth microdilution method

Evaluation of antibacterial activity of ozonated oil and ozonated water against Neisseria gonorrhoeae and Neisseria meningitidis by broth microdilution method

Promoting molecular diagnostic equity: assessing in-house real-time PCR for Neisseria gonorrhoeae in anal samples from MSM recruited in an outpatient setting in Morocco.

Gonorrhea is a prevalent sexually transmitted infection among men who have sex with men (MSM). In Morocco, the basic laboratory diagnosis of Neisseria gonorrhoeae (NG) is based on microscopy and, in some settings, on culture. However, no nucleic acid amplification test (NAAT) has been implemented for routine diagnosis of gonorrhoeae.The aim of this study is to assess the effectiveness of an in-house real-time PCR test for detecting N. gonorrhoeae DNA in anal swabs samples collected during an Integrated Behavioral and Biological survey. Samples from 245 MSM, recruited using a Respondent Driven Sampling, were collected and tested for NG infection using GeneXpert CT/NG assay (Cepheid, USA). An In-House real-time PCR technique targeting the pseudo gene porA was developed and used for a parallel investigation of the same infection. The reliability of the in-house RT-PCR was validated through tests of reproducibility, repeatability, limit of detection, and cross-reactivity with other bacteria. The intrinsic performance characteristics of the qRT-PCR were assessed, namely, the sensitivity, the specificity, the positive predictive value (PPV), and the negative predictive value (NPV). The GeneXpert CT/NG assay was adopted as a reference method. For N. gonorrhoeae detection, the in-house real-time PCR assay showed a sensitivity and specificity of 80% and 100%, respectively. The PPV of the assay was 100% and the NPV was 97.3%. The in-house real-time PCR assay has high specificity and sensitivity, and it emerges as a promising approach for detecting N. gonorrhoeae in clinical specimens, particularly in decentralized settings such as regional laboratories.

Emergence of Extensively Drug-Resistant Neisseria gonorrhoeae, France, 2023.

Since 2022, Europe has had 4 cases of extensively drug-resistant Neisseria gonorrhoeae, sequence type 16406, that is resistant to ceftriaxone and highly resistant to azithromycin. We report 2 new cases from France in 2023 involving strains genetically related to the 4 cases from Europe as well as isolates from Cambodia.

Routine Testing for Chlamydia trachomatis and Neisseria gonorrhoeae Infections within an HIV Pre-Exposure Prophylaxis Program in Hanoi, Vietnam: Implications for Low- and Middle-Income Countries.

Data on Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) infections within HIV pre-exposure prophylaxis (PrEP) programs in low- and middle-income countries (LMICs) are limited. Our study reports the prevalence, anatomical distribution, and correlates of NG and CT infections within an HIV PrEP program in Hanoi, Vietnam. From January-December 2022, HIV PrEP program clients who were male at birth, ≥16 years old, reported ≥1 male sex partner in the prior 12 months, were enrolled. A questionnaire collected sociodemographics, sexual behaviors, and clinical data. CT/NG testing was performed on self-collected urine, rectal, and pharyngeal specimens. Multivariate logistic regression was used to identify factors associated with CT and NG infections. There were 529 participants enrolled, the median age was 25.1 years. The overall prevalence of CT or NG was 28.9% (153/529). The prevalence of NG was 14.3% and highest for pharyngeal infections (11.7%), while for CT, the prevalence was 20.4% and highest for rectal infections (14.0%). Symptoms in the prior week were reported by 45.8% (70/153) of those with CT or NG infections. Condomless anal sex (aOR= 1.98; 95% CI: 1.27, 3.08) and sexualized drug use in the prior 6 months (aOR= 1.68; 95% CI: 1.07, 2.65) were associated with CT/NG infections. Our study found a high prevalence of NG and CT infections, including pharyngeal and rectal infections, within an HIV PrEP program in Hanoi, Vietnam. The findings underscore the need for further research on CT/NG prevention and the development of evidence-based guidelines for CT/NG screening in HIV PrEP programs in LMIC settings.

An updated molecular diagnostic for surveillance of tetM in Neisseria gonorrhoeae.

Doxycycline post-exposure prophylaxis (doxy-PEP) for sexually transmitted bacterial infections reduces the risk of syphilis and chlamydia, but effectiveness against gonorrhea is variable, likely attributable to varying resistance rates. As doxy-PEP is incorporated into clinical practice, an urgent unanswered question is whether increased doxycycline use will drive tetracycline-class resistance in Neisseria gonorrhoeae . Here, we report an updated RT-PCR molecular diagnostic to detect the tetM gene that confers high-level tetracycline resistance in N. gonorrhoeae .

Real-world safety profile of eculizumab: an analysis of FDA adverse event reporting system and systematic review of case reports

ABSTRACT Background Most of the safety data regarding eculizumab came from clinical trials, while its safety information in the real world is still limited. Research design and methods The data of eculizumab in the FDA Adverse Event Reporting System (FAERS) database (from the first quarter of 2007 to the first quarter of 2023) was collected and analyzed. The case reports of adverse drug reactions (ADRs) related to eculizumab in PubMed, Embase and Web of Science before May 2023 were systematically reviewed. Results A total of 464 ADRs of eculizumab were identified in the FAERS database. The top five ADRs with the highest proportional reporting ratio (PRR) are total complement activity decreased, extravascular hemolysis, hemoglobinuria, total complement activity increased and breakthrough hemolysis. Fifty-one cases of ADR related to eculizumab were identified from 44 publications. The number of reported cases of eculizumab associated Neisseria gonorrhoeae infection in case reports was observed to be comparable to the number of cases of Neisseria meningitidis infection. Conclusions Clinicians must pay close attention to the risk of infections in patients receiving eculizumab, including severe N. meningitidis infection and other potentially fatal infections such as N. gonorrhoeae infection. In addition, The possible emergence of new ADRs should be vigilant during clinical medication.

Impact of STIs on cervical cancer screening: Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae in visual inspection with acetic acid (VIA) positive women in Mozambique.

Cervical cancer, primarily from HPV, is prevalent in countries like Mozambique, with HIV individuals at higher risk. The Visual Inspection with Acetic Acid (VIA) screening method can be influenced by STIs like Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). This study examines CT and NG prevalence in HIV-positive and negative women using VIA in Mozambique's DREAM program. In this cross-sectional research conducted at a DREAM program facility in Maputo from 01/07/2021 to 31/05/2022, cervical specimens were taken from VIA-positive patients. CT/NG testing was performed using the Cobas® 4800 DNA CT/NG test. Statistical analyses focused on associations and prevalence rates, considering demographic, clinical, and exposure data. Among 117 women, we observed a CT prevalence of 6.8% (8/117) and an NG prevalence of 2.6%(3/117). No significant associations between CT/NG infection rates and factors such as age, HIV status, VIA results, or high-risk HPV (hrHPV) was observed. We found a 47% prevalence of hrHPV infections among participants with cervical lesions; no significant association between hrHPV and CT/NG infections was observed. This study highlights the prevalence of CT and NG in VIA-positive women in Mozambique, emphasizing the STI burden and suggesting integration of STI screening in cervical cancer prevention strategies.

High-level ceftriaxone resistance due to transfer of penA allele 60.001 into endemic gonococcal lineages in Hangzhou, China.

Neisseria gonorrhoeae strains associated with the high-level ceftriaxone-resistant FC428 clone or containing its main resistance determinant, penA allele 60.001, have shown global transmission. In Hangzhou, China, 10% of the isolates were associated with the FC428 clone in 2019. Here, we investigated ceftriaxone resistance and the prevalence of FC428-associated strains in Hangzhou in 2020-22. A total of 209 gonococcal isolates were investigated for antimicrobial susceptibility to ceftriaxone and other antibiotics by agar dilution method. Sequence types and penA alleles were determined by PCR and sequence analysis. Resistance to ceftriaxone (MIC > 0.125 mg/L) was observed for 16% (33/209) of the isolates, whereas 6.7% (14/209) of the isolates displayed high-level ceftriaxone resistance (MIC = 1 mg/L). These 14 high-level ceftriaxone-resistant isolates and another isolate displaying an MIC = 0.25 mg/L contained penA allele 60.001, with eight of these isolates, all from 2020 to 2021 belonging to MLST ST1903, the sequence type commonly associated with the original FC428 clone. Importantly, the six penA allele 60.001-containing isolates from 2022 belonged to MLST ST8123, ST7365 and ST7367, which are among the most frequently encountered sequence types found in China. Therefore, these results indicate that endemic lineages in China have acquired penA allele 60.001. Here, we report continued transmission of gonococcal strains associated with the FC428 clone or containing penA allele 60.001 in Hangzhou. A major concern for public health is the acquisition of penA allele 60.001 by successful endemic lineages, which might enhance the transmission of this high-level ceftriaxone resistance trait.

Atypical presentation of herpes simplex virus 2 primary infection: a case report

BackgroundCervicitis, an infectious or noninfectious inflammation of the cervix, encompasses a wide range of clinical conditions, from asymptomatic infections to severe lesions, making its diagnosis difficult. Acute cervicitis may develop into pelvic inflammatory disease. In patients with cervicitis, current guidelines recommend testing for herpes simplex virus when external genital lesions are present. Here, we present the case of a patient with an atypical primary herpes simplex virus 2 infection manifesting as cervicitis without genital lesions.Case presentationA 29-year-old Caucasian woman was hospitalized for pelvic inflammatory disease. The patient complained of severe suprapubic pain, fever, and heavy vaginal discharge. The external genitalia were unremarkable, so empirical antibiotic treatment was initiated. Despite 48 hours of well-administered antibiotic therapy, her complaints persisted. Polymerase chain reaction for possible microbial causes was negative for Chlamydia trachomatis and Neisseria gonorrhoeae. There was no bacterial vaginosis. Repeat gynecological examinations with endovaginal ultrasound revealed an enlarged cervix, and pelvic magnetic resonance imaging supported a diagnosis of cervicitis. At this point, additional screening for other sexually transmitted infections and infectious disease-related etiologies of cervicitis was performed, and the polymerase chain reaction analysis of newly isolated samples was positive for herpes simplex virus 2. No antiviral treatment was initiated given the delay in diagnosing herpes simplex virus 2 infection and the slow but spontaneous abatement of symptoms.ConclusionHerpes simplex virus infection should be considered as a possible cause of cervicitis, even in the absence of typical genital lesions. Early detection of herpes simplex virus allows early treatment, helping to reduce the duration and severity of symptoms and therefore potentially reducing recurrences and improving disease control. These data and data from future cases might spur changes in the guidelines on cervicitis testing and treatment.

CRISPR-based platform for clinical diagnosis of Neisseria gonorrhoeae and simultaneous tracking of ceftriaxone-resistant FC428 clone

The global dissemination of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone poses substantial challenges in the control and treatment of gonorrhoea, resulting in heightened morbidity rates and the increased risk of public health emergencies. However, currently, the identification of N. gonorrhoeae FC428 clone is labor-intensive and laboratory-based, which is not suitable for rapid on-site analyses and effective decision making. Herein, an Equipment-free CRISPR/Cas12a assisted RPA platform is developed for rapid duplex detection (E-card), which provides a solution for early and effective clinical diagnosis of N. gonorrhoeae, particularly the simultaneous tracking of ceftriaxone-resistant FC428 clone to allow reasonable and timely antimicrobial therapy. The E-card achieves 1 copy/reaction for N. gonorrhoeae screening and 10 copies/reaction for ceftriaxone-resistant FC428 clone detection within 40 min. The accuracy of N. gonorrhoeae and FC428 identification has been validated by concordance rates of 100 % and 98 % between the E-card platform and PCR-sequencing, respectively. This convenient CRISPR-based disposable molecular diagnostic platform can serve as a powerful tool to rapid detection of N. gonorrhoeae and its ceftriaxone-resistant FC428 clone, enabling effective assessment of infection, effective medicine decision making and the mitigation of health burdens.

Impact of varying pre-exposure prophylaxis programs on HIV and Neisseria gonorrhoeae transmission among MSM in the Netherlands: a modelling study.

In 2019, a five-year pre-exposure prophylaxis (PrEP) program started in the Netherlands, in which up to 8,500 men who have sex with men (MSM) can obtain PrEP and 3-monthly consultations with HIV/STI testing. We assessed the impact of the PrEP program on transmission of HIV and Neisseria gonorrhea (NG) among MSM in the Netherlands and examined prospective variations of the program after 2024. We used an agent-based model to estimate the effect of the PrEP program. For prospective PrEP programs from 2024, we varied the capacity (8,500; 12,000; 16,000 participants) and consultation frequency (3-monthly; 6-monthly; 70% 3-monthly and 30% 6-monthly) for t. At a capacity of 8,500 participants and 3-monthly consultations, the PrEP program could lead to 3,140 (95%CrI 1,780 - 4,780) and 27,930 (95%CrI 14,560 - 46,280) averted HIV and NG infections; requiring 316,050 (95%CrI 314,120 - 317,580) consultations. At a capacity of 16,000 participants the programs with 3-monthly consultations and 6-monthly consultations could lead to comparable numbers of averted HIV (3,940 (95%CrI 2,420 - 5,460), and 3,900 (2,320 - 5,630) respectively) and NG infections (29,970 (95%CrI 15,490 - 50,350), and 29,960 (95%CrI 13,610 - 50,620) respectively), while requiring substantially different numbers of consultations: 589,330 (95%CrI 586,240 - 591,160) and 272,590 (95%CrI 271,770 - 273,290) respectively. Continuation of a PrEP program could lead to a substantial reduction in HIV and NG transmission. More infections could be averted if the number of participants is increased. In turn, the consultations frequency could be reduced without reducing the number of averted infections if capacity is increased.

Association between saliva use for masturbation and urethral gonorrhoea in men who have sex with men: A cross-sectional study

ObjectivesThe saliva of individuals with oropharyngeal gonorrhoea can contain viable Neisseria gonorrhoeae. This study examined if using saliva as a lubricant for masturbation is a risk factor for urethral gonorrhoea among men who have sex with men (MSM). MethodsIn this cross-sectional study, MSM aged ≥18 years attending the Melbourne Sexual Health Centre between February 2021 and December 2023 were surveyed. Data were collected on sexual activities in the past 7 days, including receiving fellatio, condomless insertive anal sex, docking, and using saliva for masturbation. Multivariable logistic regression examined associations between these exposures and urethral gonorrhoea. ResultsThe median age of the 3114 men was 32 (IQR: 27-40), with 4.7% (n = 145) testing positive for urethral gonorrhoea. Urethral gonorrhoea was independently associated with an increasing number of partners for condomless insertive anal sex (P < 0.001). It was not significantly associated with receiving fellatio (P = 0.613), docking (P = 0.207), or using saliva for masturbation (P = 0.117). However, of the 110 men who only used saliva for masturbation, two (1.8%) had urethral gonorrhoea, and one (0.9%) had both urethral and oropharyngeal gonorrhoea. ConclusionCondomless insertive anal sex is the leading risk factor for urethral gonorrhoea and not using saliva as a lubricant for masturbation.

Epidemiological impact of Neisseria gonorrhoeae and Chlamydia trachomatis screening in men having sex with men: a modelling study

ObjectivesThe impact of the systematic screening of Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) in men having sex with men (MSM) on these pathogens’ epidemiology remains unclear. We conducted a...

A molecular and phenotypic analysis of the prevalence and patterns of antimicrobial resistance in Neisseria gonorrhoeae isolates from Kwazulu Natal, South Africa

Antimicrobial drug resistance in Neisseria gonorrhoeae has been documented worldwide; however, the situation in Sub-Saharan Africa has received little attention. It is critical to establish diagnostics and extend surveillance to prevent the emergence of illnesses resistant to multiple treatments. Monitoring antimicrobial susceptibility is crucial to gather data that can inform treatment recommendations, resulting in effective therapy, reduced gonorrhea-related complications and transmission, and efficient treatment. Government authorities may set research and preventive objectives, as well as treatment recommendations, using data from the Gonococcal Antimicrobial Surveillance Program (GISP). Local and state health authorities may use GISP data to make decisions about the allocation of STI prevention services and resources, to guide preventive planning, and to disseminate information about the most successful treatment practices. Using molecular and culture approaches, we investigated the occurrence of antibiotic resistance in isolates from KwaZulu Natal, South Africa. The majority of gonococcal isolates (48 %) showed absolute resistance to ciprofloxacin, with penicillin and tetracycline resistance rates of 14 % each. Only one gonococcal isolate tested positive for azithromycin resistance, with a minimum inhibitory concentration (MIC) of 1.5 µg/mL. Ceftriaxone was effective against all gonococcal isolates tested.

Neisseria gonorrhoeae isolates resistant to extended spectrum cephalosporins and macrolides isolated from symptomatic men in western Kenya.

We characterized the antimicrobial resistance (AMR) profiles of Neisseria gonorrhoeae (NG) isolated from symptomatic men at a sexually transmitted infection clinic in Kisumu, Kenya. Two urethral swabs were obtained from symptomatic men between 2020 and 2022, one for Gram's stain and the other inoculated directly onto modified Thayer-Martin media containing 1% VCNT and 1% IsoVitaleX enrichment. Culture results were confirmed by colony morphology, Gram's stain and oxidase test. Duplicate isolates were shipped to Uniformed Services University for confirmation and characterization. Susceptibility to eight drugs was assessed by E-test. Agar dilution confirmed resistance to ceftriaxone, cefixime, and azithromycin. Susceptibility, intermediate resistance (IR), and resistance (R) were determined according to published criteria. Of 154 enrolled participants, 112 were culture-positive for NG. Agar dilution results in 110 (98.2%) showed the following: azithromycin-R (1.8%), and 4.5% R or IR to ceftriaxone or cefixime: ceftriaxone-R (0.9%), ceftriaxone-IR (2.7%), and cefixime-IR (2.7%). By E-test, most isolates were IR or R to tetracycline (97.2%), penicillin (90.9%), and ciprofloxacin (95.4%). We detected NG with resistance to azithromycin and ceftriaxone, indicating a growing threat to the current Kenyan dual syndromic treatment of urethritis with cephalosporin plus macrolides. Ongoing AMR surveillance is essential for effective drug choices.

Optimization of Ethoxzolamide Analogs with Improved Pharmacokinetic Properties for In Vivo Efficacy against Neisseria gonorrhoeae.

Drug-resistant gonorrhea is caused by the bacterial pathogen Neisseria gonorrhoeae, for which there is no recommended oral treatment. We have demonstrated that the FDA-approved human carbonic anhydrase inhibitor ethoxzolamide potently inhibits N. gonorrhoeae; however, is not effective at reducing N. gonorrhoeae bioburden in a mouse model. Thus, we sought to optimize the pharmacokinetic properties of the ethoxzolamide scaffold. These efforts resulted in analogs with improved activity against N. gonorrhoeae, increased metabolic stability in mouse liver microsomes, and improved Caco-2 permeability compared to ethoxzolamide. Improvement in these properties resulted in increased plasma exposure in vivo after oral dosing. Top compounds were investigated for in vivo efficacy in a vaginal mouse model of gonococcal genital tract infection, and they significantly decreased the gonococcal burden compared to vehicle and ethoxzolamide controls. Altogether, results from this study provide evidence that ethoxzolamide-based compounds have the potential to be effective oral therapeutics against gonococcal infection.

Expanding the genetic toolbox for Neisseria meningitidis with efficient tools for unmarked gene editing, complementation, and labeling.

The efficient natural transformation of Neisseria meningitidis allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic-resistance cassettes. However, this proved to be a double-edged sword, i.e., although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic-resistance markers. In addition, efficient tools for complementation or labeling are also lacking in N. meningitidis. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors), and cell labeling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.IMPORTANCENeisseria meningitidis and Neisseria gonorrhoeae are two important human pathogens. Research focusing on these bacteria requires genetic engineering, which is facilitated by their natural ability to undergo transformation. However, the ease of mutant engineering has led the Neisseria community to neglect the development of more sophisticated tools for gene editing, particularly for N. meningitidis. In this study, we have significantly expanded the meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant construction, genetic complementation, and cell tagging. This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.