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.

Where would young people using an online STI testing service want to be treated? A survey of young Australians.

Background Although there has been growth in online STI testing services, more attention is needed to understand how to facilitate effective treatment pathways for users. This study investigated where young people want to be treated for gonorrhoea and syphilis if they test positive using an online service. Methods We conducted an online survey of Australians aged 16-29years that included multiple choice and free-text questions about their preferred location for receiving injectable antibiotics. Multivariable multinomial logistic regression examined associations between respondent characteristics and service preferences. Content analysis was used to code free-text responses. Results Among 905 survey respondents, 777 (85.9%) answered questions on treatment preferences. Respondents most commonly preferred injectable antibiotics provided by a sexual health clinic (294; 37.8%) or a nurse in a pharmacy (208; 26.8%). Gender/sexually diverse respondents were more likely to select sexual health clinics over general practice (MSM RRR 2.5, 95% CI 1.1-5.7; WSW RRR 2.6, 95% CI 1.1-5.7; trans/non-binary RRR 2.5; 95% CI 1.0-6.0). Older respondents (aged 25-29years) were more likely to choose all alternatives over general practice, with the reverse found for those who had previously tested. From open-text answers, pharmacies were valued for their convenience, and sexual health clinics for providing non-judgemental, free services by specialists. Conclusions Differences in treatment preferences by certain groups of young people suggest that different service offerings may influence treatment-seeking outcomes from online STI testing services.

Risk Factors for Delayed Treatment of Gonorrhea and Chlamydia in Active-Duty Service Members.

BACKGROUND: Sexually transmitted infections including gonorrhea and chlamydia are common in the active-duty military population, with historically higher rates than their civilian counterparts. Prevention and screening are 2 of the main strategies used to reduce the chronic medical complications and costs associated with untreated gonorrhea and chlamydia; however, there is little information in the literature regarding treatment time after a positive screening. To our knowledge, there has not yet been a study regarding delayed treatment of gonorrhea and chlamydia in the active-duty population. METHODS: We performed a population-based retrospective observational study on active-duty service members (ADSMs) diagnosed with gonorrhea and chlamydia from 2010-2019. Statistical analysis was performed to determine differences in treatment times for key demographics. This study was reviewed and approved by the Brooke Army Medical Center Institutional Review Board. RESULTS: Average treatment time was 3.5 days for individuals with chlamydia and 5 days for those with gonorrhea. Treatment within 2 weeks was met for 94% of people diagnosed with chlamydia and 91% of people diagnosed with gonorrhea. Delay in treatment times for chlamydia were seen in men, ages 25-34, full-time active-duty service members, those with a history of prior infection, and soldiers in the Army. Gonorrhea treatment times were delayed in men, members of the Coast Guard, ages 35-44, and those with a history of prior infection. CONCLUSIONS: Significant differences in treatment time were seen based on sex, age, branch of service, rank, and history of prior infection.

[Do not overuse doxycycline as prophylaxis for uncomplicated sexually transmitted infections]. / Överanvänd inte doxycyklin som profylax mot STI.

STI prophylaxis using doxycycline is discussed internationally for persons at high risk of STIs (Doxy-PEP). Doxy-PEP would probably have limited effect on gonorrhoea due to resistance to tetracyclines. Doxy-PEP may reduce the incidence of chlamydia and syphilis, but would not reduce the number of complicated infections. Further studies are needed on the effects of intermittent antibiotic use on the microbiome or antibiotic resistance in general.

Cyclization increases bactericidal activity of arginine-rich cationic cell-penetrating peptide for Neisseria gonorrhoeae.

We previously reported that a linear cationic 12-amino acid cell-penetrating peptide (CPP) was bactericidal for Neisseria gonorrhoeae. In this study, our objectives were to determine the effect of cyclization of the linear CPP on its antibacterial activity for N. gonorrhoeae and cytotoxicity for human cells. We compared the bactericidal effect of 4-hour treatment with the linear CPP to that of CPPs cyclized by a thioether or a disulfide bond on human challenge and multi-drug resistant (MDR) strains of N. gonorrhoeae grown in cell culture media with 10% fetal bovine serum (FBS). The effect of lipooligosaccharide (LOS) sialylation on bactericidal activity was analyzed. We determined the ability of the CPPs to treat human cells infected in vitro with N. gonorrhoeae, to reduce the inflammatory response of human monocytic cells to gonococci, to kill strains of three commensal Neisseria species, and to inhibit gonococcal biofilms. The cyclized CPPs killed 100% of gonococci from all strains at 100 µM and >90% at 20 µM and were more potent than the linear form. The thioether-linked but not the disulfide-linked CPP was less cytotoxic for human cervical cells compared to the linear CPP. LOS sialylation had minimal effect on bactericidal activity. In treating infected human cells, the thioether-linked CPP at 20 µM killed >60% of extra- and intracellular bacteria and reduced TNF-α expression by THP-1 cells. The potency of the CPPs for the pathogenic and the commensal Neisseria was similar. The thioether-linked CPP partially eradicated gonococcal biofilms. Future studies will focus on determining efficacy in the female mouse model of gonorrhea.IMPORTANCENeisseria gonorrhoeae remains a major cause of sexually transmitted infections with 82 million cases worldwide in 2020, and 710,151 confirmed cases in the US in 2021, up 25% from 2017. N. gonorrhoeae can infect multiple tissues including the urethra, cervix, rectum, pharynx, and conjunctiva. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease, chronic pelvic pain, and infertility in 10%-20% of women. Control of gonococcal infection is widely recognized as increasingly challenging due to the lack of any vaccine. N. gonorrhoeae has quickly developed resistance to all but one class of antibiotics and the emergence of multidrug-resistant strains could result in untreatable infections. As such, gonorrhea is classified by the Center for Disease Control (CDC) as an urgent public health threat. The research presented herein on new therapeutics for gonorrhea has identified a cyclic cell-penetrating peptide (CPP) as a potent molecule targeting N. gonorrhoeae.

Providing adolescent-friendly sexually transmitted infection screening and treatment services.

PURPOSE OF REVIEW: There are high rates of sexually transmitted infections (STIs) worldwide. Adolescents and young adults (AYA) ages 15-24 years remain one of the populations that is most vulnerable to STIs. The goal of this review is to summarize recent international updates in adolescent STI screening and treatment. RECENT FINDINGS: Normalizing sexual history taking and STI testing, and advocating for adolescents to receive comprehensive sexuality education improves stigma surrounding sexual health. The global rise in syphilis is pervasive and includes high rates of infection among AYA and women of reproductive age - universal screening may be indicated depending on local epidemiology. Gonococcal antimicrobial resistance remains a significant public health concern worldwide, thus judicious use of antimicrobials and reporting cases of resistance is crucial. Sexual health services are increasingly using virtual platforms, which may be an effective strategy for STI testing and treatment among AYA. SUMMARY: Specific areas of focus to address the STI epidemic among AYA include reducing stigma surrounding sexual health, screening, and treatment of STIs, especially with the global rise in syphilis and high rates of gonorrhea resistance, in addition to increased use of telehealth services as effective education and intervention strategies.

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.

Impact of point-of-care gonorrhea and chlamydia testing in the emergency department on reducing overtreatment rates.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections continue to increase in the United States. Advancement in technology with point-of-care (POC) testing can improve the overall treatment of sexually transmitted infections (STI) in the emergency department (ED) by shortening the time to test result and administration of accurate treatment. The purpose of this study was to assess if the POC test reduced the rate of overtreatment for CT and/or NG compared to the standard-of-care (SOC) test. METHODS: This retrospective cohort study included adult patients tested for CT and NG at two urban EDs between August 2020 and October 2022. This cohort excluded hospital admissions, elopement, pregnancy, rectal and oral samples, victims of sexual assault, and diagnoses for which antimicrobial treatment overlapped that of CT/NG. The primary outcome assessed overtreatment, defined as receiving treatment in the ED or a prescription prior to discharge for patients who tested negative for CT and/or NG. Secondary outcomes included undertreatment rates, overtreatment rates in select populations, test turnaround time, and ED length of stay (LOS). RESULTS: Of 327 patients screened, 97 patients were included in the SOC group and 100 in POC. Overtreatment for CT was provided in zero POC patients and 29 (29.9%) SOC patients (p < 0.001). NG was overtreated in 1 (1%) POC and 23 (23.7%) SOC (p < 0.001). POC was associated with undertreatment of CT and/or NG in two patients, compared to four patients tested with SOC. Overall, treatment was deemed inappropriate for 5 (5%) of those tested with POC, compared to 35 (36%) tested with SOC (p < 0.001). There was no difference in ED LOS (2.7 vs 3.01 h, p = 0.41). CONCLUSIONS: POC testing facilitated the return of results prior to patients being discharged from the ED. Compared to standard testing, POC improved appropriateness of CT and NG treatment by reducing the rates of overtreatment.

First-line treatment with ceftriaxone for Neisseria gonorrhoeae infection less likely to be prescribed to patients with a penicillin allergy label: a retrospective audit of medical records.

Background Gonorrhoea notifications have increased substantially in Australia over the past decade. Neisseria gonorrhoeae is already highly resistant to several antibiotics and so, alternatives to first-line treatment are generally strongly discouraged. The penicillin allergy label (AL) on patient medical records has previously been shown to influence prescribing practices, to the detriment of best-practice management and antimicrobial stewardship. This study aimed to understand how the penicillin AL influences antibiotic selection for gonorrhoea treatment at Canberra Sexual Health Centre. Methods A retrospective chart audit of gonorrhoea cases treated at Canberra Sexual Health Centre between January 2020 and October 2023 (n =619 patients, n =728 cases). Antibiotic selection was assessed according to penicillin AL status. Ceftriaxone selection was assessed according to penicillin allergy severity reported in the medical records and as determined using a validated antibiotic allergy assessment tool. Results Cases with a penicillin AL were more likely to receive antibiotics other than ceftriaxone (n =7/41, 17.1%) than cases without the label (n =8/687, 1.2%, P n =28/41, 68.3%) to apply the assessment tool. Those reported as low-severity in the records were more likely to receive ceftriaxone (n =21/22, 95.5%) than those reported as moderate-high (n =7/11, 63.6%) or unreported (n =6/8, 0.75%). Conclusions Treatment of gonorrhoea in outpatient settings requires an understanding of penicillin allergy, and the ability to quickly and accurately identify penicillin-AL patients who can safely tolerate ceftriaxone. Institutionally endorsed penicillin allergy de-labelling protocols and access to easy-to-navigate prescribing advice within national sexually transmitted infection management guidelines would support this.

Impact of mutations in the mtrR, rpdlVD and rrl genes on azithromycin resistance in Neisseria gonorrhoeae.

INTRODUCTION: Bacterial sexually transmitted infections (STIs) pose a major public health problem. The emergence of antibiotic-resistant strains of Neisseria gonorrhoeae represents a serious threat to successful treatment and epidemiological control. The first extensively drug-resistant (XDR) strains (ceftriaxone-resistant and high-level azithromycin-resistant [HLR AZY]) have been reported. AIMS: To identify molecular mechanisms implicated in azithromycin resistance in strains isolated from patients over a three-year period in a university hospital in Switzerland. MATERIAL AND METHODS: From January 2020 to December 2022, 34 isolates (one per patient) were recovered from samples analyzed at the University Hospital of Lausanne. Eight genes involved in azithromycin resistance were sequenced: mtrR repressor (mtrCDE operon repressor) and his promotor mtrR-pr, rplD gene (L4 ribosomal protein), rplV gene (L22 ribosomal protein) and the four alleles of the rrl gene (23S rRNA). RESULTS: With a cutoff value of 1 mg/L, 15 isolates were considered as being resistant to azithromycin, whereas the remaining 19 were susceptible. The C2597T mutation in 3 or 4 of the rrl allele confer a medium-level resistance to azithromycin (MIC = 16 mg/L, N = 2). The following mutations were significantly associated with MIC values ≥1 mg/L: the three mutations V125A, A147G, R157Q in the rplD gene (N = 10) and a substitution A->C in the mtrR promotor (N = 9). Specific mutations in the mtrR repressor and its promotor were observed in both susceptible and resistant isolates. CONCLUSIONS: Resistance to azithromycin was explained by the presence of mutations in many different copies of 23S RNA ribosomal genes and their regulatory genes. Other mutations, previously reported to be associated with azithromycin resistance, were documented in both susceptible and resistant isolates, suggesting they play little role, if any, in azithromycin resistance.

Assessing thresholds of resistance prevalence at which empiric treatment of gonorrhea should change among men who have sex with men in the US: A cost-effectiveness analysis.

BACKGROUND: Since common diagnostic tests for gonorrhea do not provide information about susceptibility to antibiotics, treatment of gonorrhea remains empiric. Antibiotics used for empiric therapy are usually changed once resistance prevalence exceeds a certain threshold (e.g., 5%). A low switch threshold is intended to increase the probability that an infection is successfully treated with the first-line antibiotic, but it could also increase the pace at which recommendations are switched to newer antibiotics. Little is known about the impact of changing the switch threshold on the incidence of gonorrhea, the rate of treatment failure, and the overall cost and quality-adjusted life-years (QALYs) associated with gonorrhea. METHODS AND FINDINGS: We developed a transmission model of gonococcal infection with multiple resistant strains to project gonorrhea-associated costs and loss in QALYs under different switch thresholds among men who have sex with men (MSM) in the United States. We accounted for the costs and disutilities associated with symptoms, diagnosis, treatment, and sequelae, and combined costs and QALYs in a measure of net health benefit (NHB). Our results suggest that under a scenario where 3 antibiotics are available over the next 50 years (2 suitable for the first-line therapy of gonorrhea and 1 suitable only for the retreatment of resistant infections), changing the switch threshold between 1% and 10% does not meaningfully impact the annual number of gonorrhea cases, total costs, or total QALY losses associated with gonorrhea. However, if a new antibiotic is to become available in the future, choosing a lower switch threshold could improve the population NHB. If in addition, drug-susceptibility testing (DST) is available to inform retreatment regimens after unsuccessful first-line therapy, setting the switch threshold at 1% to 2% is expected to maximize the population NHB. A limitation of our study is that our analysis only focuses on the MSM population and does not consider the influence of interventions such as vaccine and common use of rapid drugs susceptibility tests to inform first-line therapy. CONCLUSIONS: Changing the switch threshold for first-line antibiotics may not substantially change the health and financial outcomes associated with gonorrhea. However, the switch threshold could be reduced when newer antibiotics are expected to become available soon or when in addition to future novel antibiotics, DST is also available to inform retreatment regimens.

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.

Susceptibility of Neisseria gonorrhoeae against a dual treatment antibiotics regimen in primary health centres in Surabaya, Indonesia.

BACKGROUND AND OBJECTIVES: There were 82.4 million new gonorrhoea cases worldwide in 2020. Dual treatment with ceftriaxone or cefixime and azithromycin or doxycycline is currently recommended for gonorrhoea in Indonesia. However, reduced susceptibility and resistance to cephalosporins and azithromycin are increasing. We evaluated the susceptibility pattern of Neisseria gonorrhoeae to cefixime, ceftriaxone, azithromycin and doxycycline. METHOD: N. gonorrhoeae isolates were obtained from 19 male participants with clinically and laboratory-confirmed gonorrhoea. Antibiotic susceptibility testing was conducted by disc diffusion and interpreted according to Clinical and Laboratory Standards Institute and Centers for Disease Control and Prevention criteria. RESULTS: Reduced susceptibility or resistance was observed against doxycycline in 19 isolates (100%), cefixime in six (31.6%), ceftriaxone in three (15.8%) and azithromycin in zero (0%) isolates. DISCUSSION: A dual treatment regimen with ceftriaxone and azithromycin can still be recommended as first-line therapy for gonorrhoea in Indonesia. Antibiotic susceptibility surveillance of N. gonorrhoeae should be routinely conducted.

Data quality assessment of the Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP), Thailand, 2015-2021.

BACKGROUND: Quality assessments of gonococcal surveillance data are critical to improve data validity and to enhance the value of surveillance findings. Detecting data errors by systematic audits identifies areas for quality improvement. We designed and implemented an internal audit process to evaluate the accuracy and completeness of surveillance data for the Thailand Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP). METHODS: We conducted a data quality audit of source records by comparison with the data stored in the EGASP database for five audit cycles from 2015-2021. Ten percent of culture-confirmed cases of Neisseria gonorrhoeae were randomly sampled along with any cases identified with elevated antimicrobial susceptibility testing results and cases with repeat infections. Incorrect and incomplete data were investigated, and corrective action and preventive actions (CAPA) were implemented. Accuracy was defined as the percentage of identical data in both the source records and the database. Completeness was defined as the percentage of non-missing data from either the source document or the database. Statistical analyses were performed using the t-test and the Fisher's exact test. RESULTS: We sampled and reviewed 70, 162, 85, 68, and 46 EGASP records during the five audit cycles. Overall accuracy and completeness in the five audit cycles ranged from 93.6% to 99.4% and 95.0% to 99.9%, respectively. Overall, completeness was significantly higher than accuracy (p = 0.017). For each laboratory and clinical data element, concordance was >85% in all audit cycles except for two laboratory data elements in two audit cycles. These elements significantly improved following identification and CAPA implementation. DISCUSSION: We found a high level of data accuracy and completeness in the five audit cycles. The implementation of the audit process identified areas for improvement. Systematic quality assessments of laboratory and clinical data ensure high quality EGASP surveillance data to monitor for antimicrobial resistant Neisseria gonorrhoeae in Thailand.

Gonorrhoea treatment guideline compliance and influence factors in Guangdong province, China: a cross-sectional survey.

BACKGROUND: The indiscriminate use of antibiotics has accelerated antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG), emphasising the need to follow treatment guidelines. This study aimed to assess the rate of adherence to standard treatment among patients with gonorrhoea and identify influencing factors. METHODS: A survey was conducted in Guangdong province, China, involving uncomplicated gonorrhoea cases registered in the Chinese Information System for Disease Control and Prevention. Data on demographic characteristics and medical information were collected to determine the standard treatment rate, defined as the proportion of patients receiving treatment according to national guidelines (ie, a single dose of ceftriaxone 250 mg, spectinomycin 2 g, cefotaxime 1 g or other third-generation cephalosporins). Medication choices were documented. χ² tests and multilevel logistic regression were used to analyse factors associated with standard treatment. RESULTS: The survey included 2424 patients with gonorrhoea from 59 hospitals. The standard treatment rate was 30.7% (743/2424), with 36.2% for females and 29.6% for males. Common reasons for substandard treatment included the use of non-guideline medications (42.3%, 710/1681) and incorrect dosing (36.2%, 605/1681). Factors associated with the standard treatment rate included gender, address, educational level, department, physicians' training, number of diagnosed gonorrhoea cases and hospital level. CONCLUSION: The standard treatment rate for gonorrhoea in Guangdong province, China, is below expectations. Comprehensive measures, such as establishing a goal-directed monitoring system and implementing promotional activities, are needed to improve adherence to treatment guidelines.

Surge in Ceftriaxone-Resistant Neisseria gonorrhoeae FC428-Like Strains, Asia-Pacific Region, 2015-2022.

Ceftriaxone-resistant Neisseria gonorrhoeae FC428-like strains have disseminated across the Asia-Pacific region, with a continuous rise in prevalence during 2015-2022. To mitigate the effect of these strains, we advocate for enhanced molecular diagnostics, expanded surveillance networks, and a regionally coordinated effort to combat the global spread of FC428-like strains.