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 tricarboxylic acid (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 toward acetate overflow for Adenosine triphosphate (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.

Experimental genital tract infection demonstrates Neisseria gonorrhoeae MtrCDE efflux pump is not required for in vivo human infection and identifies gonococcal colonization bottleneck.

The MtrCDE efflux pump of Neisseria gonorrhoeae exports a wide range of antimicrobial compounds that the gonococcus encounters at mucosal surfaces during colonization and infection and is a known gonococcal virulence factor. Here, we evaluate the role of this efflux pump system in strain FA1090 during in vivo human male urethral infection with N. gonorrhoeae using a controlled human infection model. With the strategy of competitive infections initiated with mixtures of wild-type FA1090 and an isogenic mutant FA1090 strain that does not contain a functional MtrCDE pump, we found that the presence of the efflux pump is not required for an infection to be established in the human male urethra. This finding contrasts with previous studies of in vivo infection in the lower genital tract of female mice, which demonstrated that mutant gonococci of a different strain (FA19) lacking a functional MtrCDE pump had a significantly reduced fitness compared to their wild-type parental FA19 strain. To determine if these conflicting results are due to strain or human vs. mouse differences, we conducted a series of systematic competitive infections in female mice with the same FA1090 strains as in humans, and with FA19 strains, including mutants that do not assemble a functional MtrCDE efflux pump. Our results indicate the fitness advantage provided by the MtrCDE efflux pump during infection of mice is strain dependent. Owing to the equal fitness of the two FA1090 strains in men, our experiments also demonstrated the presence of a colonization bottleneck of N. gonorrhoeae in the human male urethra, which may open a new area of inquiry into N. gonorrhoeae infection dynamics and control. TRIAL REGISTRATION. Clinicaltrials.gov NCT03840811.

A Retrospective Analysis of Four STI Pathogens in Obstetrics and Gynecological Patients in Hangzhou, China.

BACKGROUND: Among sexually transmitted infection (STI) pathogens, UU, CT, NG, and MG are very commonly detected. A retrospective study was conducted to analyze the clinical situations and laboratory data of patients in-fected with the four pathogens in Hangzhou city, Zhejiang Province, China. METHODS: For this study, 4,716 female outpatients and inpatients of the Department of Obstetrics and Gynecology of Hangzhou Third People´s Hospital were randomly selected from January 2019 to December 2023. Multiple types of specimens were collected and four STI pathogens were detected. Data collection was taken from the hospital's electronic medical records and statistical analysis was processed with SPSS 25.0 software. RESULTS: In the past five years, year by year, the positive rate presented an increasing trend. Out of 4,716 patients, 2,931 were positive, and the positive rate was 62.15%. The positive rate of single infection was significantly higher than of co-infection (p < 0.05). The highest positive rate of all STI patients was found in the  20 age group, and the rate was significantly different between each age group (p < 0.05). Single infection with UU occurred significantly more often than with the other three pathogens (p < 0.05), and co-infection with UU and CT and UU, CT, and MG were significantly dominating (p < 0.05). Only three patients had an infection with all four pathogens, and it was extremely rare. The 21 - 30 and 31 - 40 age groups had high incidences of infection, with a significant difference compared with other age groups (p < 0.05). CONCLUSIONS: The positive rate shows an increasing trend in Hangzhou in the past five years. More single infection than co-infection and more younger patients were detected. Single infection with UU was dominant in all positive patients. The publicity and more screening testing items of STI for younger people should be strengthened in Hangzhou, and the people of Hangzhou should be made more aware that information technology is helpful in aiding the control of STI diseases.

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.

The incubation for urethral gonorrhoea among men who have sex with men with and without oropharyngeal gonorrhoea.

We hypothesized that the incubation for urethral gonorrhoea would be longer for men with oropharyngeal gonorrhoea than those without oropharyngeal gonorrhoea. We conducted a chart review of men who have sex with men with urethral gonorrhoea symptoms at a sexual health clinic between 2019 and 2021. The incubation period was defined as the number of days between men's last sexual contact and onset of symptoms. We used a Mann-Whitney U test to compare differences in the median incubation for urethral gonorrhoea between men with and men without oropharyngeal gonorrhoea. There were 338 men with urethral symptoms (median age = 32 years; IQR: 28-39), and of these, 307 (90.1%) were tested for oropharyngeal gonorrhoea, of whom 124 (40.4%, 95% CI: 34.9-46.1) men had oropharyngeal and urethral gonorrhoea. We analyzed incubation data available for 190 (61.9%) of the 307 men, with 38.9% (74/190) testing positive for oropharyngeal gonorrhoea. The incubation for urethral gonorrhoea did not differ between 74 men (39%) with oropharyngeal gonorrhoea (median = 4 days; IQR: 2-6) and 116 men (61%) without oropharyngeal gonorrhoea (median = 2.5 days; IQR: 1-5) (p = 0.092). Research is needed to investigate gonorrhoea transmission from the oropharynx to the urethra.

Longitudinal genomic analysis of Neisseria gonorrhoeae transmission dynamics in Australia.

N. gonorrhoeae, which causes the sexually transmissible infection gonorrhoea, remains a significant public health threat globally, with challenges posed by increasing transmission and antimicrobial resistance (AMR). The COVID-19 pandemic introduced exceptional circumstances into communicable disease control, impacting the transmission of gonorrhoea and other infectious diseases. Through phylogenomic and phylodynamic analysis of 5881 N. gonorrhoeae genomes from Australia, we investigated N. gonorrhoeae transmission over five years, including a time period during the COVID-19 pandemic. Using a novel cgMLST-based genetic threshold, we demonstrate persistence of large N. gonorrhoeae genomic clusters over several years, with some persistent clusters associated with heterosexual transmission. We observed a decline in both N. gonorrhoeae transmission and genomic diversity during the COVID-19 pandemic, suggestive of an evolutionary bottleneck. The longitudinal, occult transmission of N. gonorrhoeae over many years further highlights the urgent need for improved diagnostic, treatment, and prevention strategies for gonorrhoea.

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

BACKGROUND: We characterized the antimicrobial resistance (AMR) profiles of Neisseria gonorrhoeae (NG) isolated from symptomatic men at a sexually transmitted infection clinic in Kisumu, Kenya. METHODS: 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. RESULTS: 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%). CONCLUSIONS: 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.

Zap the clap with DNA: a novel microbicide for preventing Neisseria gonorrhoeae infection.

Each year, Neisseria gonorrhoeae (Ngo) causes over 1.5 million new infections in the United States, and >87 million worldwide. The absence of a vaccine for preventing gonorrhea, the rapid emergence of multidrug-resistant and extremely drug-resistant Ngo strains, and the limited number of antibiotics available for treating gonorrhea underscore the importance of developing new modalities for addressing Ngo infection. Here, we describe DNA-based microbicides that kill Ngo but not commensals. Previously, we showed that Ngo is killed when it takes up differentially methylated DNA with homology to its genome. We exploited this Achilles heel to develop a new class of microbicides for preventing Ngo infection. These microbicides consist of DNA molecules with specific sequences and a methylation pattern different from Ngo DNA. These DNAs kill low-passage and antibiotic-resistant clinical isolates with high efficiency but leave commensals unharmed. Equally important, the DNAs are equally effective against Ngo whether they are in buffered media or personal lubricants. These findings illustrate the potential of this new class of practical, low-cost, self-administered DNA-based microbicides for preventing Ngo transmission during sexual intercourse.

Neisseria gonorrhoeae ST-1901 in Rio de Janeiro from 2006 to 2022: Phylogeny and antimicrobial resistance evolution of a well-succeeded pathogen.

Neisseria gonorrhoeae is a global threat to public health due to the accumulation of antimicrobial resistance mechanisms. ST-1901 is an internationally important sequence type (ST) because of its high incidence and the usual occurrence of chromosomally determined resistance. In this study, we describe the evolution of the ST-1901 and its single locus variants in Rio de Janeiro from 2006 to 2022. We analyzed 82 N. gonorrhoeae isolates according to antimicrobial susceptibility profile, resistance mechanisms, molecular typing, and phylogenetics. Six different single locus variants were detected. Phylogenetic analysis identified five clades, which share similar characteristics. Resistance rates for penicillin and tetracycline decreased due to the lower occurrence of resistance plasmids, but intermediary resistance to penicillin rose. Resistance to ciprofloxacin remained high throughout all clades and the years of the study. Regarding resistance to azithromycin, alterations in mtrR promoter and gene, and 23S rRNA encoding gene rrl were detected, with a notable rise in the incidence of C2611T mutations in more recent years occurring in four of five clades. In contrast, ß-lactam resistance associated penA 34 mosaic was found only in one persisting clade (Clade D), and unique G45D and A39T mutations in mtrR gene and its promoter (Nm-Like) were found only in Clade B. Taken together, these data suggest that ST-1901, a persistently circulating lineage of N. gonorrhoeae in Rio de Janeiro, has undergone changes over the years and may evolve to develop resistance to the current recommended dual therapy adopted in Brazil, namely, ceftriaxone and azithromycin.

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.

Prevalence of Four Sexually Transmitted Infection Pathogens Detected in Urine and Genital Tract Secretion in Hangzhou, China.

BACKGROUND: Sexually transmitted infections (STIs) increase gradually and have become a public health problem in the world. UU, CT, NG, and MG are four common STI pathogens. Our retrospective study analyzed the clinical situation and the laboratory data of patients infected with the four pathogens. The prevalence of the four pathogens, detected in urine and genital tract secretion, was studied in Hangzhou, China. METHODS: A total of 3,168 male and female patients were randomly selected from February 2023 to February 2024. Urine and genital secretions were collected, and four STI pathogens were controlled for detection. Data were collected from the hospital's electronic medical records, and SPSS 25.0 software was used to perform a statistical analysis. RESULTS: Among 3,168 patients, a total of 1,527 were detected as positive, and the positive rate was 48.20%. The age of patients ranged from 13 - 98 years, with an average age of 45.6. The total of patients consisted of 2,191 males and 977 females, which had a significant difference (p < 0.05). Specimens were mainly collected from the Department of Dermatovenerology, Urological Surgery, Obstetrics and Gynecology, and so on. The positive rate was statistically different between male and female patients (p < 0.05). Single infection performed a main role and accounted for 79.57% of all of the positive patients. In the ≤ 20 age group, the positive rate was the highest and was as high as 77.65%. In detail, single infection caused by UU dominated, especially in the 21 - 30 age group. Double infection caused by UU and CT and triple infection caused by UU, CT, and NG were the majority, both especially in the 21 - 30 age group. There were significant differences in the positive rates in the different age groups and in the four pathogens (p < 0.05). Quadruple infection was very rare and had only been detected in one patient. CONCLUSIONS: The prevalence of the four pathogens in Hangzhou was different from other regions. More male than female patients, more single than multiple infections, and more single and multiple infections occurring in young people were the features in Hangzhou. The study would provide reference for prevention, diagnosis, and treatment of STI.

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.

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.

Combination of compound screening with an animal model identifies pentamidine to prevent Chlamydia trachomatis infection.

Chlamydia trachomatis (Ct) is the most common cause for bacterial sexually transmitted infections (STIs) worldwide with a tremendous impact on public health. With the aim to unravel novel targets of the chlamydia life cycle, we screen a compound library and identify 28 agents to significantly reduce Ct growth. The known anti-infective agent pentamidine-one of the top candidates of the screen-shows anti-chlamydia activity in low concentrations by changing the metabolism of host cells impairing chlamydia growth. Furthermore, it effectively decreases the Ct burden upon local or systemic application in mice. Pentamidine also inhibits the growth of Neisseria gonorrhea (Ng), which is a common co-infection of Ct. The conducted compound screen is powerful in exploring antimicrobial compounds against Ct in a medium-throughput format. Following thorough in vitro and in vivo assessments, pentamidine emerges as a promising agent for topical prophylaxis or treatment against Ct and possibly other bacterial STIs.