Prevalence and Risk Factors of Penicillinase-Type β-Lactamase Producing Neisseria gonorrhoeae Isolated from Patients Attending Health-Facilities in Yaounde, Cameroon

Background and Objectives: Mitigation of antibiotic resistant Neisseria gonorrhoeae has become a priority due to considerable health and economical disabilities it generates. In order to tackle the emergence of resistant Neisseria gonorrhoeae, this study aimed to determine the prevalence and risk factors of penicillinase type β-lactamase-producing Neisseria gonorrheae among patients consulting for genital infectious disorders in two health-facilities in Yaounde, Cameroon. Materials and Method: A cross-sectional descriptive and analytical study was conducted over a 3-month period, from July 2nd to October 2nd, 2022. Vaginal and urethral secretions were collected. Biochemical identification tests were performed on colonies grown on chocolate agar + polyvitex using the Api NH gallery. The detection of penicillinases was equally performed using the API NH gallery and confirmed using the antimicrobial susceptibility testing. The Minimum Inhibitory Concentrations of some antibiotics were determined using the E-Test. Results: The results showed that out of the 198 patients sampled, 16 (8.08%) were positive for Neisseria gonorrhoeae, among which 13/16 (81.25%) were penicillinase-type β-lactamase producers. Antimicrobial susceptibility testing results showed high co-resistances to antibiotics, mainly ciprofloxacin (100%), nalidixic acid (92.31%) and azithromycin (84.62%). Moreover, high Minimum Inhibitory Concentrations of ceftriaxone (ranging from 6 to 24 mg/L) was observed toward Neisseria gonorrhoeae isolates. The risk factors of the carriage of penicillinase-type β-lactamase producing Neisseria gonorrhoeae identified were: a history of Sexually Transmitted infections (p = 0.01) and unprotected sexual intercourse (p = 0.01). Conclusion: The emergence of penicillinase-type β-lactamase producing Neisseria gonorrhoeae is increasing and the situation is becoming worrisome. The identified risk factors can constitute a basic outlook to tackle resistant Neisseria gonorrhoeae, and therefore sustain antibiotic stewardship.

PCR Detection of Neisseria Gonorrhoeae Infection in Women by Self Obtained Low Vaginal Swabs

Objective: To evaluate the performance of polymerasechain reaction (PCR) using self obtained low vaginalswabs (SOLVS) to detect Neisseria gonorrhoeae (NG).Methods: One SOLVS and two cervical swabs werecollected from each of 298 female STD clinic attendees.PCR and culture were performed on both samples todetect NG.Results: Thirty-three cases of gonorrhoeae werediagnosed. The sensitivity of cervical culture, cervi-cal swab PCR and SOLVS PCR were 75.8% (25/33),87.9% (29/33) and 97.0% (32/33), respectively. Thespecificities of the respective methods were 100%(265/265), 99.6% (264/265) and 99.6% (264/265).The positive predictive values (PPVs) of the respectivemethods were 100% (25/25), 96.7% (29/30) and97.0% (32/33). The negative predictive values (NPVs)of the respective methods were 97.1% (265/273),98.5% (264/268) and 99.6% (264/265).Conclusions: The performance of SOLVS PCR is atleast as good as that of conventional cervical PCR tech-niques for the detection of NG. SOLVS may take theplace of cervical swabs for screening of NG infectionin women by PCR.

Typing of Neisseria gonorrhoeae Opa and NG-MAST Gene of 12 Pairs of Sexual Contact Gonorrhea Patients in China

To identify the genomic species of Neisseria gonorrhoeae, evaluate the difference between two molecular epidemiological methods and examine the relationship between sex partners and genotypes of bacteria, 24 strains of Neisseria gonorrhoeae isolated from the outpatients with gonorrhea were identified by using the Opa genotyping and NG-MAST genotyping and the relationship between genotypes and phenotypes was studied. Twenty-four strains of Neisseria gonorrhoeae fell into 10 ST genotypes by NG-MAST genotyping, whereas these strains were classified into 12 OT Opa genotypes by Opa genotyping. A new epidemic strain of ST genotype （217-86% homologisation 178） in China was identified. It is concluded that genotypes of each pair of strains from a pair of patient/sex partner besides 45/46 are the same, indicating that contagious infection take place between patient and the sex partner. Opa genotyping was more effective than NG-MAST genotyping in identifying the genomic species of Neisseria gonorrhoeae. ST genotype could be further classified into different Opa-types.

Fluoroquinolone Resistance and Mutation Patterns in gyrA and parC Genes in Neisseria gonorrhoeae Isolates from Shanghai, China

In order to study the resistance of Neisseria （N.） gonorrhoeae to the fluoroquinolone and detect mutation patterns of quinolone resistance-determining regions （QRDRs） of clinical isolates in Shanghai, China, a total of 80 clinical isolates of N. gonorrhoeae were consecutively collected from Shanghai. The MIC of fluoroquinolone for the isolates was examined by using the agar dilution method and the mutation profiles of the QRDRs of gyrA and parC were analyzed by sequencing and restriction fragment length polymorphism （RFLP）. Chi-square test was used for comparison of the t：nutation patterns. The results showed that： （1） High percentages of the 8 isolates were resistant to ciprofloxacin （95.0%）, ofloxacin （95.0%） and lomefloxacin （97.5%）, only one strain was susceptible to the ciprofloxacin. （2） Sensitive strains had a substitute of Asp95→Ala in the gyrA, and all isolates that were resistant or intermediated to the ciprofloxacin, had a double mutation in the gyrA （Ser91, Ala 92 and Asp95）. Some strains also had a mutation in the parC. （3） The MICs of these isolates were significantly associated with the mutation patterns in the gyrA and parC. A double mutation of gyrA combined with parC87 mutation was a predominant pattern in Shanghai and could mediate high level resistance to ciprofloxacin. It suggests that mutations in the QRDRs of gyrA and parC may be responsible for the fluoroquinolone resistance. And fluoroquinolone could not be used as the first line antibiotics for gonorrhea treatment any more in Shanghai, China.

Relationship between Mutation of IR in the mtr System of Neisseria Gonorrhoeae and Multiple Antibiotic Resistance

To study the relationship between mutation of the inverted repeat sequence （IR） in the multiple transferable resistant system （mtr） of Neisseria gonorrhoeae （NG） and its multiple antibiotic resistance, minimal inhibitory concentrations （MICs） for the clinically isolated strains were tested by agar-dilution-method. The mtr system＇s IR gene of NG was sequenced after amplification by polymerase chain reaction （PCR）. Either two susceptive or five penicillin-resistant strains had no base mutation in IR gene, while all of the 13 strains with multiple-antibiotic-resistance had a singlebase deletion （A/T）. The result suggests that a single-base deletion of the thirteen-base IR sequence in mtr system of NG might result in multiple antibiotic resistance but is not associated with single antibiotic resistance.

Construction of Prokaryotic Expression Plasmid of mtrC Protein of Neisseria gonorrhoeae and Its Expression in E.Coli

Summary： In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimierobial hydrophobie agents and study on the resistant mechanism of multiple transferable resistance （mtr） efflux system, plasmid pET-28a（＋） encoding mtrC gene was constructed and the related target protein was expressed in Escherichia colt （E. cold DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a（＋） with restriction endonuelease to construct recombinant pET-mtrC which was verified by restriction endonuelease and DNA sequencing. The recom- binant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuelease and DNA sequencing. hs sequence was 99.5 % homologus to that published on GeneBank （U14993）. A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli.

Study on Serotypes and Auxotypes of Neisseria Gonorrhoeae in Guangzhou

Objective:To investigate the serotypes and auxotypes distribution of Neisseria gonorrhoeae in Guangzhou. Method: 131 strains of Neisseria gonorrhoeae wereserotyped by co-agglutination test and 108 strains wereauxotyped by La Scolea's method. Results: Out of 131 strains of Neisseria gonorrhoeae,87.8% (115/131) were WⅡ/WⅢ, while 9.9% (13/131) wereWⅠ. The most important auxotypes were Proto, Pro and ILe,42.6% (46/108), 21.3% (23/108) and 12.0%, respectively. WⅡ/WⅢ was distributed among the all auxotypes aboveand WI found only in both Proto and Pro. Conclusion: The study illustrated the prevailing serotype,WⅡ/WⅢ, and higher prevalence of Ile- in Guangzhou.

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.

Effect of Atmospheric Pressure Non-equilibrium Plasmas on Neisseria gonorrhoeae

In this study, the sterilizing effect of atmospheric pressure nonequilibrium plasmas （APNPs） on Neisseria gonorrhoeae （N. gonorrhoeae） was preliminarily examined and the possible mechanisms were explored. N. gonorrhoeae FA1090, FA19 and MSll were treated by APNPs and their survival rate was analyzed by using CFUs counting and structurally studied by laser scanning confocal microscopy. The morphological changes of bacterial cell membrane and wall were studied under TEM. Our results showed that APNPs had strong sterilizing effect on N. gonorrhoeae. The survival rate of MS11 in N. gonorrhoeae liquid medium was 60.65% after disinfection with the APNPs for 5 rain, whereas, the survival rate of FA19 was 92.60% and the rate of FA1090 was 96.40%. The survival rate of MS 11 was 21.13% after exposure to APNPs for 6 rain, whereas the survival rate of FA19 was 31.60% and the rate of FA1090 was 91.00%. N. gonorrhoeae was structurally damaged after treatment with APNPs. It is concluded that APNPs is able to effectively and quickly kill the N. gonorrhoeae, and the killing effect is related to the architectural damage of cell membrane.

In vitro Recombination and Identification of Mutated Fragment Corresponding to Regulation Region of mtrR Gene of Neisseria Gonorrhoeae

A site-directed mutant DNA fragment Neisseria Gonorrhoeae （NG） stains to construct the was synthesized and transfected into clinical transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension （SOEing）, a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction （PCR）. The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.

Construtcion of Neisseria Gonorrhoeae Porin B Plasmid Recombinant and Its Expression in E.coli

A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE_3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE_3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.

<i>In vitro</i>activity of cationic peptides against <i>Neisseria gonorrhoeae</i>and vaginal <i>Lactobacillus</i>species: The effect of divalent cations

One of the new strategies for the prevention of HIV acquisition is the use of microbicides such as topical microbicides including antimicrobial and antiviral peptides. Ideally, new drug candidates should kill pathogens without determent to the normal bacterial flora considered important in health;such as hydrogen peroxide producing Lactobacillus species. The antimicrobial peptides LL-37 and LSA-5 were studied to determine their spectrum of activity against bacterial pathogens and normal flora organisms. The effects of divalent cations at biologically relevant concentrations were determined. We show the synthetic lytic peptide LSA-5 and the naturally occurring peptides LL-37 inactivate Neisseria gonorrhoeae but are less active against many normal flora members such as Lactobacillus species. Biologically relevant concentrations of calcium and magnesium prevented killing of sensitive strains. LSA-5 is more potent than LL-37, both are inhibited from killing sensitive strains by calcium and magnesium. Strains of Lactobacillus iners were killed by both microbicides even in the presence of the divalent cations. Antimicrobial peptides, such as LSA-5, have good potential for use in prevention of sexually transmitted disease, if formulated to sequester calcium and magnesium present in biological fluids.

Detection of <i>Chlamydia trachomatis</i>and <i>Neisseria gonorrhoeae</i>in Egyptian Women Suffering from Infertility

Chlamydial and gonococcal infections are recognized as two of the major causes of sexually transmissible human bacterial infection which may lead to infertility. In this cross sectional study, we aimed to determine the prevalence of Neisseria gonorrhoeae, Chlamydia trachomatis among Egyptian women using different microbiological methods. One hundred and fifty cervical swabs were collected, of which 100 were from infertile women. Culture and ELISA technique were used for screening of Neisseria gonorrhoeae and Chlamydia trachomatis individually. In addition, PCR was used for all examined samples. For C. trachomatis, 3 cases were positive for antigen detection by ELISA. Moreover, in obtained results of PCR, DNA was detected in 4 samples, and three of them from infertile group. So based on PCR results, the sensitivity and specificity of ELISA were 75% and 100% respectively. Furthermore, 3 samples were positive for gonococcal infections by PCR, and two of them were taken from infertile women. Positive results of two samples were verified by culture. The estimated sensitivity and specificity of culture method were 66.7% and 100% respectively. Results of this study indicate that PCR is a valuable method for detection of gonococcal and chlamydial infection and it is suitable for the confirmation of ELISA results for C. trachomatis diagnosis. Culture method is less sensitive than PCR for detection of N. gonorrhoeae. The prevalence of such infections is higher among infertile women.

Opacity Proteins of Neisseria Gonorrhoeae in Lipooligosaccharide Mutants Lost Ability to Interact with Neutrophil-restricted CEACAM3(CD66d)

Lipooligosacharide（LOS） of Neisseria gonorrhoeae（gonococci, GC） is involved in the interaction of GC with host cells. Deletion of the alpha-oligosaccharide（alpha-OS） moiety of LOS（lgt F mutant） significantly impairs invasion of GC into epithelial cell lines. GC opacity（Opa） proteins, such as Opa I, mediate phagocytosis and stimulate chemiluminescence responses in neutrophils in part through interaction with members of the carcinoembryonic antigen（CEA） family, which includes CEACAM3（CD66d）, a human neutrophil specific receptor for phagocytosis of bacteria. In the present work, we examined the effects of Opa I-expressing lgt F mutant on phagocytosis by He La-CEACAM3 cells and chemiluminescence responses in neutrophils. The results showed that lgt F mutant even expressing Opa I completely lost the ability to promote either phagocytosis mediated by CEACAM3 interaction in He La cells or chemiluminescence responses in neutrophils. These data indicated that Opa proteins in the lgt F mutant, which might result from the conformational change, cannot be functional.

Zoliflodacin: a hope to treat antibiotic-resistant Neisseria gonorrhoeae

Background:Neisseria gonorrhoeae is a gram-negative diplococcus that leads to sexually transmitted infection.N.gonorrhoeae is an obligate human pathogen that causes infection to the mucus-secreting epithelial cells both in males and females.In 2017 the center for disease control and the World Health Organization published the list of global priority pathogens-12 with denting therapeutic options,including antibiotic-resistant N.gonorrhoeae.Methods:we thoroughly characterized zoliflodacin antibiotic,its clinical trials and effect on human health by using different keywords like“zoliflodacin”,“COVID-19”,“clinical trials”from different data sources like Pub-Med,Google-Scholar,and Science-Direct.Results:Zoliflodacin shows a therapeutic approach against N.gonorrhoeae.It acts by inhibiting bacterial type 2 topoisomerase with the binding sites in bacterial gyrase.It shows promising results against N.gonorrhoeae.Zoliflodacin is effective in treating gonococcal urogenital and rectal infection.Conclusion:Currently,antibiotic is the only option to treat N.gonorrhoeae with no vaccine available to treat it.The new drug,zoliflodacin,specifically targets antibiotic-resistant gonorrhea and it has given a hope to researchers.This review elaborates the discovery of zoliflodacin,its mechanism of action,current clinical trials,and its effectiveness.

ANALYSIS OF THE ANTIGEN-ANTIBODY SPECIFICITY IN THE SEMEN OF PATIENTS WITH NEISSERIA GONORRHOEAE

This study was done to define the human genital immune response to infection with Neisseria gonorrhoeae. The semen specimens were obtained from 15 patients with uncomplicated gonococcal infection in the acute and convalescent phases and 15 men with uninfected control- After precipitated with amoniasulfate,the semen was tested against the outer membrane protein of gonococcal isolates from the same patients to examine antigen-antibody interactions by use of the western blot technique. The antibodies in the semen reacted with more gonococcal antigens in the acute phase than in the convalescent phase. IgA in the semen reacted with more antigens than did IgG in the same specimens. The predominant reacted antigens were protein I, protein II, 46～ 48, kD, 14 ～16 kD and 88～ 90 kD protein.

Development and performance evaluation of a culture-independent nanopore amplicon-based sequencing method for accurate typing and antimicrobial resistance profiling in Neisseria gonorrhoeae

Dear Editor,Bacterial antimicrobial resistance(AMR)poses a serious threat to global human health(Antimicrobial Resistance Collaborators,2022).Comprehensive profiling of AMR and accurate molecular typing are important for tracking and controlling the emergence and dissemination of antibiotic-resistant bacteria(Yahara et al.,2021).We previously developed a multiplex amplicon sequencing-based method for directly sequencing AMR-related loci in N.gonorrhoeae from clinical samples(Zhang et al.,2021).

Molecular Detection of Pathogens Involved in Sexually Transmitted Infections in Brazzaville, Congo

The objective of this multicentric study was to assess the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma génitalium and Trichomonas vaginalis infections in Brazzaville, in the Republic of Congo, using molecular methods. From January to December 2021, the sexually transmitted disease risk participants were recruited from six centers: The Association of Young HIV-Positive People of Congo, The Congolese Association for Family Welfare, The Association for Support to Vulnerable Groups, Talangaï hospital, Brazzaville university hospital (outpatient service) and the private clinic COGEMO (outpatient service). The real-time multiplex PCR was carried out to detect these pathogens. Each patient had at least one specimen (urine, urethral, anal and/or vaginal samples). The patients were considered infected when one of their samples was positive. 287 participants made of 227 women and 60 men were tested. The general prevalence of these infections was: Chlamydia trachomatis 2.79%, Neisseria gonorrhoeae 3.14%, Mycoplasma génitalium 3.45% and Trichomonas vaginalis 2.97. The prevalence rates according to sex were: C. trachomatis, M. génitalium, N. gonorrhea and T. vaginalis were 1.32%, 2.05%, 1.32% and 3.42% in women and 8.33%, 7.02%, 10% and 1.75% in men, respectively. Most infected patients were asymptomatic. Prevalence rates were higher in bisexual individuals, with the exception of T. vaginalis which showed higher prevalence in heterosexual patients. The bisexual and homosexual individuals represent a major public health problem in sexually active young adults, particularly among men having sex with men. These sexually transmitted infections are mainly asymptomatic, their diagnosis and management remain difficult in developing countries.

Determining antimicrobial resistance profiles and identifying novel mutations of Neisseria gonorrhoeae genomes obtained by multiplexed MinION sequencing

Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission,timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, the etiological agent of gonorrhea, has acquired nearly all known mechanisms of antimicrobial resistance(AMR), thereby compromising the efficacy of antimicrobial therapy. Treatment failure resulting from AMR has become a global public health concern. Whole-genome sequencing is an effective method to determine the AMR characteristics of N. gonorrhoeae. Compared with next-generation sequencing, the MinION sequencer(Oxford Nanopore Technologies(ONT)) has the advantages of long read length and portability. Based on a pilot study using MinION to sequence the genome of N. gonorrhoeae, we optimized the workflow of sequencing and data analysis in the current study. Here we sequenced nine isolates within one flow cell using a multiplexed sequencing strategy. After hybrid assembly with Illumina reads, nine integral circular chromosomes were obtained. By using the online tool Pathogenwatch and a BLAST-based workflow, we acquired complete AMR profiles related to seven classes of antibiotics. We also evaluated the performance of ONT-only assemblies. Most AMR determinants identified by ONT-only assemblies were the same as those identified by hybrid assemblies. Moreover, one of the nine assemblies indicated a potentially novel antimicrobial-related mutation located in mtrR which results in a frame-shift, premature stop codon, and truncated peptide.In addition, this is the first study using the MinION sequencer to obtain complete genome sequences of N. gonorrhoeae strains which are epidemic in China. This study shows that complete genome sequences and antimicrobial characteristics of N.gonorrhoeae can be obtained using the MinION sequencer in a simple and cost-effective manner, with hardly any knowledge of bioinformatics required. More importantly, this strategy provides us with a potential approach to discover new AMR determinants.

Using RAPD in Neisseria gonorrhoeae genotyping and transmission detection

The aim of this paper is to develop an applic-able Random Amplified Polymorphic DNAs(RAPD)method for genotyping Neisseria gonorrhoeae strain,and discuss the possibility of using the RAPD method to trace N.gonorrhoeae strain transmission route.Four different pretreatment methods were used on the N.gonorrhoeae genomic DNA component,and the best adaptive extract method was selected for RAPD.Different RAPD primers sequence was used for amplification and their differenti-ating capabilities for N.gonorrhoeae strains were com-pared.Applicable RAPD primer was selected for N.gonorrhoeae genotyping and then applied into transmis-sion detection.The results show that the so called cetyl-trimethylammonium bromide(CTAB)method for extracting genomic DNA could give integrated genomic DNA and give out relatively better RAPD fingerprint maps,subsequently,using selected RAPD primer could give out a group of amplification polymerase chain reac-tion bands.The fingerprint maps from different N.gonor-rhoeae strains were distinctive.Some main segments were common to all the N.gonorrhoeae strains tested.Some segments were different among the N.gonorrhoeae strains.According to the fingerprint maps and similarity index of different N.gonorrhoeae isolates,isolates from a pair of sex-partners were very similar.Based on these findings,the best extracting method and suitable RAPD primer were chosen.The RAPD fingerprint maps could type N.gonorrhoeae effectively and could be used as an additional approach in molecular epidemiology for tracing infection sources.