The NarE protein of Neisseria gonorrhoeae catalyzes ADP-ribosylation of several ADP-ribose acceptors despite an N-terminal deletion.

The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE-6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human ß-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes.

Evaluación in vitro del efecto de Neisseria gonorrhoeae y los factores solubles producto de su metabolismo sobre la calidad espermática / In vitro evaluation of the effect of Neisseria gonorrhoeae and the soluble factors produced by its metabolism on sperm quality

AANTEDENTES: N. gonorrhoeae es una de las principales causas de uretritis, prostatitis y epididimitis en hombres. En el tracto urogenital masculino, esta bacteria o los factores solubles producto de su metabolismo pueden interactuar con los espermatozoides y alterar su calidad. OBJETIVOS: Determinar el efecto de la incubación in vitro de N. gonorrhoeae y los factores solubles producto de su metabolismo sobre la calidad espermática y evaluar la interacción bacteria-espermatozoide. MÉTODO: Se realizó incubación de una concentración 0,5 McFarland de N. gonorrhoeae y de los factores solubles producto de su metabolismo bacteriano con muestras de semen de voluntarios aparentemente sanos. Se cuantificaron los parámetros espermáticos convencionales (movilidad y viabilidad) y funcionales (potencial de membrana mitocondrial, integridad y lipoperoxidación de la membrana espermática, detección de especies reactivas del oxígeno, integridad de la cromatina y expresión de Anexina V), empleando microscopía y citometría de flujo, respectivamente. Para evaluar la interacción de N. gonorrhoeae con los espermatozoides humanos se realizaron extendidos en placas para su observación al microscopio. RESULTADOS: N. gonorrhoeae puede unirse al espermatozoide y disminuir la viabilidad espermática luego de 1,5 horas de incubación con los espermatozoides humanos (84,5% vs 66,5%, p<0,05), sin afectar los parámetros espermáticos funcionales. CONCLUSIÓN: N. gonorrhoeae interactúa con los espermatozoides humanos afectando la viabilidad espermática.

BACKGROUND: N. gonorrhoeae is a major cause of urethritis, prostatitis and epididymitis in men. In the male urogenital tract, the bacteria or soluble products of their metabolism may interact with sperm, and alter their quality. AIMS: To determine the in vitro effect of incubation of N. gonorrhoeae and soluble products of their metabolism on sperm quality and assess the bacteria-sperm interaction. METHODS: Soluble products of bacterial metabolism and 0.5 McFarland concentration of N. gonorrhoeae were incubated with semen samples from healthy volunteers. Conventional (motility and viability) and functional sperm parameters (potential of mitochondrial membrane integrity and sperm membrane lipid peroxidation, detection of reactive oxygen species, chromatin integrity and expression of annexin V) were quantified using microscopy and cytometry flow, respectively. To assess the interaction of N. gonorrhoeae to human sperm microscopic observation was performed. RESULTS: N. gonorrhoeae can join the sperm and decreases sperm viability after 1.5 hours of incubation with human sperm (84.5% vs 66.5%, p<0.05), without affecting the functional sperm parameters. CONCLUSION: N. gonorrhoeae interacts with human sperm affecting sperm viability.

Neisseria gonorrhoeae induced disruption of cell junction complexes in epithelial cells of the human genital tract.

Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epithelial barriers in order to reach subepithelial tissues for host colonization. The aim of this study was to examine the effects of gonococci on cell junction complexes of genital epithelial cells of women. Polarized Ishikawa cells, a cell line derived from endometrial epithelium, were used for experimental infection. Infected cells displayed a spindle-like shape with an irregular distribution, indicating potential alteration of cell-cell contacts. Accordingly, analysis by confocal microscopy and cellular fractionation revealed that gonococci induced redistribution of the adherens junction proteins E-cadherin and its adapter protein ß-catenin from the membrane to a cytoplasmic pool, with no significant differences in protein levels. In contrast, gonococcal infection did not induce modification of either expression or distribution of the tight junction proteins Occludin and ZO-1. Similar results were observed for Fallopian tube epithelia. Interestingly, infected Ishikawa cells also showed an altered pattern of actin cytoskeleton, observed in the form of stress fibers across the cytoplasm, which in turn matched a strong alteration on the expression of fibronectin, an adhesive glycoprotein component of extracellular matrix. Interestingly, using western blotting, activation of the ERK pathway was detected after gonococcal infection while p38 pathway was not activated. All effects were pili and Opa independent. Altogether, results indicated that gonococcus, as a mechanism of pathogenesis, induced disruption of junction complexes with early detaching of E-cadherin and ß-catenin from the adherens junction complex, followed by a redistribution and reorganization of actin cytoskeleton and fibronectin within the extracellular matrix.

Syndromic management and STI control in urban Peru.

BACKGROUND: Syndromic management is an inexpensive and effective method for the treatment of symptomatic sexually transmitted infections (STIs), but its effectiveness as a method of STI control in at-risk populations is questionable. We sought to determine the potential utility of syndromic management as a public health strategy to control STI transmission in high-risk populations in urban Peru. METHODOLOGY: We surveyed 3,285 at-risk men and women from three Peruvian cities from 2003-05. Participants were asked about the presence of genital ulcers, discharge, or dysuria in the preceding six months. Participants reporting symptoms were asked about subsequent health-seeking and partner notification behavior. Urine and vaginal swab samples were tested for Neisseria gonorrhoeae and Chlamydia trachomatis by nucleic acid testing. Serum was tested for syphilis and Herpes Simplex Virus-Type 2 antibodies. FINDINGS: Recent urogenital discharge or dysuria was reported by 42.1% of participants with gonorrhea or chlamydia versus 28.3% of participants without infection. Genital ulceration was reported by 6.2% of participants with, and 7.4% of participants without, recent syphilis. Many participants reporting symptoms continued sexual activity while symptomatic, and approximately half of all symptomatic participants sought treatment. The positive and negative predictive values of urogenital discharge or genital ulcer disease in detecting STIs that are common in the study population were 14.4% and 81.5% for chlamydia in women and 8.3% and 89.5% for syphilis among gay-identified men. CONCLUSIONS: In our study, STIs among high-risk men and women in urban Peru were frequently asymptomatic and symptomatic participants often remained sexually active without seeking treatment. Additional research is needed to assess the costs and benefits of targeted, laboratory-based STI screening as part of a comprehensive STI control program in developing countries.

Evaluation of the BIOCEN GC agar medium base in antimicrobial susceptibility testing of Neisseria gonorrhoeae.

BACKGROUND: Since 1995, the Cuban Reference Laboratory for Neisseria has been monitoring the antibiotic susceptibility of gonococci, following the methodology of the National Committee for Clinical Laboratory Standards, which uses GC agar medium base supplemented with 1% Vitox. We evaluated three lots of GC agar medium base produced by BIOCEN, Cuba, in antibiotic susceptibility testing of reference and wild strains of gonococci. METHODS: The susceptibilities to five antibiotics were evaluated five times on three lots of GC agar medium base from BIOCEN. Four and one gonococcal reference strains were tested by MIC dilution and disc diffusion methods, respectively. Later, the antimicrobial susceptibilities of ten wild Neisseria gonorrhoeae strains were tested in triplicate. As internal control, a GC agar medium from Difco was used. RESULTS: All antibiotic MICs obtained on four lots of GC agar medium from different manufacturers fell within the proposed quality control limits for reference strains analyzed. The disc diffusion data for the reference strain of N. gonorrhoeae ATCC 49226 to five antibiotics provided essentially identical results in all lots of GC agar medium base. For wild strains of gonococci, identical modal MIC values and zone size diameters within a 3-mm range were observed in all the antibiotics tested. CONCLUSIONS: Excellent agreement in susceptibility testing methods among different lots of GC agar medium base from BIOCEN and Difco was obtained for all reference and wild gonococcal strains and antibiotics tested. We proposed that GC medium from BIOCEN can be used in antimicrobial susceptibility testing of N. gonorrhoeae by MIC dilution and disc diffusion tests.

[Evaluation of different methods of utilization of sugars in Neisseria gonorrhoeae]. / Evaluación de diferentes métodos de utilización de azúcares en Neisseria gonorrhoeae.

5 methods of utilization of sugars were evaluated in 25 strains previously identified as N. gonorrhoeae: CTA agar, modified CTA agar, gelatin starch agar, Mueller Hinton agar plus bromotimol blue and rapid method. 100% of the strains of N. gonorrhoeae were identified by the CTA and rapid methods, whereas 96% were identified by the modified CTA and gelatin starch agar methods. No strain of gonoccocus was identified by the Mueller Hinton agar method plus bromotimol blue. The cystine tripticase agar medium (CTA) is the elective method to confirm the isolates of Neisseria gonorrhoeae. As it is mainly made by U.S. companies, it is difficult for Cuba to acquire it. The gelatin-starch agar method and the rapid method are useful alternative of the CTA medium, so their use is proposed in this paper.

Characterization of multiresistant strains of Neisseria gonorrhoeae isolated in Nicaragua.

The extensive use of antibiotics in Nicaragua raises concerns about the resulting levels of susceptibility of pathogenic bacteria. This is the first study that characterizes 18 strains of N. gonorrhoeae isolated in Nicaragua (1989), for their antibiotic susceptibility. Strains were predominantly of the auxotype/serotype Proto/PIB. There was no difference in lipopolysaccharides profiles obtained after SDS-PAGE for all strains. Variable expression of the PII outer membrane protein was not associated to antimicrobial resistance. All strains were susceptible to ceftriaxone, spectinomycin, rifampin and cefoxitin. The strains were classified in five groups based on plasmid profiles. A total of 78% of the isolates were penicillinase-producing (PPNG) and 22% were tetracycline-resistant N. gonorrhoeae (TRNG). One PPNG strain showed a concomitant decreased of penicillin binding to penicillin-binding protein 2. These randomly chosen isolates of N. gonorrhoeae from Nicaragua possess high levels of resistance to multiple families of drugs.

PIP: In Nicaragua, in 1989, health workers obtained urethral or cervical samples from 18 people with gonorrhea attending public health clinics in Managua and sent them to the National Laboratory of Public Health in Managua for characterization of their antibiotic susceptibility. Of the 18 strains, 15 (83.3%) were of the auxotype/serotype Proto/PIB. Electrophoresis of lipopolysaccharides on SDS-polyacrylamide gels (15%) with 4 M urea revealed no difference in lipopolysaccharide profiles for all strains. The variable expression of the 31-kDa opacity outer membrane protein was not related to antimicrobial resistance. All isolates exhibited susceptibility to ceftriaxone, spectinomycin, cefazolin, cefoxitin, and rifampin. 78% of the strains produced beta-lactamase. 89% of the strains were resistant to penicillin and ampicillin, 44% were resistant to tetracycline, 28% were resistant to cefamandol, 22% were resistant to chloramphenicol, and 11% were resistant to erythromycin. There were 5 distinct groups of Neisseria gonorrhoeae isolated according to their plasmid profiles. The largest was plasmid profile group 1 (55.6%), defined as carrying the 24.5, 3.2, and 2.6 MDa plasmids. It produced beta-lactamase. Penicillinase-producing N. gonorrhoeae (PPNG) comprised 78% of the isolates, 22% of whom were tetracycline-resistant N. gonorrhoea. One PPNG strain exhibited a parallel decrease of penicillin binding to penicillin-binding protein 2. These findings confirmed the presence of multiresistant N. gonorrhoeae strains in Managua, Nicaragua. Based on these findings, the researchers recommended that penicillin and tetracycline not be used to treat gonorrhea in Nicaragua; they recommended ceftriaxone and spectinomycin.

Neisseria Gonorrhoeae productora de Betalactamasa, entre los pacientes atendidos por un Hospital Privado del área metropolitana de Caracas / Neisseria gonorrhoeae producing betalactamese among patients attended in a provate hospital of metropolitan area of Caracas

Desde 1986 se están reportando casos de infecciones por Neisseria gonorrhoeae resistente a penicilina. Estas son causas de falla de tratamiento y aumento de los costos. Nosotros estudiamos la incidencia de resistencia a la penicilina, entre todas las cepas de N.gonorrhoeae aisladas en el laboratorio de Microbiología de la Clínica Avila, desde el 1 de agosto de 1986, hasta el 31 de agosto de 1987. Los métodos utilizados para el aislamiento y las pruebas de susceptibilidad, fueron los recomendados por la Sociedad Americana de Microbiología y el Comité Nacional para la Estandarización de los Laboratorios Cínicos. El 31,6% (6/19) de las cepas aisladas fueron resistentes a la penicilina. En todas ellas, el mecanismo fue la producción de betalactamasa. Se hacen recomendaciones

Epidemiology and treatment of uncomplicated gonorrhoea caused by non-PPNG strains in Córdoba, Argentina: auxotypes, susceptibility profiles, and plasmid analyses of urethral isolates from men.

The official records of uncomplicated gonorrhoea for Córdoba state show that between 1975 and 1985, about one in 1000 sexually active people acquired gonorrhoea each year. A study was therefore undertaken to obtain information about treatment of uncomplicated gonorrhoea, as well as the nutritional requirements, plasmid analyses, and susceptibility profiles of gonococci in this geographical area. From August 1983 to April 1984, 219 men with uncomplicated gonorrhoea were treated with one of four antibiotic schedules, all of which were over 95% efficient. All 98 strains isolated and purified were non-penicillinase-producing Neisseria gonorrhoeae (non-PPNG). The minimum inhibitory concentrations (MICs) of benzylpenicillin, tetracycline, thiamphenicol, spectinomycin, kanamycin, and cefoxitin were assessed. The MIC of benzylpenicillin showed that 88% (86) of the strains were inhibited by 0.5 mg/l of the drug, and also showed a bimodal sensitivity pattern to that antibiotic. The nutritional requirements of the 62 strains tested showed that 53% (33) were of the non-requiring (wild type) auxotype, 42% (26) required proline (pro-) and 5% (3) required proline and arginine (pro- arg-). Resistance to antibiotics was more notable in the pro- than in the wild type strains.