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Sample records for neisseria gonorrhoeae por

  1. VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways.

    PubMed

    Müller, Anne; Rassow, Joachim; Grimm, Jan; Machuy, Nikolaus; Meyer, Thomas F; Rudel, Thomas

    2002-04-15

    The human pathogen Neisseria gonorrhoeae induces host cell apoptosis during infection by delivering the outer membrane protein PorB to the host cell's mitochondria. PorB is a pore-forming beta-barrel protein sharing several features with the mitochondrial voltage-dependent anion channel (VDAC), which is involved in the regulation of apoptosis. Here we show that PorB of pathogenic Neisseria species produced by host cells is efficiently targeted to mitochondria. Imported PorB resides in the mitochondrial outer membrane and forms multimers with similar sizes as in the outer bacterial membrane. The mitochondria completely lose their membrane potential, a characteristic previously observed in cells infected with gonococci or treated with purified PorB. Closely related bacterial porins of non-pathogenic Neisseria mucosa or Escherichia coli remain in the cytosol. Import of PorB into mitochondria in vivo is independent of a linear signal sequence. Insertion of PorB into the mitochondrial outer membrane in vitro depends on the activity of Tom5, Tom20 and Tom40, but is independent of Tom70. Our data show that human VDAC and bacterial PorB are imported into mitochondria by a similar mechanism. PMID:11953311

  2. Biofilm Formation by Neisseria gonorrhoeae

    PubMed Central

    Greiner, L. L.; Edwards, J. L.; Shao, J.; Rabinak, C.; Entz, D.; Apicella, M. A.

    2005-01-01

    Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could form a biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10 μM sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain 1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with water channels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growth conditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmed that organisms were embedded in a continuous matrix with membranous structures spanning the biofilm. These studies also demonstrated that N. gonorrhoeae has the capability to form a matrix in the presence and absence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and the lectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in the biofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac and sialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primary urethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Our studies demonstrated that N. gonorrhoeae can form biofilms in continuous-flow chambers and on living cells. Studies of these biofilms may have implications for understanding asymptomatic gonococcal infection. PMID:15784536

  3. L Form of Neisseria gonorrhoeae

    PubMed Central

    Roberts, Richard B.

    1966-01-01

    Roberts, Richard B. (Walter Reed Army Institute of Research, Washington, D.C.). L form of Neisseria gonorrhoeae. J. Bacteriol. 92:1609–1614. 1966.—L forms were produced by the penicillin gradient plate technique from a recently isolated strain of Neisseria gonorrhoeae. To date, these L forms have had 30 serial passages on medium containing penicillin. Stabilized L forms developed on penicillin-free medium after 10 or more passages in the presence of penicillin. Morphological characteristics of these organisms were identical to L forms of meningococci. Medium and environmental conditions necessary for optimal growth included: Brain Heart Infusion of pH 7.2 to 7.4, 1.1 to 1.3% agar, 10 to 20% sucrose, 10 to 20% horse serum, temperature at 35 to 36 C, and increased CO2 tension (candle jar). L forms were more resistant than the parent gonococcus to penicillin, ampicillin, methicillin, cycloserine, and cephalothin, whereas both organisms had similar sensitivities to bacitracin, vancomycin, ristocetin, novobiocin, tetracycline, and erythromycin. Revertant gonococci were produced on penicillin-free medium from L forms which had had 1, 5, and 10 serial passages. Morphology and fermentative reactions of revertant strains were identical to those of the parent gonococcus. Revertant strains produced L forms more readily than the parent organism; in fact, L forms from certain revertants did not require penicillin, but only serum and sucrose for their production and propagation on artificial medium. Images PMID:4959715

  4. Conjugative Plasmids of Neisseria gonorrhoeae

    PubMed Central

    Pachulec, Emilia; van der Does, Chris

    2010-01-01

    Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between

  5. Glucose Metabolism in Neisseria gonorrhoeae

    PubMed Central

    Morse, Stephen A.; Stein, Stefanie; Hines, James

    1974-01-01

    The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO2 from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-14C]acetate over that of [2-14C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent. PMID:4156358

  6. Update on Quinolone Resistance in Neisseria gonorrhoeae.

    PubMed

    Zenilman, Jonathan M.

    2002-04-01

    Quinolones are widely used for treating gonococcal infections, typically in single-dose, oral regimens. However, in the 1990s, quinolone-resistant Neisseria gonorrhoeae emerged, potentially compromising the utility of this drug class. In the past year, these strains have widely disseminated, accounting for over half of isolates in parts of Southeast Asia. The molecular mechanism of resistance has been localized to multiple mutations in genes coding for the bacterial DNA gyrase and topoisomerase enzymes. These mutations accumulate until the minimum inhibitory concentration is 4.0 g/mL or more, which in clinical studies appears to be the threshold for clinical treatment failure. Quinolone-resistant N. gonorrhoeae is independent from other plasmid- and chromosomally-mediated resistance determinants; nearly all isolates to date have been sensitive to cephalosporins and spectinomycin. Nevertheless, designing public health strategies to contain quinolone-resistant N. gonorrhoeae will be difficult. PMID:11927047

  7. Antimicrobial Resistance and Neisseria gonorrhoeae Multiantigen Sequence Typing Profile of Neisseria gonorrhoeae in New Delhi, India.

    PubMed

    Mahajan, Neeraj; Sood, Seema; Singh, Rajendra; Kapil, Arti; Das, Bimal Kumar; Sreenivas, Vishnubhatla; Kar, Hemanta Kumar; Sharma, Vinod Kumar

    2016-08-01

    Molecular epidemiology of 100 consecutive gonococcal isolates collected between April 2010 and October 2013 from New Delhi was investigated using Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) along with its association with antimicrobial resistance profiles. Neisseria gonorrhoeae isolates were assigned into 60 different sequence types and 43 (71.6%) were novel. Sole representation was seen in 76.6% sequence types. There was significant association between ST6058 and resistance to penicillin (P = 0.00) and tetracycline (P = 0.002). PMID:27414684

  8. Drug-resistant Neisseria gonorrhoeae in Michigan

    PubMed Central

    Boehme, Martha S.; Rudrik, James T.; Ganoczy, Dara; Crandell-Alden, Erin; Schneider, William A.; Somsel, Patricia A.

    2005-01-01

    The increasing prevalence of quinolone-resistant Neisseria gonorrhoeae (QRNG) in the United States is a cause for concern. Detecting resistance is complicated by the widespread use of molecular tests that do not provide isolates for susceptibility testing. The Michigan Department of Community Health developed a sentinel surveillance program to detect antimicrobial drug resistance in N. gonorrhoeae. Sentinel surveillance from 11 laboratories submitted 1,122 isolates for antimicrobial drug susceptibility testing and detected 2 clusters of QRNG from January 2003 to September 2004. These clusters were epidemiologically distinct: one involved young, heterosexual youth, and the other involved older men who have sex with men. This finding led to changes in local treatment recommendations that limited spread of resistant strains. Development of the sentinel program, collection of data, and epidemiologic analysis of the clusters are discussed. PMID:16022773

  9. Resistance to peroxynitrite in Neisseria gonorrhoeae.

    PubMed

    Barth, Kenneth R; Isabella, Vincent M; Wright, Lori F; Clark, Virginia L

    2009-08-01

    Neisseria gonorrhoeae encodes a number of important genes that aid in survival during times of oxidative stress. The same immune cells capable of oxygen-dependent killing mechanisms also have the capacity to generate reactive nitrogen species (RNS) that may function antimicrobially. F62 and eight additional gonococcal strains displayed a high level of resistance to peroxynitrite, while Neisseria meningitidis and Escherichia coli showed a four- to seven-log and a four-log decrease in viability, respectively. Mutation of gonococcal orthologues that are known or suspected to be involved in RNS defence in other bacteria (ahpC, dnrN and msrA) resulted in no loss of viability, suggesting that N. gonorrhoeae has a novel mechanism of resistance to peroxynitrite. Whole-cell extracts of F62 prevented the oxidation of dihydrorhodamine, and decomposition of peroxynitrite was not dependent on ahpC, dnrN or msrA. F62 grown in co-culture with E. coli strain DH10B was shown to protect E. coli viability 10-fold. Also, peroxynitrite treatment of F62 did not result in accumulation of nitrated proteins, suggesting that an active peroxynitrite reductase is responsible for peroxynitrite decomposition rather than a protein sink for amino acid modification. PMID:19406894

  10. Emerging resistance in Neisseria meningitidis and Neisseria gonorrhoeae.

    PubMed

    Stefanelli, Paola

    2011-02-01

    The value of monitoring antimicrobial resistance is particularly significant for Neisseria meningitidis and Neisseria gonorrhoeae diseases, even if it is for different reasons. Although there is no global alert for the spread of resistant meningococcal strains, the emergence of resistance is correlated to the outcome of treatment and the successful prophylaxis of close contacts. Few cases of resistance among meningococci have been recorded worldwide; it remains unclear what intriguing mechanism is responsible for maintaining resistance in these cases in the absence of significant antibiotic selective pressure, as in the case of penicillin; on the contrary, although rifampicin is the antibiotic of choice in the prophylaxis of close contacts, there is a very low rate of resistance. The emergence of multidrug-resistant N. gonorrhoeae is a great challenge in controlling gonorrhea as one of the main sexually transmitted bacterial diseases. International surveillance programs permit the monitoring of the susceptibility of the pathogen and allow the revision of the standardized treatment regimen when the situation changes. PMID:21342071

  11. Detection of quinolone-resistant Neisseria gonorrhoeae.

    PubMed Central

    Kam, K M; Wong, P W; Cheung, M M; Ho, N K

    1996-01-01

    The present National Committee for Clinical Laboratory Standards (NCCLS) guideline for testing Neisseria gonorrhoeae quinolone susceptibility defines only a susceptible category for ciprofloxacin, enoxacin, lomefloxacin, and ofloxacin, while susceptible, intermediate, and resistant categories are defined for fleroxacin. To further define the criteria for detection of quinolone resistance in gonococci, by standard disk diffusion and agar dilution methodologies recommended by the NCCLS, we tested 29 strains of quinolone-resistant N. gonorrhoeae (QRNG) recently isolated from ofloxacin-treated patients who were considered clinical failures. Regression analyses were performed on these results together with those of another 20 strains showing reduced susceptibility and 13 fully susceptible strains (ofloxacin MICs of < or = 0.25 microgram/ml). With 5-micrograms ofloxacin disks, resistance in 27 (93.1%) of the QRNG strains (MICs of > 1 microgram/ml) was detected by the criterion of a zone diameter of < 22 mm, while in the remaining 2 (6.9%), the disks failed to detect resistance. A cluster of 15 highly resistant strains showed ofloxacin MICs of > 4 micrograms/ml and zone diameters of < 13 mm. When tested with 5-micrograms ciprofloxacin disks, the corresponding values for resistance and high-level resistance of these QRNG strains were < 25 mm (MICs of > 0.5 micrograms/ml) and < 15 mm (MICs of > 2 micrograms /ml), respectively. Six strains for which ofloxacin MICs were > or = 8 micrograms/ml showed no zones at all with both 5-micrograms ofloxacin and 5-micrograms ciprofloxacin disks. These QRNG strains are now firmly established in the Southeast Asia region, and it is important for clinical laboratories to recognize these clinically resistant strains and to monitor their spread. PMID:8735098

  12. Neisseria gonorrhoeae and fosfomycin: Past, present and future.

    PubMed

    Tesh, Lauren D; Shaeer, Kristy M; Cho, Jonathan C; Estrada, Sandy J; Huang, Vanthida; Bland, Christopher M; DiMondi, V Paul; Potter, Alicia N; Hussein, Gamal; Bookstaver, P Brandon

    2015-09-01

    Drug-resistant Neisseria gonorrhoeae has become a global health concern that requires immediate attention. Due to increasing resistance to cephalosporins, pursuing novel alternatives for treating N. gonorrhoeae infections is paramount. Whilst new drug development is often cumbersome, reviving antiquated antibiotic agents for treatment of modern infections has become prevalent in clinical practice. Fosfomycin exhibits bactericidal activity through a unique mechanism of action, and a variety of organisms including N. gonorrhoeae are susceptible. In vitro studies have demonstrated that fosfomycin can retain activity against ceftriaxone-resistant N. gonorrhoeae; however, it remains unclear whether there is synergy between fosfomycin and other antibiotics. Clinical investigations evaluating fosfomycin for the treatment of N. gonorrhoeae infections are confounded by methodological limitations, none the less they do provide some perspective on its potential role in therapy. Future studies are needed to establish a safe, convenient and effective fosfomycin regimen for treating N. gonorrhoeae infections. PMID:26145201

  13. Diagnosis of Neisseria gonorrhoeae Using Molecular Beacon

    PubMed Central

    Patel, Achchhe Lal; Sonkar, Subash Chandra; Kumari, Indu; Saluja, Daman

    2015-01-01

    Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries. PMID:25802857

  14. Phospholipids and fatty acids of Neisseria gonorrhoeae.

    PubMed Central

    Sud, I J; Feingold, D S

    1975-01-01

    The phospholipids and fatty acids of two strains of Neisseria gonorrhoeae of different penicillin susceptibilities were examined. The phospholipids, which comprise about 8% of the dry weight of the cells, consisted of phosphatidylethanolamine (70%) and phosphatidylglycerol (20%); small amounts of phosphatidylcholine and traces of cardiolipin were also present. Growing and stationary-phase cells were similar in content and composition of phospholipids except for phosphatidylcholine, which increased two- to fivefold in the stationary-phase cells. The fatty acids of the phospholipids were characterized by two major acids, palmitic and a C16:1, with myristic and a C18:1 acid present in smaller amounts. The fatty acids present in purified phospholipid fractions varied considerably in relative proportions from fraction to fraction. No significant difference in the composition of phospholipids from the two strains was evident. Large amounts of beta-hydroxy lauric acid were detected only after saponification of the organisms. Differences in the lipid composition between the gonococcus and other gram-negative bacteria are discussed. PMID:810478

  15. Detection of Neisseria gonorrhoeae Isolates from Tonsils and Posterior Oropharynx

    PubMed Central

    Whiley, D. M.; Lee, D. M.; Snow, A. F.; Fairley, C. K.; Peel, J.; Bradshaw, C. S.; Hocking, J. S.; Lahra, M. M.; Chen, M. Y.

    2015-01-01

    We examined the factors influencing gonorrhea detection at the pharynx. One hundred men infected with Neisseria gonorrhoeae were swabbed from the tonsils and posterior oropharynx. N. gonorrhoeae was reisolated from the tonsils and posterior oropharynx in 62% and 52%, respectively (P = 0.041). Culture positivity was greater with higher gonococcal DNA loads at the tonsils (P = 0.001) and oropharynx (P < 0.001). N. gonorrhoeae can be cultured from the tonsils and posterior oropharynx with greater isolation rates where gonococcal loads are higher. PMID:26292303

  16. Epidemiological characterization of Neisseria gonorrhoeae by lectins.

    PubMed Central

    Schalla, W O; Whittington, W L; Rice, R J; Larsen, S A

    1985-01-01

    A total of 101 isolates of penicillinase-producing and non-penicillinase-producing Neisseria gonorrhoeae with known nutritional requirements, plasmid content, and serovars, were examined for lectin agglutination patterns. These isolates were from outbreaks in Georgia, California, Hawaii, and Pennsylvania. Cell suspensions made from 16- to 18-h cultures were mixed with 14 different lectins, and the resultant agglutination patterns were classified as agglutination groups. Among the 101 isolates tested, 24 different agglutination groups were demonstrated. Of the organisms tested, 55% were located in 3 of the 24 groups, and 86% of the isolates reacted with the lectins Trichosanthes kinlowii, Griffonia simplicifolia I, peanut agglutinin, soybean agglutinin, potato agglutinin, and wheat germ agglutinin. One isolate did not react with peanut or potato agglutinin, five isolates lacked reactivity with potato agglutinin, and six isolates did not react with wheat germ agglutinin. Of the wheat germ-negative isolates, four were from Pennsylvania and were identical with regard to auxotype, plasmid content, serovar, and lectin group. The other two wheat germ-negative isolates were from California and were unrelated by the same criteria to the four Pennsylvania isolates and to each other. Among the isolates tested, there were no differences in lectin groups with regard to the sex of the patient. In the Georgia collection, agglutination with one lectin group was confined to isolates of serogroup IA. This association was not observed for the other geographic areas. Some isolates showing identical auxotype, plasmid content, and serovars could be differentiated based on lectin agglutination patterns, whereas other isolates were identical by all testing criteria. PMID:3930560

  17. Transcriptional and functional analysis of the Neisseria gonorrhoeae fur regulon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator senses intracellular iron stores and acting as a repressor, directly regulates transcription of iron-responsive genes by binding to a conserve...

  18. DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

    PubMed

    Le Guern, Rémi; Miaux, Brigitte; Pischedda, Patricia; Herwegh, Stéphanie; Courcol, René

    2016-07-01

    We evaluated the DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in 55 urine samples. Crossing threshold (Ct) values were highly similar after 3 to 14 days at room temperature (+0.002, P = 0.99). Consequently, it does not seem necessary to transfer urine specimens into a transport medium in less than 24 hours as recommended by manufacturers. PMID:27130478

  19. Fluorescent monoclonal antibody for confirmation of Neisseria gonorrhoeae cultures.

    PubMed Central

    Laughon, B E; Ehret, J M; Tanino, T T; Van der Pol, B; Handsfield, H H; Jones, R B; Judson, F N; Hook, E W

    1987-01-01

    We evaluated a monoclonal fluorescent-antibody (FA) reagent (Neisseria gonorrhoeae Culture Confirmation Test; Syva Co., Palo Alto, Calif.) for confirmation of N. gonorrhoeae isolates obtained from clinics for sexually transmitted diseases in four cities. The FA test was performed in parallel with established confirmation procedures on all organisms growing on 773 primary culture plates of modified Thayer-Martin agar. All N. gonorrhoeae isolates reacted with the FA reagent and produced a bright, easily interpretable fluorescence. The FA test correctly identified 533 N. gonorrhoeae isolates from 474 patients and did not react with 90 N. meningitidis or with 213 non-Neisseria isolates. In one city (Baltimore), Gonochek II (Du Pont Co., Wilmington, Del.) failed to identify four N. gonorrhoeae isolates reactive with the FA reagent and confirmed as N. gonorrhoeae by Phadebact (Pharmacia Inc., Piscataway, N.J.) and acid production from sugars. The FA test was rapid and specific and could be performed directly from primary isolation plates. The test requires 1 h to perform and is applicable to mixed-flora cultures. PMID:3123514

  20. Characterization of a cryptic gene pair from Neisseria gonorrhoeae that is common to pathogenic Neisseria species.

    PubMed

    Seifert, H S; Wilson, D

    1992-03-01

    A pair of genes, each of which produces in Escherichia coli a 20-kDa, periplasmically localized protein that cross-reacts with anti-rpoN monoclonal antibody, was isolated from Neisseria gonorrhoeae. Homologs of the two genes were detected in pathogenic Neisseria species but not in commensal species. These genes are designated cnp1 and cnp2 (cryptic neisserial protein). PMID:1541538

  1. Will targeting oropharyngeal gonorrhoea delay the further emergence of drug-resistant Neisseria gonorrhoeae strains?

    PubMed

    Lewis, D A

    2015-06-01

    Gonorrhoea is an important sexually transmitted infection associated with serious complications and enhanced HIV transmission. Oropharyngeal infections are often asymptomatic and will only be detected by screening. Gonococcal culture has low sensitivity (<50%) for detecting oropharyngeal gonorrhoea, and, although not yet approved commercially, nucleic acid amplification tests (NAAT) are the assay of choice. Screening for oropharyngeal gonorrhoea should be performed in high-risk populations, such as men-who-have-sex-with-men(MSM). NAATs have a poor positive predictive value when used in low-prevalence populations. Gonococci have repeatedly thwarted gonorrhoea control efforts since the first antimicrobial agents were introduced. The oropharyngeal niche provides an enabling environment for horizontal transfer of genetic material from commensal Neisseria and other bacterial species to Neisseria gonorrhoeae. This has been the mechanism responsible for the generation of mosaic penA genes, which are responsible for most of the observed cases of resistance to extended-spectrum cephalosporins (ESC). As antimicrobial-resistant gonorrhoea is now an urgent public health threat, requiring improved antibiotic stewardship, laboratory-guided recycling of older antibiotics may help reduce ESC use. Future trials of antimicrobial agents for gonorrhoea should be powered to test their efficacy at the oropharynx as this is the anatomical site where treatment failure is most likely to occur. It remains to be determined whether a combination of frequent screening of high-risk individuals and/or laboratory-directed fluoroquinolone therapy of oropharyngeal gonorrhoea will delay the further emergence of drug-resistant N. gonorrhoeae strains. PMID:25911525

  2. History and epidemiology of antibiotic susceptibilities of Neisseria gonorrhoeae.

    PubMed

    Shigemura, Katsumi; Fujisawa, Masato

    2015-01-01

    Neisseria gonorrhoeae is a common causative microorganism of male urethritis. The most important problem with this infectious disease is antibiotic resistance. For instance, in the 1980's-1990's, most studies showed almost 100% susceptibility of N. gonorrhoeae to the representative cephalosporins, cefixime and cefpodoxime. By the late 1990s, the reported susceptibility decreased to 93.3-100% and further decreased to 82.9-100% in the early 2000's. However, reported susceptibility was revived to 95.8-100% in the late 2000's to 2010's. The susceptibility of N. gonorrhoeae to penicillins varied in different countries and regions. A 2002 Japanese study showed a resistance ratio of about 30% and while Laos, China and Korea showed 80-100% resistance. Fluoroquinolones have shown a dramatic change in their effect on N. gonorrhoeae. In the early 1990's, 0.3-1.3% of N. gonorrhoeae showed low susceptibility or resistance to ciprofloxacin in the US but this figure jumped to 9.5% by 1999. In Asia, N. gonorrhoeae ciprofloxacin resistance or lower susceptibility was about 80-90% in the early 2000's and this trend continues to the present day. Azithromycin is currently the possible last weapon for N. gonorrhoeae treatment per oral administration. The susceptibility of N. gonorrhoeae to azithromycin was 100% in Indonesia in 2004 and the latest study from Germany showed 6% resistance in strains from 2010-2011. This review summarizes the history and epidemiology of N. gonorrhoeae antibiotic susceptibilities, for which the most frequently used antibiotics vary between countries or regions. PMID:25410409

  3. [Purulent keratoconjunctivitis due to Neisseria gonorrhoeae and Chlamydia trachomatis coinfection].

    PubMed

    Arvai, Mariann; Ostorházi, Eszter; Mihalik, Noémi; Kárpáti, Sarolta; Marschalkó, Márta

    2013-05-26

    Gonococcal conjunctivitis is a rare infection induced by Neisseria gonorrhoeae and it usually manifests as a hyperacute purulent conjunctivitis. Ocular access of the infectious secretion during sexual intercourse is the way of transmission among adults. Inclusion conjunctivitis caused by the serovars D-K of Chlamydia trachomatis also affects the sexually active population. Authors present a case of a 33-year-old homosexual man who was treated for late latent syphilis formerly. Clinical symptoms were yellow purulent discharge for 3 weeks without any urological or upper respiratory tract symptoms. Conjunctival Neisseria gonorrhoeae and Chlamydia trachomatis infection was identified using cultures and polymerase chain reaction; pharyngeal swab culture and polymerase chain reaction showed positive results for both pathogens. The patient was probably under influence of party drugs at the time of sexual abuse when he became infected. After parenteral and oral cephalosporin and azithromycin therapy the patient had complete recovery within three weeks. PMID:23692878

  4. Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

    PubMed Central

    Peterson, S. W.; Martin, I.; Demczuk, W.; Bharat, A.; Hoang, L.; Wylie, J.; Allen, V.; Lefebvre, B.; Tyrrell, G.; Horsman, G.; Haldane, D.; Garceau, R.; Wong, T.

    2015-01-01

    The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results. PMID:25878350

  5. Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae.

    PubMed

    Peterson, S W; Martin, I; Demczuk, W; Bharat, A; Hoang, L; Wylie, J; Allen, V; Lefebvre, B; Tyrrell, G; Horsman, G; Haldane, D; Garceau, R; Wong, T; Mulvey, M R

    2015-07-01

    The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results. PMID:25878350

  6. Pili-taxis: Clustering of Neisseria gonorrhoeae bacteria

    NASA Astrophysics Data System (ADS)

    Taktikos, Johannes; Zaburdaev, Vasily; Biais, Nicolas; Stark, Holger; Weitz, David A.

    2012-02-01

    The first step of colonization of Neisseria gonorrhoeae bacteria, the etiological agent of gonorrhea, is the attachment to human epithelial cells. The attachment of N. gonorrhoeae bacteria to surfaces or other cells is primarily mediated by filamentous appendages, called type IV pili (Tfp). Cycles of elongation and retraction of Tfp are responsible for a common bacterial motility called twitching motility which allows the bacteria to crawl over surfaces. Experimentally, N. gonorrhoeae cells initially dispersed over a surface agglomerate into round microcolonies within hours. It is so far not known whether this clustering is driven entirely by the Tfp dynamics or if chemotactic interactions are needed. Thus, we investigate whether the agglomeration may stem solely from the pili-mediated attraction between cells. By developing a statistical model for pili-taxis, we try to explain the experimental measurements of the time evolution of the mean cluster size, number of clusters, and area fraction covered by the cells.

  7. Defenses against oxidative stress in Neisseria gonorrhoeae and Neisseria meningitidis: distinctive systems for different lifestyles.

    PubMed

    Seib, Kate L; Tseng, Hsing-Ju; McEwan, Alastair G; Apicella, Michael A; Jennings, Michael P

    2004-07-01

    Defenses against oxidative stress are crucial for the survival of the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. An Mn(II) uptake system is involved in manganese (Mn)-dependent resistance to superoxide radicals in N. gonorrhoeae. Here, we show that accumulation of Mn also confers resistance to hydrogen peroxide killing via a catalase-independent mechanism. An mntC mutant of N. meningitidis is susceptible to oxidative killing, but supplementation of growth media with Mn does not enhance the organism's resistance to oxidative killing. N. meningitidis is able to grow in the presence of millimolar levels of Mn ion, in contrast to N. gonorrhoeae, whose growth is retarded at Mn concentrations >100 micromol/L, indicating that Mn homeostasis in the 2 species is probably quite different. N. meningitidis superoxide dismutase B plays a role in protection against oxidative killing. However, a sodC mutant of N. meningitidis is no more sensitive to oxidative killing than is the wild type. A cytochrome c peroxidase (Ccp) is present in N. gonorrhoeae but not in N. meningitidis. Investigations of a ccp mutant revealed a role for Ccp in protection against hydrogen peroxide killing. These differences in oxidative defenses in the pathogenic Neisseria are most likely a result of their localization in different ecological niches. PMID:15195253

  8. Rapid identification of Neisseria gonorrhoeae and Neisseria meningitidis by using enzymatic profiles.

    PubMed Central

    D'Amato, R F; Eriquez, L A; Tomfohrde, K M; Singerman, E

    1978-01-01

    The enzymatic profiles of Neisseria gonorrhoeae, N. meningitidis, and related species were determined, using a total of 48 chromogenic substrates. Enzyme classes assayed for included glycosidases, aminopeptidases, phosphoamidases, proteases, lipases, esterases, and aryl sulfatase. A final test selection of 10 substrates, based upon their differential and reproducible characteristics, allowed the separation of N. gonorrhoeae and N. meningitidis from each other and from all species tested within 4 h after primary isolation on modified Thayer-Martin medium. The need for subculturing suspect colonies from modified Thayer-Martin medium to chocolate medium with a subsequent loss of 18 to 24 h of identification is eliminated. PMID:203607

  9. Neisseria gonorrhoeae and humans perform an evolutionary LINE dance.

    PubMed

    Anderson, Mark T; Seifert, H Steven

    2011-05-01

    Horizontal gene transfer is an important mechanism for generating genetic diversity. As the number of sequenced genomes continues to increase, so do the examples of horizontal genetic exchange between both related and divergent organisms. Here we discuss the recent finding that certain strains of the human pathogen Neisseria gonorrhoeae have incorporated a small fragment of human DNA sequence into their genomes. The horizontally acquired sequence exhibits 98-100% nucleotide identity to a 685 bp portion of the highly repetitive retrotransposable element L1 and its presence in the gonococcal genome has been confirmed by multiple molecular techniques. The possibility of similar L1 horizontal gene transfer events having occurred in other bacteria based on genomic sequence evidence is explored. Potential mechanisms of how N. gonorrhoeae was able to acquire and maintain this human sequence are also discussed in addition to the evolutionary implications of such an event. PMID:22016852

  10. [Antimicrobial susceptibility of Neisseria gonorrhoeae strains determined by disk diffusion].

    PubMed

    Llanes Caballero, R; Acosta Giraldo, J C; Sosa Puente, J; Guzmán Hernández, D; Gutiérrez González, O; Llop Hernández, A

    1999-01-01

    The Gonoccocus Laboratory of "Pedro Kourí" Tropical Medicine Institute carried out a study of in vitro susceptibility of Neisseria gonorrhoeae to penicillin, tetracycline, cefuroxime ceftriaxone, cefotaxine and ciprofoxacin by means of a disk diffusion method with the culture medium agar base GC plus supplement. In the first phase, the method was standardized and the reference N. gonorrhoeae ATCC 49226 strain was used whereas in the second phase, 50 gonococcal strains isolated in 8 provinces during 1995 and 1996 were examined. The results of such standardization confirmed that the antimicrobial susceptibility values were within the allowable limits. 52 and 34% of strains were resistant to penicillin and tetracycline respectively and all of them showed susceptibility to the rest of evaluated antimicrobial drugs. We recommend the use of the disk diffusion method for surveillance of gonococci resistance to these drugs in our country. PMID:10887570

  11. Pilin gene variation in Neisseria gonorrhoeae: reassessing the old paradigms

    PubMed Central

    Hill, Stuart A.; Davies, John K.

    2009-01-01

    Neisseria gonorrhoeae displays considerable potential for antigenic variation as shown in human experimental studies. Various surface antigens can change either by antigenic variation using RecA-dependent recombination schemes (e.g., PilE antigenic variation), or, alternatively, through phase variation (on/off switching) in a RecA-independent fashion (e.g., Opa and LOS phase variation). PilE antigenic variation has been well documented over the years. However, with the availability of the Nesseria gonorrhoeae FA1090 genome sequence, considerable genetic advances have recently been made regarding the mechanistic considerations of the gene conversion event leading to an altered PilE protein. This review will compare the various models that have been presented and will highlight potential mechanistic problems that may constrain any genetic model for pilE gene variation. PMID:19396954

  12. Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence.

    PubMed Central

    Seifert, H S; Ajioka, R S; Paruchuri, D; Heffron, F; So, M

    1990-01-01

    The method of shuttle mutagenesis has been extended to Neisseria gonorrhoeae. We have constructed a defective mini-Tn3 derivative that encodes chloramphenicol resistance in both N. gonorrhoeae and Escherichia coli and selected for mutations in the chloramphenicol resistance gene that express higher levels of antibiotic resistance in N. gonorrhoeae. Isogenic N. gonorrhoeae strains that differ only in pilin expression were constructed and used to test the effect of pilin null mutations on DNA transformation competence. PMID:2152910

  13. Strain-specific virulence-associated antigen of Neisseria gonorrhoeae.

    PubMed Central

    Pierce, W A; Leong, J K; Hough, D M

    1975-01-01

    A strain-specific virulence-associated antigen has been found in Neisseria gonorrhoeae strain F-62. Using immunodiffusion in agar gel, it has been shown that the antigen is distinguishable from endotoxin and the virulence-associated toxic protein. It does not appear to be derived from pili. The antigen was not detected in T1 and/or T2 colony type cultures of 10 other isolates. It exhibited a possible partial immunological relationship with an antigen found in one additional strain. It was susceptible to digestion with Pronase and trypsin. Images PMID:804445

  14. Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

    PubMed Central

    Hoyne, P A; Haas, R; Meyer, T F; Davies, J K; Elleman, T C

    1992-01-01

    Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell. Images PMID:1358873

  15. Ceftibuten Resistance and Treatment Failure of Neisseria gonorrhoeae Infection▿

    PubMed Central

    Lo, Janice Y. C.; Ho, K. M.; Leung, Anna O. C.; Tiu, Felisa S. T.; Tsang, Grand K. L.; Lo, Angus C. T.; Tapsall, John W.

    2008-01-01

    Neisseria gonorrhoeae infections have been empirically treated in Hong Kong with a single oral 400-mg dose of ceftibuten since 1997. Following anecdotal reports of the treatment failure of gonorrhea with oral extended-spectrum cephalosporins, the current study was undertaken to determine the antimicrobial susceptibility pattern and molecular characteristics of isolates of N. gonorrhoeae among patients with putative treatment failure in a sexually transmitted disease clinic setting. Between October 2006 and August 2007, 44 isolates of N. gonorrhoeae were studied from patients identified clinically to have treatment failure with empirical ceftibuten. The ceftibuten MICs for three strains were found to have been 8 mg/liter. These strains were determined by N. gonorrhoeae multiantigen sequence typing to belong to sequence type 835 (ST835) or the closely related ST2469. The testing of an additional eight archived ST835 strains revealed similarly elevated ceftibuten MICs. The penA gene sequences of these 11 isolates all had the mosaic pattern previously described as pattern X. Of note is that the ceftriaxone susceptibility results of these strains all fell within the susceptible range. It is concluded that ceftibuten resistance may contribute to the empirical treatment failure of gonorrhea caused by strains harboring the mosaic penA gene, which confers reduced susceptibility to oral extended-spectrum cephalosporins. Screening for such resistance in the routine clinical laboratory may be undertaken by the disk diffusion test. The continued monitoring of antimicrobial resistance and molecular characteristics of N. gonorrhoeae isolates is important to ensure that control and prevention strategies remain effective. PMID:18663018

  16. Functional analysis of the Gonococcal Genetic Island of Neisseria gonorrhoeae.

    PubMed

    Pachulec, Emilia; Siewering, Katja; Bender, Tobias; Heller, Eva-Maria; Salgado-Pabon, Wilmara; Schmoller, Shelly K; Woodhams, Katelynn L; Dillard, Joseph P; van der Does, Chris

    2014-01-01

    Neisseria gonorrhoeae is an obligate human pathogen that is responsible for the sexually-transmitted disease gonorrhea. N. gonorrhoeae encodes a T4SS within the Gonococcal Genetic Island (GGI), which secretes ssDNA directly into the external milieu. Type IV secretion systems (T4SSs) play a role in horizontal gene transfer and delivery of effector molecules into target cells. We demonstrate that GGI-like T4SSs are present in other β-proteobacteria, as well as in α- and γ-proteobacteria. Sequence comparison of GGI-like T4SSs reveals that the GGI-like T4SSs form a highly conserved unit that can be found located both on chromosomes and on plasmids. To better understand the mechanism of DNA secretion by N. gonorrhoeae, we performed mutagenesis of all genes encoded within the GGI, and studied the effects of these mutations on DNA secretion. We show that genes required for DNA secretion are encoded within the yaa-atlA and parA-parB regions, while genes encoded in the yfeB-exp1 region could be deleted without any effect on DNA secretion. Genes essential for DNA secretion are encoded within at least four different operons. PMID:25340397

  17. Preservation of Neisseria gonorrhoeae at -20 degrees C.

    PubMed Central

    Harbec, P S; Turcotte, P

    1996-01-01

    To explore the feasibility of preserving Neisseria gonorrhoeae at -20 degrees C, we studied its viability quantitatively and qualitatively for 12 and 18 months, respectively, in the following media: a gelatin-based medium used mainly to prepare dried gelatin discs (S. Yamai, Y. Obara, T. Nikkawa, Y Shimoda, and Y. Miyamoto, Br. J. Vener. Dis. 55:90-93, 1979), a simplified version (LSPQ preservation medium), and Trypticase soy broth with 10% (vol/vol) glycerol, a medium commonly used for preservation at -70 degrees C. The latter was studied for 4 months only. Four reference strains and two clinical isolates of N. gonorrhoeae were used. The storage temperature was rigorously preadjusted and monitored at -20 +/- 1 degree C during the entire project. After 12 months of storage, all strains remained viable in both gelatin-based media, whereas a significant loss of viability was observed in Trypticase soy broth-10% glycerol after only 4 months. After 18 months, five strains were still viable in both gelatin-based media and no significant difference was observed between antimicrobial susceptibility results and those of the original strains preserved at -70 degrees C. On the basis of these results, we believe that LSPQ preservation medium represents a good alternative for the storage of N. gonorrhoeae at -20 degrees C for at least a year. Furthermore, it is easy to prepare and use and can by stored at 4 to 8 degrees C for a year prior to use. PMID:8727891

  18. [MOLECULAR MECHANISMS OF DRUG RESISTANCE NEISSERIA GONORRHOEAE HISTORY AND PROSPECTS].

    PubMed

    Bodoev, I N; Il'ina, E N

    2015-01-01

    Neisseria gonorrhoeae (gonococcus) is a strict human pathogen, which causes gonorrhea--an infectious disease, whose origin dates back to more than two thousand years. Due to the unique plasticity of the genetic material, these bacteria have acquired the capacity to adapt to the host immune system, cause repeated infections, as well as withstand antimicrobials. Since the introduction of antibiotics in 1930s, gonococcus has displayed its propensity to develop resistance to all clinically useful antibiotics. It is important to note that the known resistance determinants of N. gonorrhoeae were acquired through horizontal gene transfer, recombination and spontaneous mutagenesis, and may be located both in the chromosome and on the plasmid. After introduction of a new antimicrobial drug, gonococcus becomes resistant within two decades and replaces sensitive bacterial population. Currently Ceftriaxone is the last remaining antibiotic for first-line treatment of gonorrhea. However, the first gonococcus displaying high-level resistance to Ceftriaxone was isolated in Japan a few years ago. Therefore, in the near future, gonorrhea may become untreatable. In the present review, we discuss the chronology of the anti-gonorrhea drugs (antibiotics) replacement, the evolution of resistance mechanisms emergence and future perspectives of N. gonorrhoeae treatment. PMID:26665738

  19. Neisseria gonorrhoeae antimicrobial susceptibility in Barcelona: penA, ponA, mtrR, and porB mutations and NG-MAST sequence types associated with decreased susceptibility to cephalosporins.

    PubMed

    Serra-Pladevall, J; Barberá, M J; Rodriguez, S; Bartolomé-Comas, R; Roig, G; Juvé, R; Andreu, A

    2016-09-01

    The aims of this study were to determine the antimicrobial susceptibility of Neisseria gonorrhoeae (NG) in our area, to analyze the molecular mechanisms involved in cephalosporins resistance, and to undertake molecular typing of our NG strains. Antimicrobial susceptibility was determined using the Etest. The genes penA, mtrR, penB, and ponA were studied. Molecular typing was performed by N. gonorrhoeae multiantigen sequence typing. Of 329 strains analyzed in 2013, none showed high-level cephalosporin resistance, but 8.2 % had resistance to cefixime [minimum inhibitory concentration (MIC) > 0.125 μg/mL] and 0.6 % to ceftriaxone (MIC > 0.125 μg/mL). Azithromycin resistance was documented in 4.3 % and ciprofloxacin resistance in 49.2 %. Among 48 strains with an MIC ≥ 0.125 μg/mL to cefixime, 58.3 % showed the penA mosaic pattern XXXIV, 98 % a Leu → Pro substitution at position 421 of the ponA gene, 100 % amino acid changes at positions 101 and 102 of the PorB1b porin, and 87.5 % of strains an adenine deletion in the promoter region of the MtrC-D-E efflux pump. A significant difference between strains with and without decreased cephalosporin susceptibility (MIC ≥ 0.125 μg/mL) was observed for these four genes. Of the 48 strains with an MIC ≥ 0.125 μg/mL to cefixime, 43.8 % belonged to the genogroup G1407 and 27.1 % belonged to the genogroup G2400. A significant association of G1407 with decreased susceptibility (MIC ≥ 0.125 μg/mL) and G2992 with susceptibility was found, and also between G1407 and mosaic pattern XXXIV and between G2400 and A501T substitution in penA. The NG resistance rate in our area is higher than the median of Europe. We have detected the emergence of G2400, which may be a source of antimicrobial resistance. PMID:27255221

  20. Resistance to penicillin and identification of penicillinase-producing Neisseria gonorrhoeae among clinical isolates in Thailand.

    PubMed Central

    Crum, J W; Duangmani, C; Vibulyasekha, S; Suthisomboon, K

    1980-01-01

    Penicillin-resistant Neisseria gonorrhoeae from 405 patients were studied by determination of penicillin minimal inhibitory concentrations an penicillinase production. Eighteen percent were identified as penicillin-producing N. gonorrhoeae and the mean minimal inhibitory concentration, for all except penicillin-producing strains, was 0.805 micrograms/ml. PMID:6778382

  1. Transformation-deficient mutants of piliated Neisseria gonorrhoeae.

    PubMed Central

    Biswas, G D; Lacks, S A; Sparling, P F

    1989-01-01

    Seven transformation-deficient mutants of piliated, competent Neisseria gonorrhoeae were isolated by screening them for their inability to be transformed by chromosomal DNA after chemical mutagenesis. Three distinct classes of mutants were obtained, each of which was piliated, as determined by electron microscopy. One class exhibited abnormal colony morphology and was unable to take up DNA into a DNase-resistant state. A second class was morphologically normal and took up DNA into a DNase-resistant state normally, but was deficient in both chromosomal and plasmid transformation; mutations in these mutants may affect entry of DNA into the cell proper. A third class was similar to the second but was fully competent for plasmid transformation, suggesting that there was a defect in a late stage of chromosomal transformation. Images PMID:2563367

  2. Possible Mechanism of Decreased Susceptibility of Neisseria gonorrhoeae to Penicillin

    PubMed Central

    Rodriguez, William; Saz, Arthur K.

    1975-01-01

    By use of 14C-labeled benzyl penicillin, it has been established that β-lactamases and/or acylases play no role in the resistance of Neisseria gonorrhoeae to penicillin. It has been found, however, that very susceptible strains of the organisms (minimal inhibitory concentration, 0.008 μg/ml) bind 10 to 15 times as much penicillin as do moderately to highly resistant strains of the gonoccoccus (minimal inhibitory concentration, 0.125 to 2.0 μg/ml). It is postulated that this degree of change in binding components of the whole cell and whole cytoplasmic membrane is sufficient to account for the decreased susceptibility of the organism to penicillin. PMID:808158

  3. A 26-base-pair repetitive sequence specific for Neisseria gonorrhoeae and Neisseria meningitidis genomic DNA.

    PubMed Central

    Correia, F F; Inouye, S; Inouye, M

    1986-01-01

    Two-dimensional heteroduplex mapping of Neisseria gonorrhoeae genomic DNA revealed a number of spots, indicating the existence of repetitive sequences. When one of the spots was extracted and used as a probe for Southern blot analysis, two HindIII bands (11.0 and 3.6 kilobases [kb]) of the genomic digest hybridized with approximately equal intensity. The 3.6-kb fragment was cloned and found to contain two different types of repeated sequence. One type was approximately 1.1 kb in length and was found at least twice in the entire genome. The other consisted of a 26-base-pair family GT(C/A)C(Py)G(Pu)TTTTTGTTAAT(Py)C(Pu)CTATA (Py, pyrimidine; Pu, purine) that was repeated at least 20 times in the entire genome. This repetitive sequence was found also in Neisseria meningitidis but not in various other gram-negative bacteria. Images PMID:3091577

  4. The obligate human pathogen, Neisseria gonorrhoeae, is polyploid.

    PubMed

    Tobiason, Deborah M; Seifert, H Steven

    2006-06-01

    We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts. PMID:16719561

  5. Porin protein of Neisseria gonorrhoeae: cloning and gene structure.

    PubMed Central

    Gotschlich, E C; Seiff, M E; Blake, M S; Koomey, M

    1987-01-01

    The outer membrane porin molecule of Neisseria gonorrhoeae is known as protein I (PI). Among different strains of gonococci there is variability of PI, and two main classes, PIA and PIB, have been recognized. A lambda gt11 bank of gonococcal DNA was screened using monoclonal antibodies directed to a PIB-type porin molecule of N. gonorrhoeae, and three immunoreactive clones were isolated. DNA sequence analysis indicated that each contained only portions of the PI structural gene, but that together they contained the complete gene, and its structure was determined. The DNA sequence predicts a protein of 348 amino acids with a typical 19 amino acid signal peptide. The PI protein resembles Escherichia coli porins in size, lack of long hydrophobic sequences, and absence of cysteine residues. Sequence homologies between PI and the E. coli porins were found, particularly in the 100 N-terminal and the 110 C-terminal amino acids. In addition to the coding sequence of PI, the complementary strand contains a large open reading frame. At the 3' end of the PI gene, immediately following an inverted repeat (probably the transcription terminator), the clone contains an unusual sequence consisting of 31 perfect repeats of the heptamer CTGTTTT. Hybridization analysis suggests that there is a single structural gene for PI and that it is homologous to the gene found in a PIA-bearing strain of gonococcus. Images PMID:2825179

  6. Transcriptional landscape and essential genes of Neisseria gonorrhoeae

    PubMed Central

    Remmele, Christian W.; Xian, Yibo; Albrecht, Marco; Faulstich, Michaela; Fraunholz, Martin; Heinrichs, Elisabeth; Dittrich, Marcus T.; Müller, Tobias; Reinhardt, Richard; Rudel, Thomas

    2014-01-01

    The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes. PMID:25143534

  7. Current and future antimicrobial treatment of gonorrhoea - the rapidly evolving Neisseria gonorrhoeae continues to challenge.

    PubMed

    Unemo, Magnus

    2015-01-01

    Neisseria gonorrhoeae has developed antimicrobial resistance (AMR) to all drugs previously and currently recommended for empirical monotherapy of gonorrhoea. In vitro resistance, including high-level, to the last option ceftriaxone and sporadic failures to treat pharyngeal gonorrhoea with ceftriaxone have emerged. In response, empirical dual antimicrobial therapy (ceftriaxone 250-1000 mg plus azithromycin 1-2 g) has been introduced in several particularly high-income regions or countries. These treatment regimens appear currently effective and should be considered in all settings where local quality assured AMR data do not support other therapeutic options. However, the dual antimicrobial regimens, implemented in limited geographic regions, will not entirely prevent resistance emergence and, unfortunately, most likely it is only a matter of when, and not if, treatment failures with also these dual antimicrobial regimens will emerge. Accordingly, novel affordable antimicrobials for monotherapy or at least inclusion in new dual treatment regimens, which might need to be considered for all newly developed antimicrobials, are essential. Several of the recently developed antimicrobials deserve increased attention for potential future treatment of gonorrhoea. In vitro activity studies examining collections of geographically, temporally and genetically diverse gonococcal isolates, including multidrug-resistant strains particularly with resistance to ceftriaxone and azithromycin, are important. Furthermore, understanding of effects and biological fitness of current and emerging (in vitro induced/selected and in vivo emerged) genetic resistance mechanisms for these antimicrobials, prediction of resistance emergence, time-kill curve analysis to evaluate antibacterial activity, appropriate mice experiments, and correlates between genetic and phenotypic laboratory parameters, and clinical treatment outcomes, would also be valuable. Subsequently, appropriately designed

  8. Mechanisms of iron acquisition by the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae.

    PubMed

    Rohde, Kyle H; Dyer, David W

    2003-09-01

    It is well established that bacterial pathogenesis is dependent on the ability to acquire iron within the host. The success of the highly adapted obligate human pathogens Neisseria meningitidis (NM) and Neisseria gonorrhoeae (NG) can be attributed in part to the efficient utilization of multiple host iron (Fe) sources, allowing replication on mucosal surfaces, in the bloodstream, and intracellularly. Most Gram-negative bacterial strategies for scavenging iron from the human host rely on the TonB protein to energize active iron transport across the outer membrane. Pathogenic Neisseria express multiple high-affinity iron transporters including a family of two-component TonB-dependent receptors as well as multiple single-component TonB-dependent Fe transporters. This review describes our current understanding of the mechanisms Neisseria have evolved to utilize various iron sources encountered during infection of the human host. Recent studies have provided insight into the interaction of neisserial outer membrane receptors with host iron carrier proteins. Emerging structural information on neisserial iron transporters will be compared with the crystal structures and biochemical data available for homologous Escherichia coli TonB-dependent Fe-siderophore receptors. In the process, we will highlight the aspects of the iron transport process that are unique and those that remain to be experimentally demonstrated in Neisseria. These include receptor structure/function, the mechanism of iron removal from protein ligands, the fate of Fe and heme-Fe after traversing the outer membrane, and the role of TonB-associated energy in receptor functions. Finally, we will discuss regulatory mechanisms that control the expression of iron scavenging systems. The investigation of iron metabolism in NM and NG is important for understanding the biochemistry of this virulence factor, the development of vaccines targeted at outer membrane iron receptors, and therapeutic interventions

  9. Neisseria gonorrhoeae cell envelope: permeability to hydrophobic molecules.

    PubMed Central

    Lysko, P G; Morse, S A

    1981-01-01

    Isogenic variants of antibiotic-resistant and -sensitive Neisseria gonorrhoeae were examined for differences in the inhibition of oxygen uptake by steroid hormones. Mutants designated as env, which possessed cell envelope mutations allowing phenotypic suppression of low-level antibiotic resistance, were more sensitive to steroid hormone inhibition of oxygen uptake than the wild-type parental strains. Possession of an mtr locus, which confers nonspecific resistance to multiple antibiotics, dyes, and detergents, was also associated with an increase in resistance to steroid hormone inhibition of oxygen uptake. The penA2 locus, which confers an eightfold increase in resistance to penicillin, was not responsible for the increased resistance to steroid hormones. Phospholipids in the outer membrane of intact env-2 cells were susceptible to digestion by phospholipase C, indicating exposure of phospholipid head groups on the outer surface. Cells of a wild-type and mtr-2 strain were not susceptible to phospholipase C digestion unless they were pretreated with mixed exoglycosidases. This pretreatment also increased the sensitivity of mtr-2 cells to progesterone inhibition of O2 uptake. These data suggest that the permeability of the gonococcus to hydrophobic antibiotic and steroid molecules is mediated by the degree of phospholipid exposure on the outer membrane. PMID:6780535

  10. Radioimmunoassay for detection of antibodies to Neisseria gonorrhoeae.

    PubMed Central

    Usategui, M; Savard, E V; Mondabaugh, S M; Keigher, N L

    1982-01-01

    A radioimmunoassay has been developed and evaluated for the serological diagnosis of gonorrhea. Purified gonococcal antigen was obtained from a culture of Neisseria gonorrhoeae (B370) and labeled with 125I for use in a double-antibody test system. The test was evaluated in populations segregated by sex and risk. The specificity of the assay in females was 90.2% (55/61) in low risk, 82.2% (2,245/ 2,732) in medium risk, and 54.1% (335/619) in high risk. The sensitivity was 69% (20/29) in medium risk and 78.3% (288/367) in high risk. In males, test specificity was 92.3% (24/26) in low risk and 50% (48/96) in high risk. The sensitivity was 70.8% (143/202) in the high-risk group. The data in this study indicate that this assay should not be employed for screening of either high- or medium-risk populations. PMID:6809784

  11. Neisseria gonorrhoeae suppresses the oxidative burst of human polymorphonuclear leukocytes

    PubMed Central

    Criss, Alison K.; Seifert, H. Steven

    2008-01-01

    Symptomatic infection with Neisseria gonorrhoeae (Gc) results in a potent polymorphonuclear leukocyte (PMN)-driven inflammatory response, but the mechanisms by which Gc withstands PMN attack are poorly defined. Here we report that Gc can suppress the PMN oxidative burst, a central component of the PMN antimicrobial arsenal. Primary human PMNs remained viable after exposure to liquid-grown, exponential-phase, opacity-associated protein (Opa)-negative Gc of strains FA1090 and MS11 but did not generate reactive oxygen species (ROS), even after bacterial opsonization. Liquid-grown FA1090 Gc expressing OpaB, an Opa protein previously correlated with PMN ROS production, elicited a minor PMN oxidative burst. PMN ROS production in response to Opa− and OpaB+ Gc was markedly enhanced if bacteria were agar-grown or if liquid-grown bacteria were heat killed. Liquid-grown Opa- Gc inhibited the PMN oxidative burst elicited by isogenic dead bacteria, formylated peptides or Staphylococcus aureus but did not inhibit PMN ROS production by OpaB+ Gc or phorbol esters. Suppression of the oxidative burst required Gc-PMN contact and bacterial protein synthesis but not phagocytosis. These results suggest that viable Gc directly inhibits PMN signaling pathways required for induction of the oxidative burst, which may contribute to gonococcal pathogenesis during inflammatory stages of gonorrheal disease. PMID:18684112

  12. Neisseria gonorrhoeae prepilin export studied in Escherichia coli.

    PubMed Central

    Dupuy, B; Taha, M K; Pugsley, A P; Marchal, C

    1991-01-01

    The pilE gene of Neisseria gonorrhoeae MS11 and a series of pilE-phoA gene fusions were expressed in Escherichia coli. The PhoA hybrid proteins were shown to be located in the membrane fraction of the cells, and the prepilin product of the pilE gene was shown to be located exclusively in the cytoplasmic membrane. Analysis of the prepilin-PhoA hybrids showed that the first 20 residues of prepilin can function as an efficient export (signal) sequence. This segment of prepilin includes an unbroken sequence of 8 hydrophobic or neutral residues that form the N-terminal half of a 16-residue hydrophobic region of prepilin. Neither prepilin nor the prepilin-PhoA hybrids were processed by E. coli leader peptidase despite the presence of two consensus cleavage sites for this enzyme just after this hydrophobic region. Comparisons of the specific molecular activities of the four prepilin-PhoA hybrids and analysis of their susceptibility to proteolysis by trypsin and proteinase K in spheroplasts allow us to propose two models for the topology of prepilin in the E. coli cytoplasmic membrane. The bulk of the evidence supports the simplest of the two models, in which prepilin is anchored in the membrane solely by the N-terminal hydrophobic domain, with the extreme N terminus facing the cytoplasm and the longer C terminus facing the periplasm. Images FIG. 2 FIG. 4 FIG. 5 FIG. 6 PMID:1938955

  13. Genetic transformation of genes for protein II in Neisseria gonorrhoeae.

    PubMed Central

    Schwalbe, R S; Cannon, J G

    1986-01-01

    The protein II (PII) outer membrane proteins of Neisseria gonorrhoeae are a family of heat-modifiable proteins that are subject to phase variation, in which the synthesis of different PII species is turned on and off at a high frequency. Transformation of PII genes from a donor gonococcal strain into a recipient strain was detected with monoclonal antibodies specific for the PII proteins of the donor. Individual PII protein-expressing transformants generally bound only one donor-specific PII monoclonal antibody. Recovery of transformants expressing a donor-specific PII protein depended on the PII protein expression state of the donor: the transformed population bound only monoclonal antibodies specific for PII proteins that were expressed in the donor. Colony variants with an altered frequency of switching of PII protein expression were isolated, but the altered switch phenotype did not cotransform with the PII structural gene. These results provide genetic evidence that PII proteins are the products of different genes and that expressed and unexpressed forms of the PII gene are different from each other. Images PMID:3087951

  14. Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae.

    PubMed

    Salimnia, H; Radia, A; Bernatchez, S; Beveridge, T J; Dillon, J R

    2000-01-01

    We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ(Ng) encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZ(Ng) is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ(Ng) on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZ(Ng) was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ(Ng) gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZ(Ng)-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZ(Ng)-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize at the middle of the cell. Expression of the FtsZ(Ng)-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZ(Ng)-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector. PMID:10648099

  15. Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria gonorrhoeae Infection.

    PubMed

    Savage, Victoria J; Charrier, Cédric; Salisbury, Anne-Marie; Box, Helen; Chaffer-Malam, Nathan; Huxley, Anthony; Kirk, Ralph; Noonan, Gary M; Mohmed, Sarfraz; Craighead, Mark W; Ratcliffe, Andrew J; Best, Stuart A; Stokes, Neil R

    2016-09-01

    There is an urgent need for new antibiotics to treat multidrug-resistant Neisseria gonorrhoeae In this report, the microbiology, in vivo pharmacokinetics, and efficacy of REDX05931, a representative novel tricyclic topoisomerase inhibitor, were evaluated. REDX05931 demonstrated high oral bioavailability in mice and reduced N. gonorrhoeae infection after a single dose in a mouse model of gonorrhea. These data support the potential of this series of small molecules as a new treatment for drug-resistant gonorrheal infections. PMID:27324777

  16. Anaerobic survival of clinical isolates and laboratory strains of Neisseria gonorrhoea: use in transfer and storage.

    PubMed Central

    Short, H B; Clark, V L; Kellogg, D S; Young, F E

    1982-01-01

    Eleven laboratory strains and 67 clinical isolates of Neisseria gonorrhoeae were tested for the ability to survive during anaerobic incubation. The survival of the laboratory strains was dependent on auxotype, temperature, and cell density on agar plates. For both the laboratory strains and the clinical isolates, anaerobic survival was better at lower temperatures. We concluded that anaerobic incubation, for as long as 7 days, is useful when transporting or storing N. gonorrhoeae. Images PMID:6808019

  17. Electron capture gas chromatographic detection of acethylmethylcarbinol produced by neisseria gonorrhoeae.

    PubMed

    Morse, C D; Brooks, J B; Kellogg, D S

    1976-01-01

    Acetylmethylcarbinol (acetoin) production by Neisseria gonorrhoeae and other Neisseria species was established by gas-liquid chromatography and by mass spectrometric data. Sixty-nine isolates of Neisseria were tested by incubating them in a chemically defined fluid medium. The medium was extracted with organic solvents and derivatized with heptafluorobutryic anhydride for gas chromatography and mass spectrometry. Cultures of 58 of the same strains were tested with the conventional Voges-Proskauer reagents, and results were compared with those of gas-liquid chromatography. When glucose was used as an energy source, N. gonorrhoeae, some N. meningitidis, and N. lactamica produced enough acetoin in 16 h to be detectable by either method, whereas other Neisseria species produce amounts detectable only by gas chromatography. The conventional acetylmethylcarbinol test with the chemically defined medium and maltose as an energy source might be used to develop methods that would differentiate certain members of the genus, including the pathogenic species. PMID:815266

  18. Crystallographic analysis of Neisseria meningitidis PorB extracellular loops potentially implicated in TLR2 recognition.

    PubMed

    Kattner, Christof; Toussi, Deana N; Zaucha, Jan; Wetzler, Lee M; Rüppel, Nadine; Zachariae, Ulrich; Massari, Paola; Tanabe, Mikio

    2014-03-01

    Among all Neisseriae species, Neisseria meningitidis and Neisseria gonorrhoeae are the only human pathogens, causative agents of bacterial meningitis and gonorrhoea, respectively. PorB, a pan-Neisseriae trimeric porin that mediates diffusive transport of essential molecules across the bacterial outer membrane, is also known to activate host innate immunity via Toll-like receptor 2 (TLR2)-mediated signaling. The molecular mechanism of PorB binding to TLR2 is not known, but it has been hypothesized that electrostatic interactions contribute to ligand/receptor binding. Strain-specific sequence variability in the surface-exposed loops of PorB which are potentially implicated in TLR2 binding, may explain the difference in TLR2-mediated cell activation in vitro by PorB homologs from the commensal Neisseriae lactamica and the pathogen N. meningitidis. Here, we report a comparative structural analysis of PorB from N. meningitidis serogroup B strain 8765 (63% sequence homology with PorB from N. meningitidis serogroup W135) and a mutant in which amino acid substitutions in the extracellular loop 7 lead to significantly reduced TLR2-dependent activity in vitro. We observe that this mutation both alters the loop conformation and causes dramatic changes of electrostatic surface charge, both of which may affect TLR2 recognition and signaling. PMID:24361688

  19. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    PubMed

    Connor, Daniel O; Zantow, Jonas; Hust, Michael; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae. PMID:26859666

  20. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display

    PubMed Central

    Connor, Daniel O.; Zantow, Jonas; Hust, Michael; Bier, Frank F.; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae. PMID:26859666

  1. An evaluation of the role of properdin in alternative pathway activation on Neisseria meningitidis and Neisseria gonorrhoeae.

    PubMed

    Agarwal, Sarika; Ferreira, Viviana P; Cortes, Claudio; Pangburn, Michael K; Rice, Peter A; Ram, Sanjay

    2010-07-01

    Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological "native" (dimeric, trimeric, and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, preincubation of bacteria with unfractionated properdin, followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of 10 Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the "conventional" mechanism of stabilizing AP C3 convertases. PMID:20530262

  2. A modified reduced transport fluid for the preservation of Neisseria gonorrhoeae during transport.

    PubMed

    Finlayson, M H; Koralewski, F F; Kindermann, R A

    1975-10-11

    Reduced transport fluid (RTF) was modified by altering its pH and by the addition of a yeast dialysate. This reduced transport yeast-containing fluid (RTYF) was shown to be superior to RTF in maintaining viability of Neisseria gonorrhoeae in cultures and in clinical material. PMID:242082

  3. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to...

  4. Decline in Decreased Cephalosporin Susceptibility and Increase in Azithromycin Resistance in Neisseria gonorrhoeae, Canada

    PubMed Central

    Sawatzky, P.; Liu, G.; Allen, V; Lefebvre, B.; Hoang, L.; Drews, S.; Horsman, G.; Wylie, J.; Haldane, D.; Garceau, R.; Ratnam, S.; Wong, T.; Archibald, C.; Mulvey, M.R.

    2016-01-01

    Antimicrobial resistance profiles were determined for Neisseria gonorrhoeae strains isolated in Canada during 2010–2014. The proportion of isolates with decreased susceptibility to cephalosporins declined significantly between 2011 and 2014, whereas azithromycin resistance increased significantly during that period. Continued surveillance of antimicrobial drug susceptibilities is imperative to inform treatment guidelines. PMID:26689114

  5. Draft Genome Sequence of Neisseria gonorrhoeae Sequence Type 1407, a Multidrug-Resistant Clinical Isolate.

    PubMed

    Anselmo, A; Ciammaruconi, A; Carannante, A; Neri, A; Fazio, C; Fortunato, A; Palozzi, A M; Vacca, P; Fillo, S; Lista, F; Stefanelli, P

    2015-01-01

    Gonorrhea may become untreatable due to the spread of resistant or multidrug-resistant strains. Cefixime-resistant gonococci belonging to sequence type 1407 have been described worldwide. We report the genome sequence of Neisseria gonorrhoeae strain G2891, a multidrug-resistant isolate of sequence type 1407, collected in Italy in 2013. PMID:26272575

  6. Microbiological Characteristics of Chlamydia trachomatis and Neisseria gonorrhoeae Infections in South African Women

    PubMed Central

    de Waaij, Dewi J.; Bos, Myrte; van der Eem, Lisette; Bébéar, Cécile; Mbambazela, Nontembeko; Ouburg, Sander; Peters, Remco P. H.

    2015-01-01

    We analyzed data of 263 women with at least one genital or anorectal sexually transmitted infection from a cross-sectional study conducted in rural South Africa. We provide new insights concerning the concurrence of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis infections as well as the characteristics of bacterial loads. PMID:26511740

  7. Alterations in Dihydropteroate Synthetase in Cell-Free Extracts of Sulfanilamide-Resistant Neisseria meningitidis and Neisseria gonorrhoeae

    PubMed Central

    Ho, Richard I.; Corman, Leonard; Morse, Stephen A.; Artenstein, Malcolm S.

    1974-01-01

    Extracts from Neisseria meningitidis and N. gonorrhoeae with varying susceptibility to sulfanilamide have been investigated for dihydropteroate synthetase activity. Sulfanilamide was a competitive inhibitor of dihydropteroate synthetase with respect to p-aminobenzoate in extracts from both species. Though the Km for p-aminobenzoate was unaffected, the Ki for sulfanilamide increased and the Vmax decreased as the strains' resistance to sulfanilamide increased. Temperature studies have revealed differences in the dihydropteroate synthetase from N. meningitidis and N. gonorrhoeae. A direct relationship was observed between the minimal inhibitory concentration of sulfanilamide determined in vitro and the ratio of Ki/Km. This ratio may be a molecular explanation of sulfanilamide resistance for both N. meningitidis and N. gonorrhoeae. PMID:15825393

  8. Draft Genome Sequence of Neisseria gonorrhoeae Strain NG_869 with Penicillin, Tetracycline and Ciprofloxacin Resistance Determinants Isolated from Malaysia.

    PubMed

    Ang, Geik Yong; Yu, Choo Yee; Yong, Delicia Ann; Cheong, Yuet Meng; Yin, Wai-Fong; Chan, Kok-Gan

    2016-06-01

    Gonorrhea is a sexually transmitted infection caused by Neisseria gonorrhoeae and the increasing reports of multidrug-resistant gonococcal isolates are a global public health care concern. Herein, we report the genome sequence of N. gonorrhoeae strain NG_869 isolated from Malaysia which may provide insights into the drug resistance determinants in gonococcal bacteria. PMID:27570316

  9. Multicenter Investigation of Gepotidacin (GSK2140944) Agar Dilution Quality Control Determinations for Neisseria gonorrhoeae ATCC 49226.

    PubMed

    Jones, Ronald N; Fedler, Kelley A; Scangarella-Oman, Nicole E; Ross, James E; Flamm, Robert K

    2016-07-01

    Gepotidacin, a novel triazaacenaphthylene antibacterial agent, is the first in a new class of type IIA topoisomerase inhibitors with activity against many biothreat and conventional pathogens, including Neisseria gonorrhoeae To assist ongoing clinical studies of gepotidacin to treat gonorrhea, a multilaboratory quality assurance investigation determined the reference organism (N. gonorrhoeae ATCC 49226) quality control MIC range to be 0.25 to 1 μg/ml (88.8% of gepotidacin MIC results at the 0.5 μg/ml mode). PMID:27161642

  10. Induction of HIV-1 long terminal repeat-mediated transcription by Neisseria gonorrhoeae.

    PubMed

    Chen, Adrienne; Boulton, Ian C; Pongoski, Jodi; Cochrane, Alan; Gray-Owen, Scott D

    2003-03-01

    Gonorrhoea enhances the transmission of HIV through increased viral shedding and the increased probability of seroconversion among previously HIV-negative individuals. However, the mechanism(s) underlying these influences remain poorly understood. We demonstrated that exposure to Neisseria gonorrhoeae induces the nuclear factor kappa B-dependent transcription from the HIV-1 long terminal repeat in derivatives of the Jurkat CD4 T cell line. These data suggest that gonococcal infection directly impacts HIV-1 transmission through the localized stimulation of viral expression. PMID:12598784

  11. Neisseria gonorrhoeae infection protects human endocervical epithelial cells from apoptosis via expression of host antiapoptotic proteins.

    PubMed

    Follows, S A; Murlidharan, J; Massari, P; Wetzler, L M; Genco, C A

    2009-09-01

    Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-kappaB activation and resulted in increased gene expression of the NF-kappaB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells. PMID:19546192

  12. Inversion of Moraxella lacunata type 4 pilin gene sequences by a Neisseria gonorrhoeae site-specific recombinase.

    PubMed Central

    Rozsa, F W; Meyer, T F; Fussenegger, M

    1997-01-01

    A plasmid library of Neisseria gonorrhoeae sequences was screened for the ability to mediate recombinations on a sequence containing the Moraxella lacunata type 4 pilin gene invertible region in Escherichia coli. A plasmid containing the N. gonorrhoeae sequence encoding the putative recombinase (gcr) was identified and sequenced. Plasmids containing gcr were able to mediate site-specific recombinations despite a weak amino acid homology to Piv, the native M. lacunata pilin gene invertase. The gcr gene is present only in pathogenic strains of Neisseria tested; however, in our assays gene knockouts of gcr did not alter the variation of surface features that play a role in the pathogenesis of N. gonorrhoeae. PMID:9079926

  13. Comparative characterization of the iga gene encoding IgA1 protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae.

    PubMed

    Lomholt, H; Poulsen, K; Kilian, M

    1995-02-01

    Cloning and sequencing of the IgA1 protease gene (iga) from Neisseria meningitidis strain HF13 showed an overall structure equivalent to iga genes from Neisseria gonorrhoeae and Haemophilus influenzae, although no region corresponding to the gonococcal alpha-peptide was evident. An additional 18 N. meningitidis and 3 H. influenzae iga genes were amplified by the polymerase chain reaction technique and sequenced corresponding approximately to the N-terminal half of the mature enzyme. Comparative analyses of a total of 29 iga genes showed that pathogenic Neisseria have iga genes with a significantly lower degree of heterogeneity than H. influenzae iga genes. Recombinational events indicated by mosaic-like structures corresponding to those found among N. gonorrhoeae protease genes were detected among N. meningitidis iga genes. One region showed characteristic differences in sequence and length which correlated with each of the different cleavage specificities. Meningococci were extremely conserved in this region with no evidence of recombination between isolates of different cleavage specificities. Sequences further downstream showed no obvious relationship with enzyme cleavage type. This region consisted of conserved areas interspersed with highly variable areas. Amino acid sequence homologies in the variable regions of meningococci reflected the antigenic types defined by using polyclonal neutralizing antibodies. PMID:7783620

  14. Non-cytotoxic nanomaterials enhance antimicrobial activities of cefmetazole against multidrug-resistant Neisseria gonorrhoeae.

    PubMed

    Li, Lan-Hui; Yen, Muh-Yong; Ho, Chao-Chi; Wu, Ping; Wang, Chien-Chun; Maurya, Pawan Kumar; Chen, Pai-Shan; Chen, Wei; Hsieh, Wan-Yu; Chen, Huei-Wen

    2013-01-01

    The emergence and spread of antibiotic-resistant Neisseria gonorrhoeae has led to difficulties in treating patients, and novel strategies to prevent and treat this infection are urgently needed. Here, we examined 21 different nanomaterials for their potential activity against N. gonorrhoeae (ATCC 49226). Silver nanoparticles (Ag NPs, 120 nm) showed the greatest potency for reducing N. gonorrhoeae colony formation (MIC: 12.5 µg/ml) and possessed the dominant influence on the antibacterial activity with their properties of the nanoparticles within a concentration range that did not induce cytotoxicity in human fibroblasts or epithelial cells. Electron microscopy revealed that the Ag NPs significantly reduced bacterial cell membrane integrity. Furthermore, the use of clinical isolates of multidrug-resistant N. gonorrhoeae showed that combined treatment with 120 nm Ag NPs and cefmetazole produced additive effects. This is the first report to screen the effectiveness of nanomaterials against N. gonorrhoeae, and our results indicate that 120 nm Ag NPs deliver low levels of toxicity to human epithelial cells and could be used as an adjuvant with antibiotic therapy, either for topical use or as a coating for biomaterials, to prevent or treat multidrug-resistant N. gonorrhoeae. PMID:23705013

  15. Non-Cytotoxic Nanomaterials Enhance Antimicrobial Activities of Cefmetazole against Multidrug-Resistant Neisseria gonorrhoeae

    PubMed Central

    Li, Lan-Hui; Yen, Muh-Yong; Ho, Chao-Chi; Wu, Ping; Wang, Chien-Chun; Maurya, Pawan Kumar; Chen, Pai-Shan; Chen, Wei; Hsieh, Wan-Yu; Chen, Huei-Wen

    2013-01-01

    The emergence and spread of antibiotic-resistant Neisseria gonorrhoeae has led to difficulties in treating patients, and novel strategies to prevent and treat this infection are urgently needed. Here, we examined 21 different nanomaterials for their potential activity against N. gonorrhoeae (ATCC 49226). Silver nanoparticles (Ag NPs, 120 nm) showed the greatest potency for reducing N. gonorrhoeae colony formation (MIC: 12.5 µg/ml) and possessed the dominant influence on the antibacterial activity with their properties of the nanoparticles within a concentration range that did not induce cytotoxicity in human fibroblasts or epithelial cells. Electron microscopy revealed that the Ag NPs significantly reduced bacterial cell membrane integrity. Furthermore, the use of clinical isolates of multidrug-resistant N. gonorrhoeae showed that combined treatment with 120 nm Ag NPs and cefmetazole produced additive effects. This is the first report to screen the effectiveness of nanomaterials against N. gonorrhoeae, and our results indicate that 120 nm Ag NPs deliver low levels of toxicity to human epithelial cells and could be used as an adjuvant with antibiotic therapy, either for topical use or as a coating for biomaterials, to prevent or treat multidrug-resistant N. gonorrhoeae. PMID:23705013

  16. Antimicrobial susceptibility and genotyping analysis of Hungarian Neisseria gonorrhoeae strains in 2013.

    PubMed

    Nemes-Nikodém, Éva; Brunner, Alexandra; Pintér, Dóra; Mihalik, Noémi; Lengyel, György; Marschalkó, Márta; Kárpáti, Sarolta; Szabó, Dóra; Ostorházi, Eszter

    2014-12-01

    Emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern worldwide. The current study aims to determine the antimicrobial resistance in N. gonorrhoeae and associated molecular typing to enhance gonococcal antimicrobial surveillance in Hungary. In the National N. gonorrhoeae Reference Laboratory of Hungary 187 N. gonorrhoeae infections were detected in 2013, antibiograms were determined for all the isolated strains, and 52 (one index strain from every sexually contact related group) of them were also analysed by the N. gonorrhoeae multi-antigen sequence typing (NG-MAST) method. Twenty-two different NG-MAST sequence types (STs) were identified, of which 8 STs had not been previously described. In Hungary, the highly diversified gonococcal population displayed high resistance to penicillin, ciprofloxacin and tetracycline (the antimicrobials previously recommended for gonorrhoea treatment). Resistance to the currently recommended extended spectrum cephalosporines were rare: only two of the expected strains, an ST 1407 and an ST 210, had cefixime MIC above the resistance breakpoint. By the revision of our National Treatment Guideline, it must be considered, that the azithromycin resistance is about 60% among the four most frequently isolated STs in Hungary. PMID:25496972

  17. Antimicrobial susceptibility and genetic characteristics of Neisseria gonorrhoeae isolates from Vietnam, 2011

    PubMed Central

    2013-01-01

    Background Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health concern worldwide. In Vietnam, knowledge regarding N. gonorrhoeae prevalence and AMR is limited, and data concerning genetic characteristics of N. gonorrhoeae is totally lacking. Herein, we investigated the phenotypic AMR (previous, current and possible future treatment options), genetic resistance determinants for extended-spectrum cephalosporins (ESCs), and genotypic distribution of N. gonorrhoeae isolated in 2011 in Hanoi, Vietnam. Methods N. gonorrhoeae isolates from Hanoi, Vietnam isolated in 2011 (n = 108) were examined using antibiograms (Etest for 10 antimicrobials), Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST), and sequencing of ESC resistance determinants (penA, mtrR and penB). Results The levels of in vitro resistance were as follows: ciprofloxacin 98%, tetracycline 82%, penicillin G 48%, azithromycin 11%, ceftriaxone 5%, cefixime 1%, and spectinomycin 0%. The MICs of gentamicin (0.023-6 mg/L), ertapenem (0.002-0.125 mg/L) and solithromycin (<0.016-0.25 mg/L) were relatively low. No penA mosaic alleles were found, however, 78% of the isolates contained an alteration of amino acid A501 (A501V (44%) and A501T (34%)) in the encoded penicillin-binding protein 2. A single nucleotide (A) deletion in the inverted repeat of the promoter region of the mtrR gene and amino acid alterations in MtrR was observed in 91% and 94% of the isolates, respectively. penB resistance determinants were detected in 87% of the isolates. Seventy-five different NG-MAST STs were identified, of which 59 STs have not been previously described. Conclusions In Vietnam, the highly diversified gonococcal population displayed high in vitro resistance to antimicrobials previously recommended for gonorrhoea treatment (with exception of spectinomycin), but resistance also to the currently recommended ESCs were found. Nevertheless, the MICs of three potential future treatment

  18. Zabofloxacin (DW-224a) activity against Neisseria gonorrhoeae including quinolone-resistant strains.

    PubMed

    Jones, Ronald N; Biedenbach, Douglas J; Ambrose, Paul G; Wikler, Matthew A

    2008-09-01

    Zabofloxacin, a new fluoroquinolone compound (DW-224a), was tested by reference agar dilution methods against 35 strains of multiresistant Neisseria gonorrhoeae. The potency of zabofloxacin (MIC(50), 0.016 microg/mL) was generally comparable with azithromycin but 8-fold superior to ciprofloxacin. This novel naphthyridine should be explored as an alternative therapy for quinolone-nonsusceptible gonorrhea and Chlamydia trachomatis infections. PMID:18620833

  19. In Vitro selection of Neisseria gonorrhoeae mutants with elevated MIC values and increased resistance to cephalosporins.

    PubMed

    Johnson, Steven R; Grad, Yonatan; Ganakammal, Satishkumar Ranganathan; Burroughs, Mark; Frace, Mike; Lipsitch, Marc; Weil, Ryan; Trees, David

    2014-11-01

    Strains of Neisseria gonorrhoeae with mosaic penA genes bearing novel point mutations in penA have been isolated from ceftriaxone treatment failures. Such isolates exhibit significantly higher MIC values to third-generation cephalosporins. Here we report the in vitro isolation of two mutants with elevated MICs to cephalosporins. The first possesses a point mutation in the transpeptidase region of the mosaic penA gene, and the second contains an insertion mutation in pilQ. PMID:25199775

  20. Studies on gonococcus infection. XV. Identification of surface proteins of Neisseria gonorrhoeae correlated with leukocyte association.

    PubMed Central

    King, G J; Swanson, J

    1978-01-01

    Neisseria gonorrhoeae which exhibit high levels of leukocyte association have a surface protein which is considerably diminished in isogenic gonococci which exhibit low levels of leukocyte association (LA). The LA protein exhibits strain variation in molecular weight and immunogenicity. Membranes derived from LA+ and LA- organisms show quantitative differences in their adsorption to leukocytes; these differences are analogous to those found for the intact organisms regarding their association with leukocytes. Images PMID:211086

  1. Construtcion of Neisseria gonorrhoeae porin B plasmid recombinant and its expression in E. coli.

    PubMed

    Song, Qifa; Liao, Fang; Ye, Siying; Cui, Bing; Xiong, Ping

    2005-01-01

    A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E. coli DE3 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 DE3 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 EISA 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. PMID:16201262

  2. Crystal structure of the open state of the Neisseria gonorrhoeae MtrE outer membrane channel.

    PubMed

    Lei, Hsiang-Ting; Chou, Tsung-Han; Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Do, Sylvia V; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2014-01-01

    Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel. PMID:24901251

  3. Antimicrobial susceptibility testing of Neisseria gonorrhoeae and implications for epidemiology and therapy.

    PubMed Central

    Fekete, T

    1993-01-01

    Antimicrobial susceptibility testing (AST) of Neisseria gonorrhoeae has been under development since the early days of antimicrobial agents. However, it is rarely applied to clinical isolates today. The history of the various in vitro tests to determine the susceptibility of N. gonorrhoeae to antibiotics is rich with evidence that these results predict response to therapy for almost all agents tested. Further, AST is a useful and important aspect of strain characterization and disease epidemiology in conjunction with the more specific but laborious techniques of auxotyping, serotyping, and plasmid analysis. Current technology has overcome many of the objections to AST for N. gonorrhoeae with standardization of test media and the development of an accurate disk diffusion AST method that is suited to most clinical laboratories regardless of volume or level of technical expertise. Ironically, the very low level of resistance to the current primary treatment strategy in the United States, ceftriaxone or another potent cephalosporin, makes the use of AST somewhat superfluous. PMID:8457978

  4. Changing antimicrobial resistance profiles among Neisseria gonorrhoeae isolates in Italy, 2003 to 2012.

    PubMed

    Carannante, Anna; Renna, Giovanna; Dal Conte, Ivano; Ghisetti, Valeria; Matteelli, Alberto; Prignano, Grazia; Impara, Giampaolo; Cusini, Marco; D'Antuono, Antonietta; Vocale, Caterina; Antonetti, Raffaele; Gaino, Marina; Busetti, Marina; Latino, Maria Agnese; Mencacci, Antonella; Bonanno, Carmen; Cava, Maria Carmela; Giraldi, Cristina; Stefanelli, Paola

    2014-10-01

    The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobial agents is a major public health concern and a serious issue related to the occurrence of further untreatable gonorrhea infections. A retrospective analysis on 1,430 N. gonorrhoeae isolates, collected from 2003 through 2012, for antimicrobial susceptibility by Etest and molecular characterization by Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) was carried out in Italy. Azithromycin-resistant gonococci decreased from 14% in 2007 to 2.2% in 2012. Similarly, isolates with high MICs to cefixime (>0.125 mg/liter) decreased from 11% in 2008 to 3.3% in 2012. The ciprofloxacin resistance rate remains quite stable, following an increasing trend up to 64% in 2012. The percentage of penicillinase-producing N. gonorrhoeae (PPNG) significantly declined from 77% in 2003 to 7% in 2012. A total of 81 multidrug-resistant (MDR) gonococci were identified, showing 11 different antimicrobial resistance patterns. These were isolated from men who have sex with men (MSM) and from heterosexual patients. Two sequence types (STs), ST661 and ST1407, were the most common. Genogroup 1407, which included cefixime-, ciprofloxacin-, and azithromycin-resistant isolates, was found. In conclusion, a change in the antimicrobial resistance profiles among gonococci was identified in Italy together with a percentage of MDR isolates. PMID:25070110

  5. Antibiotic resistance in Neisseria gonorrhoeae: origin, evolution, and lessons learned for the future.

    PubMed

    Unemo, Magnus; Shafer, William M

    2011-08-01

    The strict human pathogen Neisseria gonorrhoeae has caused gonorrhea for thousands of years, and currently gonorrhea is the second most prevalent bacterial sexually transmitted infection worldwide. Given the ancient nature of N. gonorrhoeae and its unique obligate relationship with humankind over the millennia, its remarkable ability to adapt to the host immune system and cause repeated infections, and its propensity to develop resistance to all clinically useful antibiotics, the gonococcus is an ideal pathogen on which to study the evolution of bacterial pathogenesis, including antimicrobial resistance, over the long term and within the host during infection. Recently, the first gonococcus displaying high-level resistance to ceftriaxone, identified in Japan, was characterized in detail. Ceftriaxone is the last remaining option for empirical first-line treatment, and N. gonorrhoeae now seems to be evolving into a true "superbug." In the near future, gonorrhea may become untreatable in certain circumstances. Herein, the history of antibiotics used for treatment of gonorrhea, the evolution of resistance emergence in N. gonorrhoeae, the linkage between resistance and biological fitness of N. gonorrhoeae, lessons learned, and future perspectives are reviewed and discussed. PMID:22239555

  6. Ligase chain reaction for detection of Neisseria gonorrhoeae in urogenital swabs.

    PubMed Central

    Ching, S; Lee, H; Hook, E W; Jacobs, M R; Zenilman, J

    1995-01-01

    The ligase chain reaction (LCR) is an in vitro nucleic acid amplification technique that exponentially amplifies targeted DNA sequences. In a multicenter study, we evaluated the use of a 4-h LCR-based assay for the diagnosis of Neisseria gonorrhoeae infection of the cervix and male urethra. The LCR results were compared with those of culture for N. gonorrhoeae by using selective media. This assay amplifies target sequences within the N. gonorrhoeae opacity gene. Discordant LCR-positive and culture-negative specimens were further evaluated by testing by another LCR assay which used N. gonorrhoeae-specific pilin probe sets. A total of 1,539 female endocervical specimens and 808 male urethral swab specimens were evaluated in the study. An expanded "gold standard" was defined to include all culture-positive as well as culture-negative, confirmed LCR-positive specimens. After resolution of discrepant samples, the sensitivities of the N. gonorrhoeae LCR assays for the female and male specimens were 97.3 and 98.5%, respectively, with specificities of 99.6 and 99.8%, respectively. Resolved culture sensitivities were 83.9 and 96.5% for the female and male specimens, respectively. The LCR assay for gonorrhea is a rapid, highly sensitive nonculture method for detecting gonococcal infection of the cervix and male urethra. PMID:8586683

  7. Preliminary evaluation of the ligase chain reaction for specific detection of Neisseria gonorrhoeae.

    PubMed Central

    Birkenmeyer, L; Armstrong, A S

    1992-01-01

    Rapid identification of Neisseria gonorrhoeae in clinical specimens is essential for effective control. Traditional culture requires a minimum of 24 h, and for some specimens harboring gonococci, the gonococci fail to grow or are misidentified. The recently described ligase chain reaction (LCR) is a highly specific and sensitive DNA amplification technique which was evaluated as an alternative to routine culture. Three LCR probe sets were used. Two of the probe sets were directed against the multi-copy Opa genes (Omp-II), while the third set was targeted against the multicopy Pilin genes. Each LCR probe set was evaluated with 260 microorganisms including 136 global isolates of N. gonorrhoeae, 41 isolates of N. meningitidis, and 10 isolates of N. lactamica; 26 nonpathogenic Neisseria strains; and 47 isolates of non-Neisseria species that may reside in clinical specimens. Amplification products were detected by using the IMx LCR format (Abbott Laboratories, Abbott Park, Ill.). Strains of N. gonorrhoeae were assayed at 270 cells per LCR (approximately 6.7 x 10(4) CFU/ml) with the Opa and Pilin probes, producing signals at least 21 and 15 times above background, respectively. In contrast, only background values were observed when testing the probe sets with 124 nongonococcal strains at 1.3 x 10(6) cells per LCR (approximately 3.2 x 10(8) CFU/ml). One hundred urogenital specimens were assayed by LCR, and compared with culture, the three probes were 100% sensitive (8 of 8) and 97.8% specific (90 of 92), resulting in an agreement of 98% (98 of 100). On the basis of the results of these preliminary studies, LCR has the potential to be an accurate and rapid DNA probe assay for the detection of N. gonorrhoeae in clinical specimens. PMID:1452689

  8. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners.

    PubMed

    Fingerhuth, Stephanie M; Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L

    2016-05-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y-1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y-1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y-1 in HMW and 3.12 y-1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population's treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread. PMID:27196299

  9. Oral ciprofloxacin versus ceftriaxone for the treatment of urethritis from resistant Neisseria gonorrhoeae in Zambia.

    PubMed Central

    Bryan, J P; Hira, S K; Brady, W; Luo, N; Mwale, C; Mpoko, G; Krieg, R; Siwiwaliondo, E; Reichart, C; Waters, C

    1990-01-01

    Neisseria gonorrhoeae strains resistant to treatment with penicillin, tetracycline, and/or spectinomycin are increasing in prevalence in many parts of the world. In Zambia, 52% of N. gonorrhoeae isolates produced beta-lactamase in 1986. Few oral regimens have proven effective for treatment of resistant N. gonorrhoeae. We conducted a prospective, double-blind, randomized clinical trial of 250 mg of ciprofloxacin given orally versus 250 mg of ceftriaxone given intramuscularly for treatment of uncomplicated gonococcal urethritis in adult males. Two hundred men were enrolled and treated. The two groups were comparable in age (27.5 years), prevalence of latent syphilis (14 and 10%), and human immunodeficiency virus infection (32 and 38%). Of 165 patients with cultures positive for N. gonorrhoeae who returned for follow-up, ciprofloxacin cured 83 of 83 (100%), including 26 with penicillinase-producing N. gonorrhoeae (PPNG) and 21 with N. gonorrhoeae with chromosomally mediated resistance to multiple antibiotics (CMRNG), and ceftriaxone cured 81 of 82 (98.7%), including 30 with PPNG and 19 with CMRNG. Both treatment regimens were well tolerated. Chlamydia trachomatis in urethral exudate was found by direct fluorescent-antibody microscopic examination or by culture in 10 (5%) participants. All N. gonorrhoeae isolates were inhibited by ceftriaxone at 0.06 micrograms/ml, except one which was inhibited at 0.125 micrograms/ml, while ciprofloxacin inhibited all isolates at 0.03 micrograms/ml. Ciprofloxacin is a safe and effective therapy for uncomplicated gonococcal urethritis, including that caused by PPNG and CMRNG in human immunodeficiency virus-infected men. PMID:2113796

  10. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners

    PubMed Central

    Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L.

    2016-01-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y−1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y−1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y−1 in HMW and 3.12 y−1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population’s treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread. PMID:27196299

  11. Neisseria meningitidis C114 contains silent, truncated pilin genes that are homologous to Neisseria gonorrhoeae pil sequences.

    PubMed Central

    Perry, A C; Nicolson, I J; Saunders, J R

    1988-01-01

    Neisseria meningitidis pili can be classified into two groups: those (referred to here as class I pili) which are similar to gonococcal pili in that they react with monoclonal antibody SM1 and those that are dissimilar to gonococcal pili in that they lack the SM1-reactive epitope (class II pili). Pilus expression in N. meningitidis C114, a class II pilus-producing isolate, was investigated. The sole genomic segment of this strain that bore extensive homology with the pilE locus of Neisseria gonorrhoeae P9 was cloned in Escherichia coli. The production of the pilus structural subunit (pilin) from this meningococcal segment could not be detected by immunological and coupled in vitro transcription-translation analyses. Nucleotide sequence analysis revealed the presence in the C114 genome of two variant, tandemly arranged pilin genes (copies 1 and 2). Copies 1 and 2 are partial pilin genes that constitute part of a silent meningococcal pilin gene (pil gene) region, designated pilS. Both copies are truncated, corresponding to variable domains of the gonococcal pilE gene but lacking homologous N-terminal coding sequences. Located within sequences surrounding copies 1 and 2 were several classes of repeated elements that are associated with pil loci in N. gonorrhoeae. Images PMID:2895102

  12. Molecular characterization of the interaction between sialylated Neisseria gonorrhoeae and factor H.

    PubMed

    Shaughnessy, Jutamas; Ram, Sanjay; Bhattacharjee, Arnab; Pedrosa, Joana; Tran, Connie; Horvath, Gabor; Monks, Brian; Visintin, Alberto; Jokiranta, T Sakari; Rice, Peter A

    2011-06-24

    Human factor H (HufH), a key inhibitor of the alternative pathway of complement, binds to Neisseria gonorrhoeae and constitutes an important mechanism of human-specific complement evasion. The C-terminal domain 20 of HufH contains the binding site for sialylated gonococci. We exploited differences in amino acid sequences between human and non-binding chimpanzee fH domain 20 to create cross-species mutations to define amino acids important for binding to sialylated gonococci. We used fH/Fc fusion constructs that contained contiguous fH domains 18-20 fused to Fc fragments of murine IgG2a. The Fc region was used both as a tag for detection of each fusion molecule on the bacterial surface and as an indicator for complement-dependent killing. Arg-1203 was critical for binding to both porin (Por) B.1A and PorB.1B strains. Modeling of the R1203N human-to-chimpanzee mutation using the crystal structure of HufH19-20 as a template showed a loss of positive charge that protrudes at the C terminus of domain 20. We tested the functional importance of Arg-1203 by incubating sialylated gonococci with normal human serum, in the presence of wild-type HufH18-20/Fc or its R1203A mutant. Gonococci bound and were killed by wild-type HufH18-20/Fc but not by the R1203A mutant. A recombinant fH/Fc molecule that contained chimpanzee domain 20, humanized only at amino acid 1203 (N1203R) also bound to sialylated gonococci and restored killing. These findings provide further insights into the species specificity of gonococcal infections and proof-of-concept of a novel therapeutic approach against gonorrhea, a disease rapidly becoming resistant to conventional antibiotics. PMID:21531728

  13. Azithromycin resistance and its mechanism in Neisseria gonorrhoeae strains in Hyogo, Japan.

    PubMed

    Shigemura, Katsumi; Osawa, Kayo; Miura, Makiko; Tanaka, Kazushi; Arakawa, Soichi; Shirakawa, Toshiro; Fujisawa, Masato

    2015-05-01

    Therapeutic options are limited for Neisseria gonorrhoeae infection, especially for oral drugs. The purpose of this study was to investigate the susceptibility of N. gonorrhoeae to oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains of N. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59 N. gonorrhoeae strains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 μg/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 μg/ml or less) (4/22; 18.2%) (P < 0.0001). However, a mutation of codon 40 (Ala → Asp) in the mtrR gene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P < 0.0001). In N. gonorrhoeae multiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or -intermediate strains, as previously described. In conclusion, a deletion mutation in the mtrR promoter region may be a significant indicator for higher MIC (0.5 μg/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance in N. gonorrhoeae infection. PMID:25712352

  14. Mutation in ribosomal protein S5 leads to spectinomycin resistance in Neisseria gonorrhoeae.

    PubMed

    Ilina, Elena N; Malakhova, Maya V; Bodoev, Ivan N; Oparina, Nina Y; Filimonova, Alla V; Govorun, Vadim M

    2013-01-01

    Spectinomycin remains a useful reserve option for therapy of gonorrhea. The emergence of multidrug-resistant Neisseria gonorrhoeae strains with decreased susceptibility to cefixime and to ceftriaxone makes it the only medicine still effective for treatment of gonorrhea infection in analogous cases. However, adoption of spectinomycin as a routinely used drug of choice was soon followed by reports of spectinomycin resistance. The main molecular mechanism of spectinomycin resistance in N. gonorrhoeae was C1192T substitution in 16S rRNA genes. Here we reported a Thr-24→Pro mutation in ribosomal protein S5 (RPS5) found in spectinomycin resistant clinical N. gonorrhoeae strain, which carried no changes in 16S rRNA. In a series of experiments, the transfer of rpsE gene allele encoding the mutant RPS5 to the recipient N. gonorrhoeae strains was analyzed. The relatively high rate of transformation [ca. 10(-5) colony-forming units (CFUs)] indicates the possibility of spread of spectinonycin resistance within gonococcal population due to the horizontal gene transfer (HGT). PMID:23847609

  15. Cloning and organization of seven arginine biosynthesis genes from Neisseria gonorrhoeae.

    PubMed Central

    Picard, F J; Dillon, J R

    1989-01-01

    A genomic library for Neisseria gonorrhoeae, constructed in the lambda cloning vector EMBL4, was screened for clones carrying arginine biosynthesis genes by complementation of Escherichia coli mutants. Clones complementing defects in argA, argB, argE, argG, argIF, carA, and carB were isolated. An E. coli defective in the acetylornithine deacetylase gene (argE) was complemented by the ornithine acetyltransferase gene (argJ) from N. gonorrhoeae. This heterologous complementation is reported for the first time. The carAB operon from E. coli hybridized with the gonococcal clones that carried carA or carB genes under conditions of high stringency, detecting 80% or greater similarity and showing that the nucleotide sequence of the carbamoylphosphate synthetase genes is very similar in these two organisms. Under these conditions for hybridization, the gonococcal clones carrying argB or argF genes did not hybridize with plasmids containing the corresponding E. coli gene. Cocomplementation experiments established gene linkage between carA and carB. Clones complementing a gene defect in argE were also able to complement an argA mutation. This suggests that the enzyme ornithine acetyltransferase from N. gonorrhoeae (encoded by argJ) may be able to complement both argA and argE mutations in E. coli. The arginine biosynthesis genes in N. gonorrhoeae appear to be scattered as in members of the family Pseudomonadaceae. Images PMID:2493452

  16. Population structure of Neisseria gonorrhoeae based on whole genome data and its relationship with antibiotic resistance.

    PubMed

    Ezewudo, Matthew N; Joseph, Sandeep J; Castillo-Ramirez, Santiago; Dean, Deborah; Del Rio, Carlos; Didelot, Xavier; Dillon, Jo-Anne; Selden, Richard F; Shafer, William M; Turingan, Rosemary S; Unemo, Magnus; Read, Timothy D

    2015-01-01

    Neisseria gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted infection (STI) of major importance. As a result of antibiotic resistance, there are now limited options for treating patients. We collected draft genome sequence data and associated metadata data on 76 N. gonorrhoeae strains from around the globe and searched for known determinants of antibiotics resistance within the strains. The population structure and evolutionary forces within the pathogen population were analyzed. Our results indicated a cosmopolitan gonoccocal population mainly made up of five subgroups. The estimated ratio of recombination to mutation (r/m = 2.2) from our data set indicates an appreciable level of recombination occurring in the population. Strains with resistance phenotypes to more recent antibiotics (azithromycin and cefixime) were mostly found in two of the five population subgroups. PMID:25780762

  17. Evaluation of the Microcult system for isolating and identifying Neisseria gonorrhoeae.

    PubMed Central

    Williams, R J; Ratnatunga, C S; Hamilton-Miller, J M; Brumfitt, W

    1978-01-01

    Specimens from 95 patients attending a venereal diseases clinic were examined for gonococci by three methods--a conventional culture technique using modified Thayer-Martin medium, microscopy of a Gram-stained direct smear, and the Microcult system. For 56% of the specimens the results by all three methods agreed. Assuming the results obtained by culture on Thayer-Martin medium to be correct, the largest source of error was due to false-positive results: microscopy gave 26 and Microcult gave 15 such results. False-negative results were less common: Microcult gave 14, microscopy six. Microcult gave positive results more quickly than the conventional Thayer-Martin cultural method, but the gonococci were difficult to isolate by subculture from the Microcult culture pads. The Microcult medium was not absolutely specific for Neisseria gonorrhoeae. Nevertheless, the Microcult test may well prove to be a useful adjunct to the diagnosis of gonorrhoea, especially when laboratory facilities are not readily available. PMID:417090

  18. High-level azithromycin-resistant Neisseria gonorrhoeae clinical isolate in France, March 2014.

    PubMed

    Bercot, B; Belkacem, A; Goubard, A; Mougari, F; Sednaoui, P; La Ruche, G; Cambau, E

    2014-01-01

    We report the first case in France of a high-level azithromycin-resistant Neisseria gonorrhoeae (minimum inhibitory concentration (MIC) = 96 mg/L) assigned to MLST7363 (NG-MAST ST6360), also resistant to ciprofloxacin and tetracycline but susceptible to ceftriaxone. The patient was a 51 year-old heterosexual man who returned following 1g azithromycin monotherapy. Mechanisms of azithromycin resistance were a C2599T mutation in the four copies of the rrl gene and a novel mutation in the promoter of the mtrR gene. PMID:25394255

  19. Recommendations for the Laboratory-Based Detection of Chlamydia trachomatis and Neisseria gonorrhoeae — 2014

    PubMed Central

    Papp, John R.; Schachter, Julius; Gaydos, Charlotte A.; Van Der Pol, Barbara

    2014-01-01

    Summary This report updates CDC's 2002 recommendations regarding screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections (CDC. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections—2002. MMWR 2002;51[No. RR-15]) and provides new recommendations regarding optimal specimen types, the use of tests to detect rectal and oropharyngeal C. trachomatis and N. gonorrhoeae infections, and circumstances when supplemental testing is indicated. The recommendations in this report are intended for use by clinical laboratory directors, laboratory staff, clinicians, and disease control personnel who must choose among the multiple available tests, establish standard operating procedures for collecting and processing specimens, interpret test results for laboratory reporting, and counsel and treat patients. The performance of nucleic acid amplification tests (NAATs) with respect to overall sensitivity, specificity, and ease of specimen transport is better than that of any of the other tests available for the diagnosis of chlamydial and gonococcal infections. Laboratories should use NAATs to detect chlamydia and gonorrhea except in cases of child sexual assault involving boys and rectal and oropharyngeal infections in prepubescent girls and when evaluating a potential gonorrhea treatment failure, in which case culture and susceptibility testing might be required. NAATs that have been cleared by the Food and Drug Administration (FDA) for the detection of C. trachomatis and N. gonorrhoeae infections are recommended as screening or diagnostic tests because they have been evaluated in patients with and without symptoms. Maintaining the capability to culture for both N. gonorrhoeae and C. trachomatis in laboratories throughout the country is important because data are insufficient to recommend nonculture tests in cases of sexual assault in prepubescent boys and extragenital anatomic site exposure in prepubescent girls. N

  20. In Vitro Antibiotic Susceptibility of Neisseria gonorrhoeae in Jakarta, Indonesia

    PubMed Central

    Lesmana, Murad; Lebron, Carlos I.; Taslim, Djufri; Tjaniadi, Periska; Subekti, Decy; Wasfy, Momtaz O.; Campbell, James R.; Oyofo, Buhari A.

    2001-01-01

    Antibiotic susceptibilities were determined for 122 Neisseria gonorrheae isolates obtained from 400 sex workers in Jakarta, Indonesia, and susceptibilities to ciprofloxacin, cefuroxime, cefoxitin, cefotaxime, ceftriaxone, chloramphenicol, and spectinomycin were found. All isolates were resistant to tetracycline. A number of the isolates demonstrated decreased susceptibilities to erythromycin (MIC ≥ 1.0 μg/ml), thiamphenicol (MIC ≥ 1.0 μg/ml), kanamycin (MIC ≥ 16.0 μg/ml), penicillin (MIC ≥ 2.0 μg/ml), gentamicin (MIC ≥ 16.0 μg/ml), and norfloxacin (MIC = 0.5 μg/ml). These data showed that certain antibiotics previously used in the treatment of gonorrhea are no longer effective. PMID:11120999

  1. Common antigenic domains in transferrin-binding protein 2 of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae type b.

    PubMed

    Stevenson, P; Williams, P; Griffiths, E

    1992-06-01

    There is now considerable evidence to show that in the Neisseria and Haemophilus species, membrane receptors specific for either transferrin or lactoferrin are involved in the acquisition of iron from these glycoproteins. In Neisseria meningitidis, the transferrin receptor appears to consist of two proteins, one of which (TBP 1) has an M(r) of 95,000 and the other of which (TBP 2) has an M(r) ranging from 68,000 to 85,000, depending on the strain; TBP 2 binds transferrin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, but TBP 1 does not do so. The relative contributions of these two proteins to the binding reaction observed with intact cells and to iron uptake are presently unknown. However, they are being considered as potential components of a group B meningococcal vaccine. Analogous higher- and lower-molecular-weight proteins associated with transferrin binding have been found in N. gonorrhoeae and Haemophilus influenzae. Previous work with polyclonal antibodies raised in mice with whole cells of iron-restricted N. meningitidis showed that the meningococcal TBP 2 exhibits considerable antigenic heterogeneity. Here, we report that antiserum against purified TBP 2 from one strain of N. meningitidis cross-reacts on immunoblotting with the TBP 2 of all meningococcal isolates examined, as well as with the TBP 2 of N. gonorrhoeae. This antiserum also cross-reacted with the TBP 2 of several strains of H. influenzae type b, thus showing the presence of common antigenic domains among these functionally equivalent proteins in different pathogens; no cross-reaction was detected with a purified sample of the human transferrin receptor. PMID:1587606

  2. Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile.

    PubMed

    Ortiz, María Carolina; Lefimil, Claudia; Rodas, Paula I; Vernal, Rolando; Lopez, Mercedes; Acuña-Castillo, Claudio; Imarai, Mónica; Escobar, Alejandro

    2015-01-01

    Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies. PMID:26125939

  3. The effectiveness of gentamicin in the treatment of Neisseria gonorrhoeae: a systematic review

    PubMed Central

    2014-01-01

    Background A high level of resistance in Neisseria gonorrhoeae has developed against penicillins, sulphonamides, tetracyclines and quinolones, and recent surveillance data have shown a gradual reduction in sensitivity to current first-line agents with an upward drift in the minimum inhibitory concentration of ceftriaxone. Laboratory sensitivity testing suggests that gentamicin, an aminoglycoside, may be an effective treatment option for gonorrhoea infection when used as a single intramuscular dose. Methods A search of electronic reference databases and grey literature was used to identify randomised trials and well-conducted prospective studies with concurrent controls evaluating single-dose gentamicin against placebo or a comparator regimen in the treatment of uncomplicated gonorrhoea infection in men and women aged 16 years and over. The primary outcome was microbiological cure of N. gonorrhoeae. Results Eight hundred and thirty-nine studies were identified, of which five (1,063 total participants) were included. All five studies administered single-dose gentamicin via intramuscular injection to men with uncomplicated gonococcal urethritis. Three studies were randomised trials, one was quasi-randomised and one was non-randomised but included a comparator arm. Comparator antibiotics included an alternative aminoglycoside or antibiotic used in the syndromic management of male urethritis. Methodology was poorly described in all five included studies. The high risk of bias within studies and clinical heterogeneity between studies meant that it was inappropriate to pool data for meta-analysis. Cure rates of 62% to 98% were reported with gentamicin treatment. The relative risk of cure was comparable between gentamicin and comparator antibiotics. Conclusions The studies identified provide insufficient data to support or refute the efficacy and safety of single-dose intramuscular gentamicin in the treatment of uncomplicated gonorrhoea infection. Additional randomised

  4. Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile

    PubMed Central

    Ortiz, María Carolina; Lefimil, Claudia; Rodas, Paula I.; Vernal, Rolando; Lopez, Mercedes; Acuña-Castillo, Claudio; Imarai, Mónica; Escobar, Alejandro

    2015-01-01

    Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies. PMID:26125939

  5. Proteomics of Neisseria gonorrhoeae: the treasure hunt for countermeasures against an old disease.

    PubMed

    Baarda, Benjamin I; Sikora, Aleksandra E

    2015-01-01

    Neisseria gonorrhoeae is an exquisitely adapted, strictly human pathogen and the causative agent of the sexually transmitted infection gonorrhea. This ancient human disease remains a serious problem, occurring at high incidence globally and having a major impact on reproductive and neonatal health. N. gonorrhoeae is rapidly evolving into a superbug and no effective vaccine exists to prevent gonococcal infections. Untreated or inadequately treated gonorrhea can lead to severe sequelae, including pelvic inflammatory disease and infertility in women, epididymitis in men, and sight-threatening conjunctivitis in infants born to infected mothers. Therefore, there is an immediate need for accelerated research toward the identification of molecular targets for development of drugs with new mechanisms of action and preventive vaccine(s). Global proteomic approaches are ideally suited to guide these studies. Recent quantitative proteomics (SILAC, iTRAQ, and ICAT) have illuminated the pathways utilized by N. gonorrhoeae to adapt to different lifestyles and micro-ecological niches within the host, while comparative 2D SDS-PAGE analysis has been used to elucidate spectinomycin resistance mechanisms. Further, high-throughput examinations of cell envelopes and naturally released membrane vesicles have unveiled the ubiquitous and differentially expressed proteins between temporally and geographically diverse N. gonorrhoeae isolates. This review will focus on these different approaches, emphasizing the role of proteomics in the search for vaccine candidates. Although our knowledge of N. gonorrhoeae has been expanded, still far less is known about this bacterium than the closely related N. meningitidis, where genomics- and proteomics-driven studies have led to the successful development of vaccines. PMID:26579097

  6. Proteomics of Neisseria gonorrhoeae: the treasure hunt for countermeasures against an old disease

    PubMed Central

    Baarda, Benjamin I.; Sikora, Aleksandra E.

    2015-01-01

    Neisseria gonorrhoeae is an exquisitely adapted, strictly human pathogen and the causative agent of the sexually transmitted infection gonorrhea. This ancient human disease remains a serious problem, occurring at high incidence globally and having a major impact on reproductive and neonatal health. N. gonorrhoeae is rapidly evolving into a superbug and no effective vaccine exists to prevent gonococcal infections. Untreated or inadequately treated gonorrhea can lead to severe sequelae, including pelvic inflammatory disease and infertility in women, epididymitis in men, and sight-threatening conjunctivitis in infants born to infected mothers. Therefore, there is an immediate need for accelerated research toward the identification of molecular targets for development of drugs with new mechanisms of action and preventive vaccine(s). Global proteomic approaches are ideally suited to guide these studies. Recent quantitative proteomics (SILAC, iTRAQ, and ICAT) have illuminated the pathways utilized by N. gonorrhoeae to adapt to different lifestyles and micro-ecological niches within the host, while comparative 2D SDS-PAGE analysis has been used to elucidate spectinomycin resistance mechanisms. Further, high-throughput examinations of cell envelopes and naturally released membrane vesicles have unveiled the ubiquitous and differentially expressed proteins between temporally and geographically diverse N. gonorrhoeae isolates. This review will focus on these different approaches, emphasizing the role of proteomics in the search for vaccine candidates. Although our knowledge of N. gonorrhoeae has been expanded, still far less is known about this bacterium than the closely related N. meningitidis, where genomics- and proteomics-driven studies have led to the successful development of vaccines. PMID:26579097

  7. A Novel Factor H-Fc Chimeric Immunotherapeutic Molecule against Neisseria gonorrhoeae.

    PubMed

    Shaughnessy, Jutamas; Gulati, Sunita; Agarwal, Sarika; Unemo, Magnus; Ohnishi, Makoto; Su, Xia-Hong; Monks, Brian G; Visintin, Alberto; Madico, Guillermo; Lewis, Lisa A; Golenbock, Douglas T; Reed, George W; Rice, Peter A; Ram, Sanjay

    2016-02-15

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including N. gonorrhoeae, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the use of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to N. gonorrhoeae, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc but, unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical N. gonorrhoeae isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10 of 15 (67%) strains, and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant N. gonorrhoeae. PMID:26773149

  8. Mechanism of Action of Neisseria gonorrhoeae O-Acetylpeptidoglycan Esterase, an SGNH Serine Esterase*

    PubMed Central

    Pfeffer, John M.; Weadge, Joel T.; Clarke, Anthony J.

    2013-01-01

    O-Acetylpeptidoglycan esterase from Neisseria gonorrhoeae functions to release O-acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan, thereby permitting the continued metabolism of this essential cell wall heteropolymer. It has been demonstrated to be a serine esterase with sequence similarity to the family CE-3 carbohydrate esterases of the CAZy classification system. In the absence of a three-dimensional structure for any Ape, further knowledge of its structure and function relationship is dependent on modeling and kinetic studies. In this study, we predicted Neisseria gonorrhoeae Ape1a to be an SGNH hydrolase with an adopted α/β-hydrolase fold containing a central twisted four-stranded parallel β-sheet flanked by six α-helices with the putative catalytic triad, Asp-366, His-369, and Ser-80 appropriately aligned within a pocket. The role of eight invariant and highly conserved residues localized to the active site was investigated by site-directed replacements coupled with kinetic characterization and binding studies of the resultant engineered enzymes. Based on these data and theoretical considerations, Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism, whereas Gly-78, Asp-79, His-81, Asn-235, Thr-267, and Val-368 are proposed to position appropriately the catalytic residues and participate in substrate binding. PMID:23209280

  9. Pili-mediated Interactions between Neisseria Gonorrhoeae Bacteria are the Driving Mechanism of Microcolony Merging

    NASA Astrophysics Data System (ADS)

    Poenisch, Wolfram; Weber, Christoph; Alzurqa, Khaled; Nasrollahi, Hadi; Biais, Nicolas; Zaburdaev, Vasily; Collective Dynamics of Cells Team; Mechano-Micro-Biology Lab Team

    2015-03-01

    During the early infection with Neisseria gonorrhoeae the bacteria form microcolonies consisting of a few hundreds to a few thousands of cells. The formation of colonies is mediated by type IV pili, thin and long filaments that are also involved in the motion of single cells over a substrate. A related process causes attractive cell-cell-interactions. While the motion of single cells has been extensively studied during the past years, the physical principles driving the growth of these colonies are poorly understood. One key mechanism of colony growth is coalescence of smaller colonies. Therefore we experimentally examine the process of merging of two Neisseria gonorrhoeae colonies. We develop a theoretical microscopic model of single cells interacting solely by their pili. The experimental data and the results obtained from our model are in excellent quantitative agreement. We observe a fast initial approach of the two merging colonies within a few minutes, that is followed by a slow relaxation of the colony shape with a characteristic time of several hours. These findings suggest that pili-mediated interactions are the primary driving mechanism of the microcolony merging process.

  10. Activation of NOD receptors by Neisseria gonorrhoeae modulates the innate immune response.

    PubMed

    Mavrogiorgos, Nikolaos; Mekasha, Samrawit; Yang, Yibin; Kelliher, Michelle A; Ingalls, Robin R

    2014-05-01

    NOD1 and NOD2 are members of the NOD-like receptor family of cytosolic pattern recognition receptors that recognize specific fragments of the bacterial cell wall component peptidoglycan. Neisseria species are unique amongst Gram-negative bacteria in that they turn over large amounts of peptidoglycan during growth. We examined the ability of NOD1 and NOD2 to recognize Neisseria gonorrhoeae, and determined the role of NOD-dependent signaling in regulating the immune response to gonococcal infection. Gonococci, as well as conditioned medium from mid-logarithmic phase grown bacteria, were capable of activating both human NOD1 and NOD2, as well as mouse NOD2, leading to the activation of the transcription factor NF-κB and polyubiquitination of the adaptor receptor-interacting serine-threonine kinase 2. We identified a number of cytokines and chemokines that were differentially expressed in wild type versus NOD2-deficient macrophages in response to gonococcal infection. Moreover, NOD2 signaling up-regulated complement pathway components and cytosolic nucleic acid sensors, suggesting a broad impact of NOD activation on innate immunity. Thus, NOD1 and NOD2 are important intracellular regulators of the immune response to infection with N. gonorrhoeae. Given the intracellular lifestyle of this pathogen, we believe these cytosolic receptors may provide a key innate immune defense mechanism for the host during gonococcal infection. PMID:23884094

  11. Mechanism of action of Neisseria gonorrhoeae O-acetylpeptidoglycan esterase, an SGNH serine esterase.

    PubMed

    Pfeffer, John M; Weadge, Joel T; Clarke, Anthony J

    2013-01-25

    O-Acetylpeptidoglycan esterase from Neisseria gonorrhoeae functions to release O-acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan, thereby permitting the continued metabolism of this essential cell wall heteropolymer. It has been demonstrated to be a serine esterase with sequence similarity to the family CE-3 carbohydrate esterases of the CAZy classification system. In the absence of a three-dimensional structure for any Ape, further knowledge of its structure and function relationship is dependent on modeling and kinetic studies. In this study, we predicted Neisseria gonorrhoeae Ape1a to be an SGNH hydrolase with an adopted α/β-hydrolase fold containing a central twisted four-stranded parallel β-sheet flanked by six α-helices with the putative catalytic triad, Asp-366, His-369, and Ser-80 appropriately aligned within a pocket. The role of eight invariant and highly conserved residues localized to the active site was investigated by site-directed replacements coupled with kinetic characterization and binding studies of the resultant engineered enzymes. Based on these data and theoretical considerations, Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism, whereas Gly-78, Asp-79, His-81, Asn-235, Thr-267, and Val-368 are proposed to position appropriately the catalytic residues and participate in substrate binding. PMID:23209280

  12. Incidence, epidemiology and evolution of reduced susceptibility to ciprofloxacin in Neisseria gonorrhoeae in Korea.

    PubMed

    Lee, Kyungwon; Chong, Yunsop; Erdenechemeg, L.; Soon Song, Kyung; Hun Shin, Kwang

    1998-01-01

    OBJECTIVE: To verify the decrease of susceptibility to ciprofloxacin in Neisseria gonorrhoeae, determine the size of the recently reported new beta-lactamase plasmid and explain the high prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG). METHODS: Gonococci were isolated from prostitutes in Korea. Antimicrobial susceptibility was tested by NCCLS disk diffusion and agar dilution methods. Plasmid was isolated by an alkaline lysis method. Patterns of Nhel-digested genomic DNA were compared after pulsed-field gel electrophoresis (PFGE). RESULTS: The minimum inhibitory concentration of ciprofloxacin for 50% of the isolates rose from 0.015 mg/L in 1993 to 0.12 mg/L in 1996. The proportion of PPNG remained at 70% or over during the 5-year period. The size of a novel beta-lactamase plasmid, first reported in 1994, was determined to be approximately 3.2 MDa, and 48% of the PPNG isolates contained it. Twelve of 50 isolates had the same PFGE pattern and nine others another pattern. CONCLUSION: The rapid decrease of fluoroquinolone-susceptible gonococci suggests that in the near future the drug may become less useful for gonorrhea treatment. The new 3.2-MDa plasmid may have been introduced as a result of the recent increase in overseas travel. The PFGE pattern suggests that high prevalence of PPNG may be due to dissemination of a few resistant clones among the high-risk groups. PMID:11864261

  13. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-03-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis. PMID:2106493

  14. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed Central

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-01-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis. Images PMID:2106493

  15. Serotype and serovar distribution of Neisseria gonorrhoeae isolated from high-risk populations in Bangladesh.

    PubMed

    Alam, M A; Chowdhury, M Z; Ahmed, F; Alam, A; Hossain, M A

    2012-12-01

    Neisseria gonorrhoeae, the causative agent of gonococcal infection, is known to frequently change their characteristics to evade host immune mechanism. Characterization of the clinical isolates of the organism can lead to identification of the circulating strains and often a sexual network in a community to help in designing the control strategy. Keeping in mind the above consideration, a total of 239 N. gonorrhoeae, isolated from high-risk populations, were characterized for serotypes and serovars by monoclonal antibodies against protein 1 of the organism. Majority of the serotypes were serotype B (142, 59.4%). Majority of the isolates showing resistance to at least one of the antibiotics tested were also serotype B (139, 59.2%), whereas, majority of the isolates showing resistance to any three of the antibiotics (multidrug resistant, MDR) (63%) was serotype A. A total of 41 different serovars were also identified and five of which (Arst, Bropt, Bopt, Arost, and Brop) included the highest percent (49.3%) of the isolates. Many serovars (23/41, 56.1%) were new emergent and included 58 (24.3%) of the isolates investigated. All of the new serovars were resistant to at least one of the antibiotics tested and the highest rate (40/102, 39.2%) was MDR. Serotyping and serovar determination was found contributory to understand the microepidemics of the N. gonorrhoeae isolates. Further studies including antibiogram and contact tracing can efficiently help in control of the disease. PMID:23540188

  16. Bacteriocins and other bioactive substances of probiotic lactobacilli as biological weapons against Neisseria gonorrhoeae.

    PubMed

    Ruíz, Francisco O; Pascual, Liliana; Giordano, Walter; Barberis, Lucila

    2015-04-01

    In the search of new antimicrobial agents against Neisseria gonorrhoeae, the bacteriocins-producing probiotic lactobacilli deserve special attention. The inhibitory effects of biosubstances such as organic acids, hydrogen peroxide and each bacteriocin-like inhibitory substance (BLIS) L23 and L60 on the growth of different gonococcal strains were investigated. Different non-treated and treated cell-free supernatants of two probiotic lactobacilli containing these metabolites were used. The aims of this work were (i) to evaluate the antimicrobial activity of the biosubstances produced by two probiotic lactobacilli, estimating the proportion in which each of them is responsible for the inhibitory effect, (ii) to define their minimum inhibitory concentrations (MICs) and, (iii) to determine the potential interactions between these biosubstances against N. gonorrhoeae. The main antimicrobial metabolites were the BLIS-es L23 and L60 in comparison with other biosubstances. Proportionally, their contributions to the inhibition on the gonococcal growth were 87.28% and 80.66%, respectively. The MIC values of bacteriocins were promising since these substances, when diluted, showed considerable inhibitory activity for all gonococci. In the interaction between bacteriocins, 100% of synergism was found on the gonococcal growth. In summary, this study indicates that both L23 and L60 could potentially serve to design new bioproducts against N. gonorrhoeae. PMID:25673666

  17. Neisseria gonorrhoeae Modulates Iron-Limiting Innate Immune Defenses in Macrophages

    PubMed Central

    Zughaier, Susu M.; Kandler, Justin L.; Shafer, William M.

    2014-01-01

    Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted infection termed gonorrhea. The gonococcus can survive extracellularly and intracellularly, but in both environments the bacteria must acquire iron from host proteins for survival. However, upon infection the host uses a defensive response by limiting the bioavailability of iron by a number of mechanisms including the enhanced expression of hepcidin, the master iron-regulating hormone, which reduces iron uptake from the gut and retains iron in macrophages. The host also secretes the antibacterial protein NGAL, which sequesters bacterial siderophores and therefore inhibits bacterial growth. To learn whether intracellular gonococci can subvert this defensive response, we examined expression of host genes that encode proteins involved in modulating levels of intracellular iron. We found that N. gonorrhoeae can survive in association (tightly adherent and intracellular) with monocytes and macrophages and upregulates a panel of its iron-responsive genes in this environment. We also found that gonococcal infection of human monocytes or murine macrophages resulted in the upregulation of hepcidin, NGAL, and NRAMP1 as well as downregulation of the expression of the gene encoding the short chain 3-hydroxybutyrate dehydrogenase (BDH2); BDH2 catalyzes the production of the mammalian siderophore 2,5-DHBA involved in chelating and detoxifying iron. Based on these findings, we propose that N. gonorrhoeae can subvert the iron-limiting innate immune defenses to facilitate iron acquisition and intracellular survival. PMID:24489950

  18. Regulation of catalase in Neisseria gonorrhoeae. Effects of oxidant stress and exposure to human neutrophils.

    PubMed

    Zheng, H Y; Hassett, D J; Bean, K; Cohen, M S

    1992-09-01

    We studied the effects of oxidant stress on the catalase activity and hydrogen peroxide sensitivity of Neisseria gonorrhoeae. N. gonorrhoeae is an obligate pathogen of man that evokes a remarkable but ineffective neutrophil response. Gonococci make no superoxide dismutase but express high catalase activity. Gonococcal catalase activity increased threefold when organisms were subjected to 1.0 mM hydrogen peroxide. This increase in catalase activity was marked by a parallel increase in protein concentration recognized by a rabbit polyclonal antibody raised against the purified gonococcal enzyme. Catalase was primarily localized to the gonococcal cytoplasm in the presence or absence of stress; only a single isoenzyme of catalase could be identified. Exposure of gonococci to neutrophil-derived oxidants was accomplished by stimulating neutrophils with phorbol myristate acetate or by using gonococcal Opa variants that interacted with neutrophils with different degrees of efficiency. Gonococci exposed to neutrophils demonstrated a twofold increase in catalase activity in spite of some reduction in viability. Exposure of gonococci to 1.0 mM hydrogen peroxide made the organisms significantly more resistant to higher concentrations of hydrogen peroxide and to neutrophils than control organisms. These results suggest that catalase is an important defense for N. gonorrhoeae during attack by human neutrophils. The rapid response of this enzyme to hydrogen peroxide should be taken into consideration in studies designed to evaluate the interaction between neutrophils and gonococci. PMID:1522209

  19. CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.

    PubMed

    Gray-Owen, S D; Dehio, C; Haude, A; Grunert, F; Meyer, T F

    1997-06-16

    Colonization of urogenital tissues by the human pathogen Neisseria gonorrhoeae is characteristically associated with purulent exudates of polymorphonuclear phagocytes (PMNs) containing apparently viable bacteria. Distinct variant forms of the phase-variable opacity-associated (Opa) outer membrane proteins mediate the non-opsonized binding and internalization of N. gonorrhoeae by human PMNs. Using overlay assays and an affinity isolation technique, we demonstrate the direct interaction between Opa52-expressing gonococci and members of the human carcinoembryonic antigen (CEA) family which express the CD66 epitope. Gonococci and recombinant Escherichia coli strains synthesizing Opa52 showed specific binding and internalization by transfected HeLa cell lines expressing the CD66 family members BGP (CD66a), NCA (CD66c), CGM1 (CD66d) and CEA (CD66e), but not that expressing CGM6 (CD66b). Bacterial strains expressing either no opacity protein or the epithelial cell invasion-associated Opa50 do not bind these CEA family members. Consistent with their different receptor specificities, Opa52-mediated interactions could be inhibited by polyclonal anti-CEA sera, while Opa50 binding was instead inhibited by heparin. Using confocal laser scanning microscopy, we observed a marked recruitment of CD66 antigen by Opa52-expressing gonococci on both the transfected cell lines and infected PMNs. These data indicate that members of the CEA family constitute the cellular receptors for the interaction with, and internalization of, N. gonorrhoeae. PMID:9218786

  20. Reference map and comparative proteomic analysis of Neisseria gonorrhoeae displaying high resistance against spectinomycin.

    PubMed

    Nabu, Sunanta; Lawung, Ratana; Isarankura-Na-Ayudhya, Patcharee; Isarankura-Na-Ayudhya, Chartchalerm; Roytrakul, Sittiruk; Prachayasittikul, Virapong

    2014-03-01

    A proteome reference map of Neisseria gonorrhoeae was successfully established using two-dimensional gel electrophoresis in conjunction with matrix-assisted laser desorption ionization-time of flight mass spectrometry. This map was further applied to compare protein expression profiles of high-level spectinomycin-resistant (clinical isolate) and -susceptible (reference strain) N. gonorrhoeae following treatment with subminimal inhibitory concentrations (subMICs) of spectinomycin. Approximately 200 protein spots were visualized by Coomassie brilliant blue G-250 staining and 66 spots representing 58 unique proteins were subsequently identified. Most of the identified proteins were analysed as cytoplasmic proteins and belonged to the class of energy metabolism. Comparative proteomic analysis of whole protein expression of susceptible and resistant gonococci showed up to 96% similarity while eight proteins were found to be differentially expressed in the resistant strain. In the presence of subMICs of spectinomycin, it was found that 50S ribosomal protein L7/L12, an essential component for ribosomal translocation, was upregulated in both strains, ranging from 1.5- to 3.5-fold, suggesting compensatory mechanisms of N. gonorrhoeae in response to antibiotic that inhibits protein synthesis. Moreover, the differential expression of proteins involved in energy metabolism, amino acid biosynthesis, and the cell envelope was noticeably detected, indicating significant cellular responses and adaptation against antibiotic stress. Such knowledge provides valuable data, not only fundamental proteomic data, but also knowledge of the mode of action of antibiotic and secondary target proteins implicated in adaptation and compensatory mechanisms. PMID:24567501

  1. Genetic characterization of the nucleotide excision repair system of Neisseria gonorrhoeae.

    PubMed

    LeCuyer, Brian E; Criss, Alison K; Seifert, H Steven

    2010-02-01

    Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a functional NER system. In order to comprehensively examine the role of NER in Neisseria gonorrhoeae DNA recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC, uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and uvrD mutants were complemented with a copy of each gene in an ectopic locus. Each uvr mutant strain was highly sensitive to UV irradiation and also showed sensitivity to hydrogen peroxide killing, confirming that all of the NER genes in N. gonorrhoeae are functional. The effect of RecA expression on UV survival was minor in uvr mutants but much larger in the mfd mutant. All of the NER mutants demonstrated wild-type levels of pilin antigenic variation and DNA transformation. However, the uvrD mutant exhibited higher frequencies of PilC-mediated pilus phase variation and spontaneous mutation, a finding consistent with a role for UvrD in mismatch repair. We conclude that NER functions are conserved in N. gonorrhoeae and are important for the DNA repair capabilities of this strict human pathogen. PMID:19933360

  2. Review and International Recommendation of Methods for Typing Neisseria gonorrhoeae Isolates and Their Implications for Improved Knowledge of Gonococcal Epidemiology, Treatment, and Biology

    PubMed Central

    Unemo, Magnus; Dillon, Jo-Anne R.

    2011-01-01

    Summary: Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding. PMID:21734242

  3. Identification of sRNAs expressed by the human pathogen Neisseria gonorrhoeae under disparate growth conditions.

    PubMed

    McClure, Ryan; Tjaden, Brian; Genco, Caroline

    2014-01-01

    In the last several years, bacterial gene regulation via small RNAs (sRNAs) has been recognized as an important mechanism controlling expression of essential proteins that are critical to bacterial growth and metabolism. Technologies such as RNA-seq are rapidly expanding the field of sRNAs and are enabling a global view of the "sRNAome" of several bacterial species. While numerous sRNAs have been identified in a variety of both Gram-negative and Gram-positive bacteria, only a very small number have been fully characterized in the human pathogen Neisseria gonorrhoeae, the etiological agent of the STD gonorrhea. Here we present the first analysis of N. gonorrhoeae specifically focused on the identification of sRNAs through RNA-seq analysis of the organism cultured under different in vitro growth conditions. Using a new computational program, Rockhopper, to analyze prokaryotic RNA-seq data obtained from N. gonorrhoeae we identified several putative sRNAs and confirmed their expression and size through Northern blot analysis. In addition, RNA was collected from four different growth conditions (iron replete and deplete, as well as with and without co-culture with human endocervical cells). Many of the putative sRNAs identified shoed varying expression levels relative to the different growth conditions examine or were detected only under certain conditions but not others. Comparisons of identified sRNAs with the regulatory pattern of putative mRNA targets revealed possible functional roles for these sRNAs. These studies are the first to carry out a global analysis of N. gonorrhoeae specifically focused on sRNAs and show that RNA-mediated regulation may be an important mechanism of gene control in this human pathogen. PMID:25221548

  4. Alpha-2,3-sialyltransferase enhances Neisseria gonorrhoeae survival during experimental murine genital tract infection.

    PubMed

    Wu, Hong; Jerse, Ann E

    2006-07-01

    The addition of host-derived sialic acid to Neisseria gonorrhoeae lipooligosaccharide is hypothesized to be an important mechanism by which gonococci evade host innate defenses. This hypothesis is based primarily on in vitro assays of complement-mediated and phagocytic killing. Here we report that a nonpolar alpha-2,3-sialyltransferase (lst) mutant of N. gonorrhoeae was significantly attenuated in its capacity to colonize the lower genital tract of 17-beta estradiol-treated female BALB/c mice during competitive infection with the wild-type strain. Genetic complementation of the lst mutation restored recovery of the mutant to wild-type levels. Studies with B10.D2-HC(o)H2(d)H(2)-T18c/OSN (C5-deficient) mice showed that attenuation of the lst mutant was not due to increased sensitivity to complement-mediated bacteriolysis, a result that is consistent with recently reported host restrictions in the complement cascade. However, Lst-deficient gonococci were killed more rapidly than sialylated wild-type gonococci following intraperitoneal injection into normal mice, which is consistent with sialylation conferring protection against killing by polymorphonuclear leukocytes (PMNs). As reported for human PMNs, sialylated gonococci were more resistant to killing by murine PMNs, and sialylation led to reduced association with and induction of a weaker respiratory burst in PMNs from estradiol-treated mice. In summary, these studies suggest sialylation confers a survival advantage to N. gonorrhoeae in mice by increasing resistance to PMN killing. This report is the first direct demonstration that alpha-2,3-sialyltransferase contributes to N. gonorrhoeae pathogenesis in an in vivo model. This study also validates the use of experimental murine infection to study certain aspects of gonococcal pathogenesis. PMID:16790783

  5. Inhibition of Neisseria gonorrhoeae Type II Topoisomerases by the Novel Spiropyrimidinetrione AZD0914.

    PubMed

    Kern, Gunther; Palmer, Tiffany; Ehmann, David E; Shapiro, Adam B; Andrews, Beth; Basarab, Gregory S; Doig, Peter; Fan, Jun; Gao, Ning; Mills, Scott D; Mueller, John; Sriram, Shubha; Thresher, Jason; Walkup, Grant K

    2015-08-21

    We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and β. PMID:26149691

  6. Binding of vitronectin to opa-expressing Neisseria gonorrhoeae mediates invasion of HeLa cells.

    PubMed Central

    Gómez-Duarte, O G; Dehio, M; Guzmán, C A; Chhatwal, G S; Dehio, C; Meyer, T F

    1997-01-01

    Neisseria gonorrhoeae induces local infections in the human genitourinary tract and can disseminate to other organs to cause severe disease. Blood-derived factors present in the genital mucosa have been suggested to facilitate the spread of N. gonorrhoeae in disseminated gonococcal infections. Using gentamicin invasion assays and confocal microscopy, we observed a strong stimulatory effect of fetal calf serum (FCS) on the gonococcal invasion of HeLa cells. FCS-mediated invasion was dependent on the expression of the epithelial cell invasion-associated Opa protein (plasmid-encoded Opa50 or its chromosomal homolog Opa30), while N. gonorrhoeae expressing noninvasive Opa proteins (Opa(51-60)) or no Opa protein (Opa-) was not invasive even in the presence of FCS. Incubation of N. gonorrhoeae MS11 with biotinylated FCS revealed a 78-kDa protein as the prominent protein binding to Opa50- or Opa30-expressing gonococci. This protein was recognized by antibodies against vitronectin (VN) in Western blots. Purified human or bovine VN efficiently bound to Opa50-expressing gonococci, while binding to noninvasive Opa- or Opa52-expressing gonococci was significantly lower. Binding of VN was inhibited by heparin in a concentration-dependent manner, indicating that the heparin binding sites present in VN or Opa50 may play an essential role in this interaction. Based on gentamicin invasion assays and confocal microscopy studies, VN binding was associated with an increased invasion of Opa50- and Opa30-expressing gonococci into HeLa cells. The ability of VN to mediate entry into epithelial cells may constitute an important event in the pathogenesis of local as well as disseminated gonococcal infections. PMID:9284164

  7. Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae.

    PubMed

    Piekarowicz, Andrzej; Kłyż, Aneta; Majchrzak, Michał; Stein, Daniel C

    2016-01-01

    All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage DNA sequences. To ascertain if phage encoded proteins could form the basis of a gonococcal vaccine, rabbits were orally infected with S. enterica Typhimurium strain χ3987 harboring phagemid NgoΦ6 fm. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by indirect fluorescent analysis and flow cytometry. The elicited sera was able to bind to several phage proteins. The sera also had bactericidal activity. These data demonstrate that N. gonorrhoeae filamentous phage can induce antibodies with anti-gonococcal activity and that phage proteins may be a candidate for vaccine development. PMID:26939573

  8. Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae

    PubMed Central

    Piekarowicz, Andrzej; Kłyż, Aneta; Majchrzak, Michał; Stein, Daniel C.

    2016-01-01

    All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage DNA sequences. To ascertain if phage encoded proteins could form the basis of a gonococcal vaccine, rabbits were orally infected with S. enterica Typhimurium strain χ3987 harboring phagemid NgoΦ6 fm. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by indirect fluorescent analysis and flow cytometry. The elicited sera was able to bind to several phage proteins. The sera also had bactericidal activity. These data demonstrate that N. gonorrhoeae filamentous phage can induce antibodies with anti-gonococcal activity and that phage proteins may be a candidate for vaccine development. PMID:26939573

  9. The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

    PubMed Central

    Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

    2014-01-01

    Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

  10. In vitro growth of multidrug-resistant Neisseria gonorrhoeae isolates is inhibited by ETX0914, a novel spiropyrimidinetrione.

    PubMed

    Papp, John R; Lawrence, Kenneth; Sharpe, Samera; Mueller, John; Kirkcaldy, Robert D

    2016-09-01

    Antimicrobial resistance in Neisseria gonorrhoeae has severely limited the number of treatment options, and the emergence of extended-spectrum cephalosporin resistance threatens the effectiveness of the last remaining recommended treatment regimen. This study assessed the in vitro susceptibility of N. gonorrhoeae to ETX0914, a novel spiropyrimidinetrione that inhibits DNA biosynthesis. In vitro activity was determined by agar dilution against 100 N. gonorrhoeae isolates collected from men presenting with urethritis in the USA during 2012-2013 through the Gonococcal Isolate Surveillance Project. The minimum inhibitory concentration (MIC) that inhibited growth in 50% (MIC50) and 90% (MIC90) of isolates was calculated for each antimicrobial agent. ETX0914 demonstrated a high level of antimicrobial activity against N. gonorrhoeae, including isolates with decreased susceptibility or resistance to currently available agents. The ability of ETX0914 to inhibit the growth of N. gonorrhoeae was similar to ceftriaxone, which is currently recommended in combination with azithromycin to treat gonorrhoea. The data presented in this study strongly suggest that ETX0914 should be evaluated in a clinical trial for the treatment of N. gonorrhoeae. PMID:27499432

  11. Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2

    PubMed Central

    Nudel, Kathleen; Massari, Paola

    2015-01-01

    Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated that Neisseria gonorrhoeae, a Gram-negative pathogen responsible for the sexually transmitted infection gonorrhea, protects against exogenous induction of apoptosis in human cervical epithelial cells. However, induction of cell death by N. gonorrhoeae has also been reported in other cell types. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed that N. gonorrhoeae induces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early after N. gonorrhoeae stimulation, which was followed by a marked decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes and an overall increase in production of exosomes. Inhibition of cIAP2 in N. gonorrhoeae-stimulated epithelial cells resulted in increased cell death and interleukin-1β (IL-1β) production. Collectively these results indicate that N. gonorrhoeae stimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling. PMID:26077759

  12. Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2.

    PubMed

    Nudel, Kathleen; Massari, Paola; Genco, Caroline A

    2015-09-01

    Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated that Neisseria gonorrhoeae, a Gram-negative pathogen responsible for the sexually transmitted infection gonorrhea, protects against exogenous induction of apoptosis in human cervical epithelial cells. However, induction of cell death by N. gonorrhoeae has also been reported in other cell types. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed that N. gonorrhoeae induces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early after N. gonorrhoeae stimulation, which was followed by a marked decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes and an overall increase in production of exosomes. Inhibition of cIAP2 in N. gonorrhoeae-stimulated epithelial cells resulted in increased cell death and interleukin-1β (IL-1β) production. Collectively these results indicate that N. gonorrhoeae stimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling. PMID:26077759

  13. Supplementary testing is not required in the cobas 4800 CT/NG test for Neisseria gonorrhoeae weak-positive urogenital samples.

    PubMed

    Bromhead, Collette; Liyanarachchy, Nadika; Mayes, Julia; Upton, Arlo; Balm, Michelle

    2015-01-01

    Weak-positive Neisseria gonorrhoeae nucleic acid amplification test results are difficult to interpret. We show that the frequency of unconfirmed N. gonorrhoeae results from the cobas 4800 test rises exponentially after 38.0 cycles, where the likelihood of an unconfirmed result exceeds 29%. Supplementary testing of such samples should be avoided; instead, treatment should be based on clinical pretest probability. PMID:25392357

  14. Supplementary Testing Is Not Required in the cobas 4800 CT/NG Test for Neisseria gonorrhoeae Weak-Positive Urogenital Samples

    PubMed Central

    Liyanarachchy, Nadika; Mayes, Julia; Upton, Arlo; Balm, Michelle

    2014-01-01

    Weak-positive Neisseria gonorrhoeae nucleic acid amplification test results are difficult to interpret. We show that the frequency of unconfirmed N. gonorrhoeae results from the cobas 4800 test rises exponentially after 38.0 cycles, where the likelihood of an unconfirmed result exceeds 29%. Supplementary testing of such samples should be avoided; instead, treatment should be based on clinical pretest probability. PMID:25392357

  15. Evidence for a reserpine-affected mechanism of resistance to tetracycline in Neisseria gonorrhoeae.

    PubMed

    Ruiz, Joaquim; Ribera, Anna; Jurado, Angels; Marco, Francesc; Vila, Jordi

    2005-10-01

    The presence of a reserpine-affected mechanism of tetracycline resistance was investigated in 17 Neisseria gonorrhoeae clinical isolates. To establish this fact the MIC of tetracycline in the presence and absence of reserpine was determined, and, in addition, mechanisms of tetracycline resistance were analyzed by PCR. The results showed that reserpine affects the MIC of tetracycline at least 4-fold in all isolates, including those containing the tetM gene. An inhibitory effect of reserpine against the MtrCDE efflux system was ruled out by using strains either with an inactive or with an unrepressed MtrCDE system. The results suggest the presence of a constitutive system of resistance to tetracycline, by a possible efflux pump, which may be inhibited by reserpine. Further studies are required to determine the exact nature of the action of reserpine on the MIC of tetracycline. PMID:16309425

  16. Neisseria gonorrhoeae enhances infection of dendritic cells by HIV type 1.

    PubMed

    Zhang, Jizhong; Li, Geling; Bafica, Andre; Pantelic, Milica; Zhang, Pei; Broxmeyer, Hal; Liu, Ying; Wetzler, Lee; He, Johnny J; Chen, Tie

    2005-06-15

    Clinical studies indicate that Neisseria gonorrhoeae (gonococci (GC)) has the capacity to enhance HIV type 1 (HIV-1) infection. We studied whether GC enhances HIV infection of activated dendritic cells (DCs). The results show that GC can dramatically enhance HIV replication in human DCs during coinfection. The GC component responsible for HIV infection enhancement may be peptidoglycan, which activates TLR2. TLR2 involvement is suggested by bacterial lipoprotein, a TLR2-specific inducer, which stimulates a strong enhancement of HIV infection by human DCs. Moreover, participation of TLR2 is further implicated because GC is unable to stimulate expression of HIV in DCs of TLR2-deficient HIV-1-transgenic mice. These results provide one potential mechanism through which GC infection increases HIV replication in patients infected with both GC and HIV. PMID:15944306

  17. Proteins that appear to be associated with pili in Neisseria gonorrhoeae.

    PubMed Central

    Muir, L L; Strugnell, R A; Davies, J K

    1988-01-01

    Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated. Images PMID:2898429

  18. Characteristics of antisera against periodate-resistant membrane antigens from Neisseria gonorrhoeae.

    PubMed

    Røe, S F; Eggset, G; Iversen, O J; Maeland, J A

    1980-12-01

    Crude outer membrane (OM) was prepared by extraction of bacteria of the Neisseria gonorrhoeae strains 8551. V, and VII, with an EDTA-containing buffer. The preparations contained the lipopolysaccharide (LPS) and at least 10 proteins as shown by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with untreated OM resulted in production of antibodies against several antigens, including LPS. Antisera raised against periodate-treated OM did not contain antibodies against LPS. These latter antisera agglutinated heat-treated (100 degrees C, 60 min) gonoccal cells by means of antibodies to one or more common agglutinogens and against a strain-specific agglutinogen that was susceptible to digestion with proteolytic enzymes. Both side agglutination and a plate agglutination test could be used to detect antibodies against these agglutinogens. PMID:6261525

  19. [Electron microscopic representation of the pili structure of Neisseria gonorrhoeae (author's transl)].

    PubMed

    Müller, G; Klug, H

    1979-01-01

    The technique of negative staining and ultra-thin section has been used for investigations of 30 Neisseria gonorrhoeae strains in order to represent the structure of pili (fimbriae) electron microscopically. The staining of the gonococci was effected by phosphotungstic acid (0,5%). The pili ascertained were 30 to 60 A thick. In course of in vitro passages up to 10. subculture morphological changes of the pili have been observed. The application of trisbuffer or solution of Hylase (hyaluronidase) showed not any improved results in comparison with buffered NaCl-solution as suspension medium. The investigation of ultra-thin sections showed that the structure of the pili could be exhibited not clearly. Therefore, these technique seems to be not suitable for qualitative representative of the pili. PMID:86464

  20. Evaluation of the phadebact gonococcus test, a coagglutination procedure for confirmation of Neisseria gonorrhoeae.

    PubMed Central

    Lewis, J S; Martin, J E

    1980-01-01

    Rapid and accurate immunological confirmation of presumptively positive gonococci could be facilitated with the Phadebact Gonococcus Test, a slide coagglutination procedure. The test was compared with carbohydrate utilization and fluorescent-antibody tests on 235 clinical isolates. With the coagglutination procedure, 97.1% of the isolates were identified as compared with 93.1% by carbohydrate utilization and 98.7% by fluorescent antibody. The Phadebact test was highly specific, showing no cross-reactions with 55 other Neisseria species or with 50 miscellaneous organisms occasionally found growing on selective culture media. Because of its high sensitivity and specificity, ease of performance, and ability to provide results in 2 to 3 min, this procedure provides a suitable alternative to the carbohydrate utilization and fluorescent-antibody tests for confirmation of N. gonorrhoeae. PMID:6766952

  1. Ofloxacin susceptibilities of 5,667 Neisseria gonorrhoeae strains isolated in Hong Kong.

    PubMed Central

    Kam, K M; Lo, K K; Lai, C F; Lee, Y S; Chan, C B

    1993-01-01

    Of 5,667 strains of Neisseria gonorrhoeae isolated from the Government Social Hygiene (sexually transmitted disease) Clinics in Hong Kong from 1990 to 1992, there was a trend toward an increase in the percentage of strains resistant in vitro to 0.01 and 0.1 microgram of ofloxacin per ml, with 54.3 and 5.5% resistant strains, respectively, in January 1990, rising to 95.3 and 41.5%, respectively, in December 1992. The percentage of strains for which the MIC is > 1 microgram/ml remains stable, and no clinical failure has yet been seen. This trend of decreasing susceptibility warrants close monitoring when ofloxacin is used as first-line treatment for gonorrhea. PMID:8239622

  2. Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils.

    PubMed

    Johnson, M Brittany; Criss, Alison K

    2013-08-01

    Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome-primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome-granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. Ectopically increasing primary granule-phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule-phagosome fusion, thus preventing formation of a degradative phagolysosome. PMID:23374609

  3. Enhancement of adaptive immunity to Neisseria gonorrhoeae by local intravaginal administration of microencapsulated interleukin 12.

    PubMed

    Liu, Yingru; Egilmez, Nejat K; Russell, Michael W

    2013-12-01

    Gonorrhea remains one of the most frequent infectious diseases, and Neisseria gonorrhoeae is emerging as resistant to most available antibiotics, yet it does not induce a state of specific protective immunity against reinfection. Our recent studies have demonstrated that N. gonorrhoeae proactively suppresses host T-helper (Th) 1/Th2-mediated adaptive immune responses, which can be manipulated to generate protective immunity. Here we show that intravaginally administered interleukin 12 (IL-12) encapsulated in sustained-release polymer microspheres significantly enhanced both Th1 and humoral immune responses in a mouse model of genital gonococcal infection. Treatment of mice with IL-12 microspheres during gonococcal challenge led to faster clearance of infection and induced resistance to reinfection, with the generation of gonococcus-specific circulating immunoglobulin G and vaginal immunoglobulin A and G antibodies. These results suggest that local administration of microencapsulated IL-12 can serve as a novel therapeutic and prophylactic strategy against gonorrhea, with implications for the development of an effective vaccine. PMID:24048962

  4. In silico identification of candidate drug and vaccine targets from various pathways in Neisseria gonorrhoeae.

    PubMed

    Barh, Debmalya; Kumar, Anil

    2009-01-01

    Neisseria gonorrhoeae is responsible for causing gonorrhea, one of the most common sexually transmitted diseases prevailing globally. Although extensive researches are in progress in order to control the transmission of the disease and to develop drug(s) against the pathogen, till date no effective vaccine or specific drug could be developed and only antibiotic treatment is in use. Perhaps, due to excess use of antibiotics, several resistant strains have been found. In the present study, metabolic pathways-related candidate drug and vaccine targets have been identified in N. gonorrhoeae virulent strain FA 1090 using an in silico subtractive genomics approach. 106 putative drug targets out of 537 essential genes have been predicted. 67 cytoplasmic and 9 membrane enzymes, along with 10 membrane transporters are found to be the potential drug targets from the host-pathogen common metabolic pathways. Among these targets, competence lipoproteins (NGO0277) and cysW have been identified as candidate vaccine targets. 20 drug targets have been identified from pathogen specific unique metabolic pathways. Out of these, 6 enzymes are involved in dual metabolic pathways and 2 are expressed in cell wall and fimbrium. These gonococci-specific proteins are expected to be better possible drug targets. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against antibiotic resistant strains. PMID:20109152

  5. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future.

    PubMed

    Unemo, Magnus; Shafer, William M

    2014-07-01

    Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection. PMID:24982323

  6. Use of New York City medium for improved recovery of Neisseria gonorrhoeae from clinical specimens.

    PubMed Central

    Granato, P A; Schneible-Smith, C; Weiner, L B

    1981-01-01

    New York City (NYC) and Martin-Lewis (ML) media were evaluated comparatively for their ability to support the growth of Neisseria gonorrhoeae from clinical specimens. A total of 1,010 urethral, cervical, pharyngeal, and rectal specimens were collected from walk-in patients attending a clinic for sexually transmitted diseases. A total of 187 and 165 isolates of gonococci were cultivated on NYC and ML media, respectively, with 161 of these isolates being recovered on both media. Overall, the use of NYC medium resulted in a 13.3% increased recovery rate of gonococci. When gonococci were recovered on both media from primary isolation, the NYC medium supported a more luxuriant growth and a greater number of colonies, which usually resulted in the detection of positive cultures 1 day sooner than on ML medium. Both media were comparable in their ability to suppress the growth of saprophytic microorganisms. The results of this study demonstrated that the use of NYC medium markedly enhanced the recovery of N. gonorrhoeae from clinical specimens as compared to ML medium. Images PMID:6787078

  7. Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century: Past, Evolution, and Future

    PubMed Central

    Unemo, Magnus

    2014-01-01

    SUMMARY Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection. PMID:24982323

  8. Characterisation of Neisseria gonorrhoeae in semen during urethral infection in men.

    PubMed Central

    Isbey, S F; Alcorn, T M; Davis, R H; Haizlip, J; Leone, P A; Cohen, M S

    1997-01-01

    OBJECTIVE: To determine the number of Neisseria gonorrhoeae organisms in urine and semen in men with gonococcal urethritis, and to compare selected phenotypic characteristics of organisms harvested from the urethra and semen. DESIGN: Samples from two groups of subjects were examined. Patients with symptomatic urethritis receiving treatment at an STD clinic, as well as six subjects with experimental urethritis. Semen and urine specimens were obtained after the urethral exudate was sampled. RESULTS: Using quantitative cultures, we found an average of 6 x 10(6) gonococci in urine or semen of 17 men with symptomatic urethritis seeking treatment at an STD clinic, and 2 x 10(4) gonococci in secretions of six male subjects with early experimental infection. Gonococcal outer membrane opacity (Opa) proteins and lipo-oligosaccharide (LOS) recovered from urine and semen of these subjects were very similar. CONCLUSIONS: Men with symptomatic gonorrhoea excrete a large number of gonococci in semen which is not affected by the duration of symptoms. The similar phenotype of organisms in urine and semen suggests the bacteria come from the same compartment. These data help to explain the efficiency of gonococcal transmission from men to their partners, and identify an appropriate target for a preventative vaccine or immunotherapy designed to reduce the inoculum in infected patients. Images PMID:9534748

  9. Pilin regulation in the pilT mutant of Neisseria gonorrhoeae strain MS11

    PubMed Central

    Dietrich, Manuela; Mollenkopf, Hans; So, Magdalene; Friedrich, Alexandra

    2009-01-01

    The ATPase protein PilT mediates retraction of type IV pili (Tfp). Tfp retraction of Neisseria gonorrhoeae causes many signal transduction events and changes in gene expression in infected epithelial cells. To find out whether a pilT mutation and lack of Tfp retraction, respectively, lead also to gene regulation in bacteria we performed microarrays comparing the transcriptional profiles of the N. gonorrhoeae parent strain MS11 and its isogenic pilT mutant during growth in vitro. A loss-of-function-mutation in pilT led to altered transcript levels of 63 open reading frames. Levels of pilE transcripts and its deduced protein the major Tfp subunit pilin, were increased most markedly by a mutation in pilT. Further studies revealed that pilE expression was also controlled by two other genes encoding Tfp biogenesis proteins, pilD and pilF. Our studies strongly suggest that pilE expression is a finely-tuned process. PMID:19486161

  10. Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae.

    PubMed Central

    Clark, V L; Campbell, L A; Palermo, D A; Evans, T M; Klimpel, K W

    1987-01-01

    Neisseria gonorrhoeae is generally considered to be an obligate aerobe; it can, however, grow in the absence of oxygen by anaerobic respiration by using nitrite as a terminal electron acceptor. The outer membrane protein compositions of aerobically and anaerobically grown N. gonorrhoeae strains were compared by one- and two-dimensional polyacrylamide gel electrophoresis. Anaerobically grown strains expressed at least three proteins (Pan 1 to Pan 3) at much higher levels than did aerobically grown cells. Conversely, at least five other proteins (Pox 1 to Pox 5) were found to be expressed at significantly higher levels in aerobically grown cells. None of the Pan or Pox proteins were heat modifiable, and none of the heat-modifiable protein IIs or other major outer membrane proteins (protein I, protein III, pilin, or H-8 protein) were significantly altered in expression by anaerobic growth. There were also no apparent differences in lipopolysaccharide composition in aerobically and anaerobically grown gonococci. The regulation of protein expression by oxygen availability suggests that anaerobic growth is a physiologically significant state for this organism. Images PMID:3106220

  11. Activation of NOD receptors by Neisseria gonorrhoeae modulates the innate immune response

    PubMed Central

    Mavrogiorgos, Nikolaos; Mekasha, Samrawit; Yang, Yibin; Kelliher, Michelle A.; Ingalls, Robin R.

    2013-01-01

    Nucleotide-binding oligomerization domain (NOD)-1 and NOD2 are members of the NOD-like receptor family of cytosolic pattern recognition receptors that recognize specific fragments of the bacterial cell wall component peptidoglycan. Neisseria species are unique amongst Gram-negative bacteria in that they turn over large amounts of peptidoglycan during growth. In this study we examined the ability of NOD1 and NOD2 to recognize N. gonorrhoeae, and determined the role of NOD-dependent signaling in regulating the immune response to gonococcal infection. We found that gonococci, as well as conditioned medium from mid-logarithmic phase grown bacteria, were capable of activating both human NOD1 and NOD2, as well as mouse NOD2, leading to the activation of the transcription factor NF-κB and polyubiquitination of the adaptor receptor-interacting serine-threonine kinase 2 (RIPK2). We identified a number of cytokines and chemokines that were differentially expressed in wild type vs. NOD2 deficient macrophages in response to gonococcal infection. Moreover, NOD2 signaling upregulated complement pathway components and cytosolic nucleic acid sensors, suggesting a broad impact of NOD activation on innate immunity. These data demonstrate that NOD1 and NOD2 are important intracellular regulators of the immune response to infection with N. gonorrhoeae. Given the intracellular lifestyle of this pathogen, we believe these cytosolic receptors may provide a key innate immune defense mechanism for the host during gonococcal infection. PMID:23884094

  12. Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps.

    PubMed

    Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Chou, Tsung-Han; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2015-04-01

    Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs. PMID:25818299

  13. Identification, localization, and distribution of the PilT protein in Neisseria gonorrhoeae.

    PubMed Central

    Brossay, L; Paradis, G; Fox, R; Koomey, M; Hébert, J

    1994-01-01

    A monoclonal antibody (MAb) directed against a highly conserved protein of Neisseria gonorrhoeae with a molecular size of 40 kDa was isolated and characterized. The protein antigen detected by this MAb was detected by enzyme-linked immunosorbent assay and immunoblotting in all strains of N. gonorrhoeae tested across a wide range of serovars. The 40-kDa protein was found to be expressed at relatively low levels and localized to both the cytosolic and cytoplasmic membrane fractions. Screening of a lambda gt11 expression library derived from gonococcal genomic DNA with the anti-40-kDa MAb and DNA sequence analysis suggested that the 40-kDa protein and the product of the gonococcal pilT gene were identical. Immunoblotting analysis of gonococcal mutants carrying defined mutations in the pilT gene confirmed that the 40-kDa protein was indeed PilT. The N-terminal sequence derived by microsequencing of the protein purified from gonococci led to the correction of the previously published pilT gene sequence. Sequencing of the pilT gene from three different strains revealed an extremely high degree of conservation at both the amino acid and DNA levels. Images PMID:8188352

  14. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae.

    PubMed Central

    Goodman, S D; Scocca, J J

    1988-01-01

    DNA segments from Neisseria gonorrhoeae, cloned and propagated in Escherichia coli, were tested for the ability to competitively inhibit gonococcal transformation. The nucleotide sequences of active segments were determined and compared; these sequences contained the sequence 5' GCCGTCTGAA 3' in common. Subcloning studies confirmed the identity of this sequence as the gonococcal DNA recognition site. The three instances of the recognition sequence isolated from N. gonorrhoeae chromosomal DNA contain the sequence in the immediate neighborhood of its inverted repeat. Because a single copy of the sequence functions as a recognition site, the inverted duplication is not required for specific binding. The dyad symmetric arrangements of the chromosomal recognition sequences may form stable stem-loop structures that can function as terminators or attenuators of transcription. These inverted repeats are located at the boundaries of long open reading frames. The recognition sequence also constitutes part of two other probable terminators of gonococcal genes. We conclude that the signal for recognition of transforming DNA by gonococci is a frequent component of transcriptional terminator sequences. This regulatory function might account for the origin and maintenance of recognition sequences in the chromosomes of Gram-negative transformable bacteria. PMID:3137581

  15. Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin.

    PubMed Central

    Paruchuri, D K; Seifert, H S; Ajioka, R S; Karlsson, K A; So, M

    1990-01-01

    We recently identified a set of mammalian cell receptors for Neisseria gonorrhoeae that are glycolipids. These receptors, lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriosylceramide [GalNAc( beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], were shown to be specifically bound by a gonococcal outer membrane protein distinct from pilin and protein II. Here we report the isolation of the gene encoding the gangliotetraosylceramide-binding adhesin from a N. gonorrhoeae MS11 gene bank in Escherichia coli. Transposon mutagenesis studies in E. coli indicate that the adhesion is a protein with a molecular mass of 36,000 Da. The gene encoding the 36-kDa protein is duplicated in MS11 since two transposon insertions were required to abolish expression of the gene in this bacterium. This protein is present on the surface of the gonococcus and is not associated with the pilus. Images PMID:2153292

  16. Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin.

    PubMed

    Paruchuri, D K; Seifert, H S; Ajioka, R S; Karlsson, K A; So, M

    1990-01-01

    We recently identified a set of mammalian cell receptors for Neisseria gonorrhoeae that are glycolipids. These receptors, lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriosylceramide [GalNAc( beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], were shown to be specifically bound by a gonococcal outer membrane protein distinct from pilin and protein II. Here we report the isolation of the gene encoding the gangliotetraosylceramide-binding adhesin from a N. gonorrhoeae MS11 gene bank in Escherichia coli. Transposon mutagenesis studies in E. coli indicate that the adhesion is a protein with a molecular mass of 36,000 Da. The gene encoding the 36-kDa protein is duplicated in MS11 since two transposon insertions were required to abolish expression of the gene in this bacterium. This protein is present on the surface of the gonococcus and is not associated with the pilus. PMID:2153292

  17. Comparison of Direct Inoculation and Copan Transport Systems for Isolation of Neisseria gonorrhoeae from Endocervical Specimens

    PubMed Central

    Olsen, C. C.; Schwebke, J. R.; Benjamin, W. H.; Beverly, A.; Waites, K. B.

    1999-01-01

    Two commercial swab transport systems, Copan Amies gel agar with and without charcoal (Copan Diagnostics, Corona, Calif.), were compared to direct inoculation onto modified Thayer-Martin medium for detection of Neisseria gonorrhoeae in 1,490 endocervical specimens obtained from women attending a sexually transmitted disease clinic. Copan swabs were held in the transport system for 24 h at room temperature prior to inoculation onto modified Thayer-Martin medium. All cultures were incubated at 35°C in 5% CO2, and bacteria were identified on the basis of Gram stain, oxidase, and biochemical reactions. Copan Amies gel agar transport system without charcoal detected 77 of 81 (95%) direct inoculation culture-positive specimens, and Copan Amies gel agar transport system with charcoal detected 53 of 56 (95%) directly inoculated culture-positive specimens. Copan Amies gel agar without charcoal inoculated after 6 h supported growth of 56 (98%) positive cultures out of only 55 directly inoculated culture-positive specimens. This study demonstrates that Copan swabs represent a reasonable alternative, providing convenience, low cost, and ease of use while still maintaining a satisfactory recovery rate of N. gonorrhoeae from clinical specimens, if specimens can be inoculated onto selective media within a relatively short time period not involving overnight shipment. PMID:10523556

  18. Coupling electrochemical response of a DNA biosensor with PCR for Neisseria gonorrhoeae detection.

    PubMed

    Verma, Rachna; Sood, Seema; Singh, Renu; Sumana, Gajjala; Bala, Manju; Sharma, Vinod K; Samantaray, Jyotish C; Pandey, Ravindra M; Malhotra, Bansi D

    2014-01-01

    Early diagnosis of gonococcal infections is important with regard to a patient's health and stage of infection. In this context, we report the development of an opa-gene-based electrochemical DNA biosensor for detection of Neisseria gonorrhoeae by monitoring redox peak of methylene blue indicator. The fabricated biosensor has been shown to be highly sensitive and specific when evaluated with complementary, non-complementary, and 1-base mismatch DNA sequences and polymerase chain reaction (PCR) amplified products (amplicons) of standard strain of N. gonorrhoeae (ATCC49226). The biosensor has been further evaluated using amplicons of known positive and negative clinical samples, and cut-off for positives has been determined using receiver operating characteristic curve. The sensitivity (SN), specificity (SP), positive predictive value, and negative predictive value of the biosensor have been found to be 96.2%, 88.2%, 92.6%, and 93.8%, respectively. We conclude that the combination of PCR amplification with electrochemical detection shows distinct advantage of high SN and increased SP for gonococcal detection. PMID:24207077

  19. Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea?: detailed characterization of the first strain with high-level resistance to ceftriaxone.

    PubMed

    Ohnishi, Makoto; Golparian, Daniel; Shimuta, Ken; Saika, Takeshi; Hoshina, Shinji; Iwasaku, Kazuhiro; Nakayama, Shu-ichi; Kitawaki, Jo; Unemo, Magnus

    2011-07-01

    Recently, the first Neisseria gonorrhoeae strain (H041) that is highly resistant to the extended-spectrum cephalosporin (ESC) ceftriaxone, the last remaining option for empirical first-line treatment, was isolated. We performed a detailed characterization of H041, phenotypically and genetically, to confirm the finding, examine its antimicrobial resistance (AMR), and elucidate the resistance mechanisms. H041 was examined using seven species-confirmatory tests, antibiograms (30 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of ESC resistance determinants (penA, mtrR, penB, ponA, and pilQ). Transformation, using appropriate recipient strains, was performed to confirm the ESC resistance determinants. H041 was assigned to serovar Bpyust, MLST sequence type (ST) ST7363, and the new NG-MAST ST4220. H041 proved highly resistant to ceftriaxone (2 to 4 μg/ml, which is 4- to 8-fold higher than any previously described isolate) and all other cephalosporins, as well as most other antimicrobials tested. A new penA mosaic allele caused the ceftriaxone resistance. In conclusion, N. gonorrhoeae has now shown its ability to also develop ceftriaxone resistance. Although the biological fitness of ceftriaxone resistance in N. gonorrhoeae remains unknown, N. gonorrhoeae may soon become a true superbug, causing untreatable gonorrhea. A reduction in the global gonorrhea burden by enhanced disease control activities, combined with wider strategies for general AMR control and enhanced understanding of the mechanisms of emergence and spread of AMR, which need to be monitored globally, and public health response plans for global (and national) perspectives are important. Ultimately, the development of new drugs for efficacious gonorrhea treatment is necessary. PMID:21576437

  20. In Vitro Activity of Delafloxacin against Clinical Neisseria gonorrhoeae Isolates and Selection of Gonococcal Delafloxacin Resistance.

    PubMed

    Soge, Olusegun O; Salipante, Stephen J; No, David; Duffy, Erin; Roberts, Marilyn C

    2016-05-01

    We evaluated the in vitro activity of delafloxacin against a panel of 117 Neisseria gonorrhoeae strains, including 110 clinical isolates collected from 2012 to 2015 and seven reference strains, compared with the activities of seven antimicrobials currently or previously recommended for treatment of gonorrhea. We examined the potential for delafloxacin to select for resistant mutants in ciprofloxacin-susceptible and ciprofloxacin-resistant N. gonorrhoeae We characterized mutations in the gyrA, gyrB, parC, and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) by PCR and sequencing and by whole-genome sequencing. The MIC50, MIC90, and MIC ranges of delafloxacin were 0.06 μg/ml, 0.125 μg/ml, and ≤0.001 to 0.25 μg/ml, respectively. The frequency of spontaneous mutation ranged from 10(-7) to <10(-9) The multistep delafloxacin resistance selection of 30 daily passages resulted in stable resistant mutants. There was no obvious cross-resistance to nonfluoroquinolone comparator antimicrobials. A mutant with reduced susceptibility to ciprofloxacin (MIC, 0.25 μg/ml) obtained from the ciprofloxacin-susceptible parental strain had a novel Ser91Tyr alteration in the gyrA gene. We also identified new mutations in the gyrA and/or parC and parE genes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) of two mutant strains with elevated delafloxacin MICs of 1 μg/ml. Although delafloxacin exhibited potent in vitro activity against N. gonorrhoeae isolates and reference strains with diverse antimicrobial resistance profiles and demonstrated a low tendency to select for spontaneous mutants, it is important to establish the correlation between these excellent in vitro data and treatment outcomes through appropriate randomized controlled clinical trials. PMID:26976873

  1. Reduced uptake and accumulation of norfloxacin in resistant strains of Neisseria gonorrhoeae isolated in Japan.

    PubMed Central

    Tanaka, M; Fukuda, H; Hirai, K; Hosaka, M; Matsumoto, T; Kumazawa, J

    1994-01-01

    OBJECTIVE--To investigate the alteration of cell permeability toward fluoroquinolones in Neisseria gonorrhoeae, which is a major quinolone-resistance mechanism along with the alteration of DNA gyrase in gram-negative bacteria. The prevalence of fluoroquinolone-resistant N gonorrhoeae strains is rapidly increasing in Japan. MATERIALS AND METHODS--The uptake and accumulation of norfloxacin by gonococcal cells, including six clinical and five World Health Organization (WHO) reference strains, were measured. Of the six clinical strains, two were highly resistant to norfloxacin (MIC 8.0 and 4.0 micrograms/ml), two were moderately resistant (MIC 1.0 and 0.5 microgram/ml), and two were sensitive (MIC 0.063 and 0.004 microgram/ml). All five WHO reference strains were sensitive to norfloxacin (MIC < or = 0.001 to 0.063 microgram/ml). RESULTS--Mean initial norfloxacin uptake in the four resistant strains (104 ng/mg of dry cells) was significantly lower than that in the seven sensitive strains (158 ng/mg of dry cells) (p < 0.05). The mean uptake after 20 minutes was also significantly lower in the four resistant strains (130 ng/mg of dry cells) than in the seven sensitive strains (194 ng/mg of dry cells) (p < 0.05). However, there was no significant difference in mean norfloxacin accumulation after 20 minutes between the four resistant strains (26 ng/mg of dry cells) and the seven sensitive strains (36 ng/mg of dry cells). The accumulation of norfloxacin after 20 minutes was almost zero in two of the four resistant strains, while the remaining two strains accumulated norfloxacin as well as the sensitive strains. CONCLUSIONS--These findings suggest that alteration of bacterial cell permeability is a quinolone-resistance mechanism in N gonorrhoeae isolated in Japan, and that this bacteria may exhibit other mechanisms such as alteration of DNA gyrase. PMID:7959709

  2. Assessment of Etest as an alternative to agar dilution for antimicrobial susceptibility testing of Neisseria gonorrhoeae.

    PubMed

    Liu, Hsi; Taylor, Thomas H; Pettus, Kevin; Trees, David

    2014-05-01

    We studied whether the Etest can be used as an alternative to agar dilution to determine antimicrobial susceptibilities of ceftriaxone, cefixime, and cefpodoxime in Neisseria gonorrhoeae surveillance. One hundred fifteen clinical and laboratory isolates of N. gonorrhoeae were tested following the Clinical Laboratory Improvement Amendments (CLIA)-approved CLSI standard agar dilution method and, separately, by the Etest according to the manufacturer's recommendations. The MICs were determined and compared. Ten laboratory-generated mutants were used to simulate substantially nonsusceptible specimens. The Etest and agar dilution methods were well correlated. Statistical tests produced regression R2 values of 88%, 82%, and 85% and Pearson correlation coefficients of 92%, 91%, and 92% for ceftriaxone, cefixime, and cefpodoxime, respectively. When paired comparisons were made, the two tests were 88.7%, 80%, and 87% within 1 log2 dilution from each other for ceftriaxone, cefixime, and cefpodoxime, respectively. The within-2-log2 agreements were 99.1%, 98.3%, and 94.8% for ceftriaxone, cefixime, and cefpodoxime, respectively. Notwithstanding the good correlations and the within-2-log2 general agreement, the Etest results produced slightly lower MICs than the agar dilution results. In conclusion, we found that the Etest can be effectively used as an alternative to agar dilution testing to determine the susceptibility of N. gonorrhoeae to ceftriaxone, cefixime, and cefpodoxime, although we recommend further research into extremely resistant isolates. For isolates within the typical range of clinical MICs, reexamination of the Etest interpretation of susceptible and nonsusceptible categories would likely allow for successful transition from agar dilution to the Etest. PMID:24554750

  3. Assessment of Etest as an Alternative to Agar Dilution for Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae

    PubMed Central

    Taylor, Thomas H.; Pettus, Kevin; Trees, David

    2014-01-01

    We studied whether the Etest can be used as an alternative to agar dilution to determine antimicrobial susceptibilities of ceftriaxone, cefixime, and cefpodoxime in Neisseria gonorrhoeae surveillance. One hundred fifteen clinical and laboratory isolates of N. gonorrhoeae were tested following the Clinical Laboratory Improvement Amendments (CLIA)-approved CLSI standard agar dilution method and, separately, by the Etest according to the manufacturer's recommendations. The MICs were determined and compared. Ten laboratory-generated mutants were used to simulate substantially nonsusceptible specimens. The Etest and agar dilution methods were well correlated. Statistical tests produced regression R2 values of 88%, 82%, and 85% and Pearson correlation coefficients of 92%, 91%, and 92% for ceftriaxone, cefixime, and cefpodoxime, respectively. When paired comparisons were made, the two tests were 88.7%, 80%, and 87% within 1 log2 dilution from each other for ceftriaxone, cefixime, and cefpodoxime, respectively. The within-2-log2 agreements were 99.1%, 98.3%, and 94.8% for ceftriaxone, cefixime, and cefpodoxime, respectively. Notwithstanding the good correlations and the within-2-log2 general agreement, the Etest results produced slightly lower MICs than the agar dilution results. In conclusion, we found that the Etest can be effectively used as an alternative to agar dilution testing to determine the susceptibility of N. gonorrhoeae to ceftriaxone, cefixime, and cefpodoxime, although we recommend further research into extremely resistant isolates. For isolates within the typical range of clinical MICs, reexamination of the Etest interpretation of susceptible and nonsusceptible categories would likely allow for successful transition from agar dilution to the Etest. PMID:24554750

  4. Stemming the tide of drug-resistant Neisseria gonorrhoeae: the need for an individualized approach to treatment.

    PubMed

    Buono, Sean A; Watson, Tyler D; Borenstein, Lee A; Klausner, Jeffrey D; Pandori, Mark W; Godwin, Hilary A

    2015-02-01

    Drug-resistant Neisseria gonorrhoeae poses a significant public health challenge. In recent years, gonococci resistant to first- and second-line antibiotics have spread worldwide and new strains have developed that are increasingly resistant to third-generation cephalosporins, which are currently our last line of available treatments. Given the timeline required to develop new drugs or an effective vaccine for N. gonorrhoeae, a top priority is to use the drugs that are available as effectively as possible. Currently, clinical management of gonorrhoea is based upon treatment guidelines informed by international gonococcal antimicrobial susceptibility surveillance programmes. This approach, although currently the most practical, is subject to a number of limitations since surveillance data inherently provide population-level information. As a result, basing treatment guidelines on these data can result in the prescription of more aggressive or broader treatment than is needed by individual patients and hence inadvertently contribute to the development and spread of resistance to important drugs. Clearly, methods are needed that provide patient-specific drug susceptibility information in a time frame that would allow clinicians to prescribe individualized treatment regimens for gonorrhoea. Fortunately, in recent years, there have been a number of advances in the development of rapid methods for characterizing both the genotype and the drug resistance phenotype of N. gonorrhoeae strains. Here, we review these advances and propose additional studies that would help facilitate a transition towards an individualized treatment approach for gonorrhoea. PMID:25331059

  5. Single 1 g dose of cefotaxime in the treatment of infections due to penicillinase-producing strains of Neisseria gonorrhoeae.

    PubMed Central

    de Koning, G A; Tio, D; van den Hoek, J A; van Klingeren, B

    1983-01-01

    One hundred and two patients with an uncomplicated infection due to penicillinase-producing strains of Neisseria gonorrhoeae (PPNG) were treated with a single 1 g dose of cefotaxime. At follow-up within 15 days all genital and rectal infections were cured. Pharyngeal infections also seemed to respond to this treatment. A relatively high proportion (30.9%) of patients, however, developed post-gonococcal urethritis. PMID:6299449

  6. Trends in antimicrobial resistance in Neisseria gonorrhoeae isolated from Guangzhou, China, 2000 to 2005 and 2008 to 2013.

    PubMed

    Cao, Wen-Ling; Liang, Jing-Yao; Li, Xiao-Dong; Bi, Chao; Yang, Ri-Dong; Liang, Yan-Hua; Li, Ping; Zhong, Dao-Qing; Ye, Xing-Dong; Zhang, Xi-Bao

    2015-01-01

    A total of 1224 Neisseria gonorrhoeae isolates from Guangzhou in 2 periods (2000-2005 and 2008-2013) were subjected to antimicrobial susceptibility testing. The percentage of penicillin- and ciprofloxacin-resistant isolates increased from 71.1% (473/665) to 90.9% (508/559) and 88.9% (591/665) to 98.0% (548/559), respectively. All isolates remain susceptible to spectinomycin and ceftriaxone, with increasing minimum inhibitory concentrations. PMID:25504297

  7. Construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae and its expression in E. coli.

    PubMed

    Chen, Hongxiang; Tu, Yating; Lin, Nengxing; Huang, Changzheng

    2005-01-01

    In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic 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 coli (E. coli) 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 endonuclease to construct recombinant pET-mtrC which was verified by restriction endonuclease and DNA sequencing. The recombinant 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 endonuclease and DNA sequencing. Its 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. PMID:16463681

  8. A Role for Lactate Dehydrogenases in the Survival of Neisseria gonorrhoeae in Human Polymorphonuclear Leukocytes and Cervical Epithelial Cells

    PubMed Central

    Atack, John M.; Ibranovic, Ines; Ong, Cheryl-Lynn Y.; Djoko, Karrera Y.; Chen, Nathan H.; vanden Hoven, Rachel; Jennings, Michael P.; Edwards, Jennifer L.; McEwan, Alastair G.

    2014-01-01

    Lactate is an abundant metabolite, produced by host tissues and commensal organisms, and it represents an important potential carbon source for bacterial pathogens. In the case of Neisseria spp., the importance of the lactate permease in colonization of the host has been demonstrated, but there have been few studies of lactate metabolism in pathogenic Neisseria in the postgenomic era. We describe herein the characterization of genome-annotated, respiratory, and substrate-level lactate dehydrogenases (LDHs) from the obligate human pathogen Neisseria gonorrhoeae. Biochemical assays using N. gonorrhoeae 1291 wild type and isogenic mutant strains showed that cytoplasmic LdhA (NAD+-dependent D-lactate dehydrogenase) and the membrane-bound respiratory enzymes, LdhD (D-lactate dehydrogenase) and LldD (L-lactate dehydrogenase) are correctly annotated. Mutants lacking LdhA and LdhD showed greatly reduced survival in neutrophils compared with wild type cells, highlighting the importance of D-lactate metabolism in gonococcal survival. Furthermore, an assay of host colonization using the well-established human primary cervical epithelial cell model revealed that the two respiratory enzymes make a significant contribution to colonization of and survival within the microaerobic environment of the host. Taken together, these data suggest that host-derived lactate is critical for the growth and survival of N. gonorrhoeae in human cells. PMID:24737798

  9. Mutation of the Conserved Calcium-Binding Motif in Neisseria gonorrhoeae PilC1 Impacts Adhesion but Not Piliation

    PubMed Central

    Cheng, Yuan; Johnson, Michael D. L.; Burillo-Kirch, Christine; Mocny, Jeffrey C.; Anderson, James E.; Garrett, Christopher K.; Redinbo, Matthew R.

    2013-01-01

    Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae. PMID:24002068

  10. Identification of an EF-Tu protein that is periplasm-associated and processed in Neisseria gonorrhoeae.

    PubMed

    Porcella, S F; Belland, R J; Judd, R C

    1996-09-01

    A 44 kDa protein is a dominant component of periplasmic extracts of Neisseria gonorrhoeae. Peptide sequence generated from a cyanogen-bromide-cleaved fragment of this protein indicated sequence homology with elongation factor-Tu (EF-Tu). Polyclonal antiserum was made against the 44 kDa protein purified from periplasm extracts of N. gonorrhoeae. The preabsorbed antiserum was immunoblotted against whole-cell lysates on two-dimensional gels. A 44 kDa protein and a smaller 37 kDa protein were recognized by this antiserum. A N. gonorrhoeae gamma phage DNA library was screened and a clone expressing a 44 kDa protein was identified. The DNA insert in this clone contained several genes homologous to genes contained in the str operon of Escherichia coli. One ORF product with a calculated molecular mass of 43 kDa was highly homologous to the EF-TuA of E. coli. A synthetic peptide antiserum specific for a portion of the C terminus of EF-Tu confirmed that the 37 kDa protein in whole-cell lysates of N. gonorrhoeae was a processed form of EF-Tu. Deletion of the tufA gene homologue in N. gonorrhoeae was attempted but was unsuccessful. PMID:8828215

  11. Specificity of antibodies against Neisseria gonorrhoeae that stimulate neutrophil chemotaxis. Role of antibodies directed against lipooligosaccharides.

    PubMed Central

    Densen, P; Gulati, S; Rice, P A

    1987-01-01

    Five strains each of Neisseria gonorrhoeae sensitive or resistant to complement (C) dependent killing by normal human serum (NHS) were examined for their ability to stimulate chemotaxis of polymorphonuclear leukocytes (PMNs) after preincubation with NHS; or IgM or IgG derived from NHS. Serum-sensitive N. gonorrhoeae stimulated C-dependent chemotaxis when opsonized with IgM, but not IgG, however, serum-resistant strains, taken as a whole, failed to promote chemotaxis when opsonized with either isotype. IgM titers in NHS against lipooligosaccharide (LOS) antigens from individual serum-sensitive, but not serum-resistant strains, correlated with the magnitude of chemotaxis generated by the corresponding opsonized strains (r = 0.99). Western blots demonstrated that IgM and IgG from NHS recognized different antigenic determinants on LOS from serum-sensitive gonococci. IgM from NHS immunopurified against serum-sensitive LOS accounted for two-thirds of the chemotaxis promoting activity present in whole serum. IgG titers in NHS against LOS antigens from individual serum-resistant strains also correlated with magnitude of chemotaxis generated by the corresponding opsonized strains (r = 0.87), although most opsonized serum-resistant strains did not generate significantly higher magnitudes of chemotaxis than controls. In contrast, a serum-resistant isolate from a patient with disseminated gonococcal infection (DGI) stimulated chemotaxis when opsonized with IgG obtained from the patient's convalescent serum. By Western blot, convalescent IgG antibody recognized an additional determinant on serum-resistant LOS not seen by normal IgG. Images PMID:2439546

  12. Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies.

    PubMed

    Forest, K T; Bernstein, S L; Getzoff, E D; So, M; Tribbick, G; Geysen, H M; Deal, C D; Tainer, J A

    1996-02-01

    The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber. PMID:8550220

  13. Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies.

    PubMed Central

    Forest, K T; Bernstein, S L; Getzoff, E D; So, M; Tribbick, G; Geysen, H M; Deal, C D; Tainer, J A

    1996-01-01

    The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber. PMID:8550220

  14. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. PMID:27225407

  15. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    PubMed

    Obergfell, Kyle P; Seifert, H Steven

    2016-05-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants

  16. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation

    PubMed Central

    2016-01-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic

  17. A Systematic Review of Point of Care Testing for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis

    PubMed Central

    Herbst de Cortina, Sasha; Bristow, Claire C.; Joseph Davey, Dvora; Klausner, Jeffrey D.

    2016-01-01

    Objectives. Systematic review of point of care (POC) diagnostic tests for sexually transmitted infections: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Methods. Literature search on PubMed for articles from January 2010 to August 2015, including original research in English on POC diagnostics for sexually transmitted CT, NG, and/or TV. Results. We identified 33 publications with original research on POC diagnostics for CT, NG, and/or TV. Thirteen articles evaluated test performance, yielding at least one test for each infection with sensitivity and specificity ≥90%. Each infection also had currently available tests with sensitivities <60%. Three articles analyzed cost effectiveness, and five publications discussed acceptability and feasibility. POC testing was acceptable to both providers and patients and was also demonstrated to be cost effective. Fourteen proof of concept articles introduced new tests. Conclusions. Highly sensitive and specific POC tests are available for CT, NG, and TV, but improvement is possible. Future research should focus on acceptability, feasibility, and cost of POC testing. While pregnant women specifically have not been studied, the results available in nonpregnant populations are encouraging for the ability to test and treat women in antenatal care to prevent adverse pregnancy and neonatal outcomes. PMID:27313440

  18. A Systematic Review of Point of Care Testing for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.

    PubMed

    Herbst de Cortina, Sasha; Bristow, Claire C; Joseph Davey, Dvora; Klausner, Jeffrey D

    2016-01-01

    Objectives. Systematic review of point of care (POC) diagnostic tests for sexually transmitted infections: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Methods. Literature search on PubMed for articles from January 2010 to August 2015, including original research in English on POC diagnostics for sexually transmitted CT, NG, and/or TV. Results. We identified 33 publications with original research on POC diagnostics for CT, NG, and/or TV. Thirteen articles evaluated test performance, yielding at least one test for each infection with sensitivity and specificity ≥90%. Each infection also had currently available tests with sensitivities <60%. Three articles analyzed cost effectiveness, and five publications discussed acceptability and feasibility. POC testing was acceptable to both providers and patients and was also demonstrated to be cost effective. Fourteen proof of concept articles introduced new tests. Conclusions. Highly sensitive and specific POC tests are available for CT, NG, and TV, but improvement is possible. Future research should focus on acceptability, feasibility, and cost of POC testing. While pregnant women specifically have not been studied, the results available in nonpregnant populations are encouraging for the ability to test and treat women in antenatal care to prevent adverse pregnancy and neonatal outcomes. PMID:27313440

  19. Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection.

    PubMed

    Melendez, Johan H; Santaus, Tonya M; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A; Geddes, Chris D

    2016-10-01

    Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

  20. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

    PubMed

    Gong, Zheng; Tang, M Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. PMID:26808268

  1. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    PubMed

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  2. DC-SIGN (CD209) recognition of Neisseria gonorrhoeae is circumvented by lipooligosaccharide variation.

    PubMed

    Zhang, Pei; Schwartz, Olivier; Pantelic, Milica; Li, Geling; Knazze, Quita; Nobile, Cinzia; Radovich, Milan; He, Johnny; Hong, Soon-Cheol; Klena, John; Chen, Tie

    2006-04-01

    Neisseria gonorrhoeae (GC) or Escherichia coli HB101 (hereafter referred to as E. coli) expressing opacity (Opa) proteins adhere to human host cells and stimulate phagocytosis as a result of the interaction of certain Opa proteins to carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1; CD66a) receptors. Our experiments show that the Opa-CEACAM1 interaction does not play a significant role in adherence between these bacteria and dendritic cells (DCs). Instead, phagocytosis of GC and E. coli by DCs is mediated by the DC-specific intercellular adhesion molecule-grabbing nonintegrin, (SIGN; CD209) receptor. DC-SIGN recognition and subsequent phagocytosis of GC are limited, however, to a lipooligosaccharide (LOS) mutant (lgtB) of GC. This conclusion is supported by experiments demonstrating that HeLa cells expressing human DC-SIGN (HeLa-DC-SIGN) bind exclusively to and engulf an lgtB mutant of GC, and this interaction is blocked specifically by an anti-DC-SIGN antibody. The experiments suggest that LOS variation may have evolved as a mechanism for GC to avoid phagocytosis by DCs. PMID:16461738

  3. Isolation of Neisseria gonorrhoeae on selective and nonselective media in a sexually transmitted disease clinic.

    PubMed Central

    Bonin, P; Tanino, T T; Handsfield, H H

    1984-01-01

    To assess the practical significance of reported increases in the prevalence of vancomycin-susceptible strains of Neisseria gonorrhoeae on isolation of this organism, antibiotic-free chocolate agar (CA), modified Thayer-Martin medium (MTM), and a vancomycin-free selective medium (VFSM) were compared in a sexually transmitted disease clinic. Among 326 cervical gonococcal infections detected in a comparison of CA with MTM, 92.0% were detected on CA, compared with 98.2% on MTM (P less than 0.001). Similarly, among 306 cervical infections detected in a comparison of MTM and VFSM, 95.8% of infections were detected with VFSM, compared with 98.4% for MTM (P = 0.10). For 1,632 urethral infections in men, all three media were equivalent, with none detecting fewer than 98% of the infections. Compared with a single inoculation, dual inoculation of MTM increased the diagnostic yield by 1.5% for 206 urethral infections and 2.4% for 83 cervical infections. In our clinic population, MTM is superior to CA or VFSM for the diagnosis of genital gonococcal infections, especially in women. The increased yield that accrued from inoculation of both MTM and either of the other media was not sufficiently high to warrant routine use of this practice in our clinic. PMID:6421872

  4. Trends in susceptibility of Neisseria gonorrhoeae to ceftriaxone from 1985 through 1991.

    PubMed Central

    Schwebke, J R; Whittington, W; Rice, R J; Handsfield, H H; Hale, J; Holmes, K K

    1995-01-01

    The antimicrobial susceptibilities of 16,441 gonococcal isolates from Seattle-King County were determined for ceftriaxone, cefoxitin, penicillin G, and tetracycline. From 1985 to 1989, ceftriaxone, in combination with doxycycline, was increasingly used for treatment of gonorrhea, and by 1989, it was used as therapy for > 80% of cases in Seattle-King County. MICs of ceftriaxone correlated significantly (P < 0.001) with those of the other beta-lactam antibodies included in this study. Geometric mean MICs of penicillin G for isolates that did not produce beta-lactamase increased from 1985 to 1991. The geometric mean MICs of cefoxitin, ceftriaxone, and tetracycline began to decline in 1987 but increased in 1990 and 1991. The percentage of strains with decreased susceptibility to ceftriaxone (MIC, 0.06 to 0.25 microgram/ml) rose from 0.3% in 1985 to 5.3% in 1987 but subsequently declined steadily to 2.6% in 1991, despite increased use of ceftriaxone as routine therapy for gonorrhea. Changes in patterns of antimicrobial susceptibility may be related not only to antimicrobial selection pressures but also to less well understood population shifts among Neisseria gonorrhoeae strains within a community. PMID:7785995

  5. Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein.

    PubMed Central

    Buchanan, T M; Hildebrandt, J F

    1981-01-01

    Principal outer membrane protein (protein I) of Neisseria gonorrhoeae was prepared nearly free of lipopolysaccharide (LPS) and substantially purified from other membrane proteins by chromatography of partially purified gonococcal outer membranes over Sepharose 6B in the presence of deoxycholate at pH 9.0. This protein I of nine separate antigenic types was coated to polystyrene tubes and used in the enzyme-linked immunosorbent assay (ELISA) to measure antibody to protein I or in inhibition tests to quantitate protein I antigen. No significant inhibition of the ELISA test was produced by purified LPS from the strain used to prepare each of the protein I types or by whole gonococci bearing the same LPS but different protein I antigens as the strain used to produce a given protein I antigen. Of 125 strains of gonococci used as whole organisms to inhibit the protein I ELISA, 124 (99%) typed with one or more of the nine protein I types, and 35% of these typed with a single protein I serotype. Sixty-one of 65 (94%) strains from Seattle and Atlanta patients with disseminated gonococcal infection contained protein I serotype 1, and 16 of 24 (64%) strains from Seattle patients with salpingitis bore one or both of protein I serotypes 1 and 2. Images PMID:6166568

  6. Manganese regulation of virulence factors and oxidative stress resistance in Neisseria gonorrhoeae

    PubMed Central

    Wu, Hsing-Ju; Seib, Kate L.; Srikhanta, Yogitha N.; Edwards, Jennifer; Kidd, Stephen P.; Maguire, Tina L .; Hamilton, Amanda; Pan, Kuan-Tin; Hsiao, He-Hsuan; Yao, Chen-Wen; Grimmond, Sean M.; Apicella, Michael A.; McEwan, Alastair G.; Wang, Andrew H-J.; Jennings, Michael P.

    2014-01-01

    Neisseria gonorrhoeae has evolved a complex and novel network of oxidative stress responses, including defense mechanisms that are dependent on manganese (Mn). We performed systematic analyses at the transcriptomic and proteomic (1D SDS-PAGE and Isotope-Coded Affinity Tag [ICAT]) levels to investigate the global expression changes that take place in a high Mn environment, which results in a Mn-dependent oxidative stress resistance phenotype. These studies revealed that 97 proteins are regulated at the post-transcriptional level under conditions of increased Mn concentration, including proteins involved in virulence (eg. Pilin, a key adhesin), oxidative stress defence (eg. superoxide dismutase), cellular metabolism, protein synthesis, RNA processing and cell division. Mn regulation of inorganic pyrophosphatase (Ppa) indicated the potential involvement of phosphate metabolism in the Mn-dependent oxidative stress defense. A detailed analysis of the role of Ppa and polyphosphate kinase (Ppk) in the gonococcal oxidative stress response revealed that ppk and ppa mutant strains showed increased resistance to oxidative stress. Investigation of these mutants grown with high Mn suggests that phosphate and pyrophosphate are involved in Mn-dependent oxidative stress resistance. PMID:20004262

  7. Neisseria gonorrhoeae pilin glycan contributes to CR3 activation during challenge of primary cervical epithelial cells

    PubMed Central

    Jennings, Michael P.; Jen, Freda E.-C.; Roddam, Louise F.; Apicella, Michael A.; Edwards, Jennifer L.

    2013-01-01

    Summary Expression of type IV pili by Neisseria gonorrhoeae plays a critical role in mediating adherence to human epithelial cells. Gonococcal pilin is modified with an O-linked glycan, which may be present as a di- or monosaccharide because of phase variation of select pilin glycosylation genes. It is accepted that bacterial proteins may be glycosylated; less clear is how the protein glycan may mediate virulence. Using primary, human, cervical epithelial (i.e. pex) cells, we now provide evidence to indicate that the pilin glycan mediates productive cervical infection. In this regard, pilin glycan-deficient mutant gonococci exhibited an early hyper-adhesive phenotype but were attenuated in their ability to invade pex cells. Our data further indicate that the pilin glycan was required for gonococci to bind to the I-domain region of complement receptor 3, which is naturally expressed by pex cells. Comparative, quantitative, infection assays revealed that mutant gonococci lacking the pilin glycan did not bind to the I-domain when it is in a closed, low-affinity conformation and cannot induce an active conformation to complement receptor 3 during pex cell challenge. To our knowledge, these are the first data to directly demonstrate how a protein-associated bacterial glycan may contribute to pathogenesis. PMID:21371235

  8. Multiple protein differences exist between Neisseria gonorrhoeae type 1 and type 4.

    PubMed Central

    Klimpel, K W; Clark, V L

    1988-01-01

    Neisseria gonorrhoeae undergoes a spontaneous conversion from a form which is virulent, competent for DNA-mediated transformation, and piliated (type 1) to a form which is avirulent and neither piliated nor competent (type 4). This phase variation has become thought of as simply a conversion from piliated to nonpiliated. Using the techniques of cell fractionation, two-dimensional electrophoresis, and nonequilibrium pH gradient gel electrophoresis, we identified differences in the expression levels of multiple proteins between type 1 and type 4 cells. A total of 26 type 1-specific (T1S) and 23 type 4-specific (T4S) cytoplasmic or cytoplasmic membrane proteins were identified in O'Farrell two-dimensional gels. Using nonequilibrium pH gradient gel electrophoresis, we detected a minimum of eight T1S outer membrane proteins and four T4S outer membrane proteins which were not detected in the O'Farrell gels. Thus, the conversion from type 1 to type 4 is a complex event involving many different proteins of all cellular locations. Images PMID:3126144

  9. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae

    PubMed Central

    Ducey, Thomas F.; Jackson, Lydgia; Orvis, Joshua; Dyer, David W.

    2016-01-01

    Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to repress gene expression, Fur may also work indirectly by controlling additional regulatory elements. Using in silico analysis, we identified a putative small RNA (sRNA) homolog of the meningococcal nrrF locus, and demonstrate that this sRNA is iron-repressible, suggesting that this is the gonococcal analog of the rhyB locus in Escherichia coli. Quantitative real-time RT-PCR analysis indicates that this transcript may also be temporally regulated. Transcript analysis identified the 5′ start of the transcript, using a single reaction, fluorescent-based, primer extension assay. This protocol allows for the rapid identification of transcriptional start sites of RNA transcripts, and could be used for high-throughput transcript mapping. PMID:19162160

  10. Phosphoethanolamine Modification of Neisseria gonorrhoeae Lipid A Reduces Autophagy Flux in Macrophages.

    PubMed

    Zughaier, Susu M; Kandler, Justin L; Balthazar, Jacqueline T; Shafer, William M

    2015-01-01

    Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen's ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses. PMID:26641098

  11. Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae.

    PubMed Central

    Pannekoek, Y; van Putten, J P; Dankert, J

    1992-01-01

    Iron limitation, glucose deprivation, and growth under low oxygen supply (environmental stress) increased the expression of several proteins of Neisseria gonorrhoeae, including a 63-kilodalton protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This gonococcal stress protein (GSP63) was detected in the cytosol and copurified with lithium acetate-derived outer membranes. Successful purification of the protein was achieved by sucrose density gradient centrifugation and by chromatography on phenyl-Sepharose. Gel filtration of the purified protein revealed a molecular weight of approximately 450,000, suggesting that in its native state, the protein consists of a multimer of six to eight subunits. Isoelectric focusing indicated a pI of 5.2. Immunoblotting experiments using a polyclonal antiserum raised against the purified protein demonstrated cross-reactivity with a protein of the same electrophoretic mobility as GSP63 in all eight gonococcal isolates tested. N-terminal amino acid sequencing of the protein revealed up to 65% homology with members of the Hsp60 heat shock protein family, suggesting that GSP63 is related to this group of proteins. This relationship was further substantiated by the immunological cross-reactivity of GSP63 with mycobacterial Hsp60 and the ATP-binding activity of the gonococcal stress protein. Images PMID:1400243

  12. Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae.

    PubMed

    Pannekoek, Y; van Putten, J P; Dankert, J

    1992-11-01

    Iron limitation, glucose deprivation, and growth under low oxygen supply (environmental stress) increased the expression of several proteins of Neisseria gonorrhoeae, including a 63-kilodalton protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This gonococcal stress protein (GSP63) was detected in the cytosol and copurified with lithium acetate-derived outer membranes. Successful purification of the protein was achieved by sucrose density gradient centrifugation and by chromatography on phenyl-Sepharose. Gel filtration of the purified protein revealed a molecular weight of approximately 450,000, suggesting that in its native state, the protein consists of a multimer of six to eight subunits. Isoelectric focusing indicated a pI of 5.2. Immunoblotting experiments using a polyclonal antiserum raised against the purified protein demonstrated cross-reactivity with a protein of the same electrophoretic mobility as GSP63 in all eight gonococcal isolates tested. N-terminal amino acid sequencing of the protein revealed up to 65% homology with members of the Hsp60 heat shock protein family, suggesting that GSP63 is related to this group of proteins. This relationship was further substantiated by the immunological cross-reactivity of GSP63 with mycobacterial Hsp60 and the ATP-binding activity of the gonococcal stress protein. PMID:1400243

  13. Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

    PubMed

    Harvey, H A; Ketterer, M R; Preston, A; Lubaroff, D; Williams, R; Apicella, M A

    1997-06-01

    In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected. PMID:9169783

  14. Functional analysis of NsrR, a nitric oxide sensing Rrf2 repressor in Neisseria gonorrhoeae

    PubMed Central

    Isabella, Vincent M.; Lapek, John D.; Kennedy, Edward M.; Clark, Virginia L.

    2008-01-01

    Nitric oxide has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2-type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe-2S] cluster. NsrR/DNA interactions were thoroughly analyzed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA, and nsrR upstream regions with a Kd of 7 nM, 19 nM, and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97, or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in coordination of a NO-sensitive Fe-S center required for DNA binding. PMID:19007408

  15. Neisseria gonorrhoeae PilC expression provides a selective mechanism for structural diversity of pili.

    PubMed Central

    Jonsson, A B; Pfeifer, J; Normark, S

    1992-01-01

    Pili of Neisseria gonorrhoeae undergo both phase and structural variation. Phase variation of gonococcal pili can be caused by a RecA-independent on/off switch in PilC, a protein involved in pilus biogenesis. We show here that spontaneous nonpiliated PilC- derivatives as well as PilC- insertional mutants have also acquired sequence alterations in pilE relative to the pilE gene of the piliated MS11mk(P+)-u parent, so that the pilin produced is processed to soluble S-pilin and can be released into the medium. It is proposed that pilin alterations are selected for in PilC- bacteria if the parental nonassembled pilin is toxic to the cells--i.e., is not degradable to S-pilin at rates sufficient to allow viability of the cells. Toxicity is indicated by the extreme instability of certain unassembled pilin sequences and by the low frequency of nonpiliated, pilin+, PilC- variants that emerge from piliated recA- cells. The presence of a point mutation changing leucine-39 to phenylalanine at the cleavage site for S-pilin in one nonpiliated, PilC-, recA- variant relative to its piliated parent is a further argument for a selective mechanism of structural diversity of the gonococcal pilin. Images PMID:1348857

  16. Identification of carbohydrate structures that are possible receptors for Neisseria gonorrhoeae.

    PubMed Central

    Stromberg, N; Deal, C; Nyberg, G; Normark, S; So, M; Karlsson, K A

    1988-01-01

    Different strains and isogenic variants of Neisseria gonorrhoeae were assayed for their ability to bind glycolipids extracted from various sources. Among a large number of reference glycolipids, binding was observed only to lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], isoglobotriaosylceramide [Gal(alpha 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriaosylceramide [GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer]. The latter two glycolipids bound gonococci with the highest affinity. Lactosylceramide and gangliotriaosylceramide were found in glycolipid preparations from ME180 cells, an epithelial cell line derived from a human cervical carcinoma, and thus are possible receptors for gonococci. The gonococcal surface component that bound the above glycolipids is a protein distinct from pilin and protein II. Images PMID:2898784

  17. Release of soluble pilin antigen coupled with gene conversion in Neisseria gonorrhoeae.

    PubMed Central

    Haas, R; Schwarz, H; Meyer, T F

    1987-01-01

    Gene conversion appears to be the frequent mechanism in Neisseria gonorrhoeae that leads to an altered expression of pilin, the subunit component of the pili. In this process segments of variable sequence information, the minicassettes, are transferred from silent storage loci into an expression locus. As a putative consequence of the rearrangement in the pilE gene, gonococci can enter a different phase of pilin production. Although the removal of a 7-amino acid leader peptide results in the production of typical P+ pilin used to form pili, the loss of an additional 39 amino acids yields S-pilin, a soluble form of pilin that is efficiently secreted into the extracellular environment. Both pilin types can coexist in an apparently homogeneous culture. Ps cells usually are piliated, although less extensively with regard to the length and the number of the pili when compared with P+ cells. Ps cells form T3/T4-type colonies also typical of nonpiliated cells (P-). The observations further suggest that the classical nonsecretory P- phenotype is not generated as a rule by precise gene conversion but rather by genetic changes that cause the production of an over-length pilin (L-pilin). Images PMID:2892194

  18. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae

    PubMed Central

    Gong, Zheng; Tang, M. Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A.; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. PMID:26808268

  19. Phosphoethanolamine Modification of Neisseria gonorrhoeae Lipid A Reduces Autophagy Flux in Macrophages

    PubMed Central

    Zughaier, Susu M.; Kandler, Justin L.; Balthazar, Jacqueline T.; Shafer, William M.

    2015-01-01

    Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen’s ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses. PMID:26641098

  20. The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species

    PubMed Central

    Skaar, Eric P.; Tobiason, Deborah M.; Quick, J.; Judd, Ralph C.; Weissbach, Herbert; Etienne, Frantzy; Brot, Nathan; Seifert, H. Steven

    2002-01-01

    The PilB protein of Neisseria gonorrhoeae has been reported to be involved in the regulation of pilin gene transcription, but it also possesses significant homology to the peptide methionine sulfoxide reductase family of enzymes, specifically MsrA and MsrB from Escherichia coli. MsrA and MsrB in E. coli are able to reduce methionine sulfoxide residues in proteins to methionines. In addition, the gonococcal PilB protein encodes for both MsrA and MsrB activity associated with the repair of oxidative damage to proteins. In this work, we demonstrate that the PilB protein of Neisseria gonorrhoeae is not involved in pilus expression. Additionally, we show that wild-type N. gonorrhoeae produces two forms of this polypeptide, one of which contains a signal sequence and is secreted from the bacterial cytoplasm to the outer membrane; the other lacks a signal sequence and is cytoplasmic. Furthermore, we show that the secreted form of the PilB protein is involved in survival in the presence of oxidative damage. PMID:12096194

  1. Purification and biochemical characterization of DnaK and its transcriptional activator RpoH from Neisseria gonorrhoeae.

    PubMed

    Narayanan, Shalini; Beckham, Simone A; Davies, John K; Roujeinikova, Anna

    2014-12-01

    DnaK plays a central role in stress response in the important human pathogen Neisseria gonorrhoeae. The genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ(32))-dependent promoters. In this study, we cloned, purified and biochemically characterised N. gonorrhoeae DnaK (NgDnaK) and RpoH. The NgDnaK and RpoH sequences are 73 and 50 % identical to the sequences of their respective E. coli counterparts. Similar to EcDnaK, nucleotide-free NgDnaK exists as a mix of monomers, dimers and higher oligomeric species in solution, and dissociates into monomers on addition of ATP. Like E. coli σ(32), RpoH of N. gonorrhoeae is monomeric in solution. Kinetic analysis of the basal ATPase activity of purified NgDnaK revealed a V max of 193 pmol phosphate released per minute per microgram DnaK (which is significantly higher than reported basal ATPase activity of EcDnaK), and the turnover number against ATP was 0.4 min(-1) under our assay conditions. Nucleotide-free NgDnaK bound a short model substrate, NR-peptide, with micromolar affinity close to that reported for EcDnaK. Our analysis showed that interaction between N. gonorrhoeae RpoH and DnaK appears to be ATP-dependent and non-specific, in stark contrast to the E. coli DnaK system where σ(32) and DnaK interact as monomers even in the absence of ATP. Sequence comparison showed that the DnaK-binding site of σ(32) is not conserved in RpoH. Our findings suggest that the mechanism of DnaK/RpoH recognition in N. gonorrhoeae is different from that in E. coli. PMID:25156536

  2. Accurate detection of Neisseria gonorrhoeae ciprofloxacin susceptibility directly from genital and extragenital clinical samples: towards genotype-guided antimicrobial therapy

    PubMed Central

    Pond, Marcus J.; Hall, Catherine L.; Miari, Victoria F.; Cole, Michelle; Laing, Ken G.; Jagatia, Heena; Harding-Esch, Emma; Monahan, Irene M.; Planche, Timothy; Hinds, Jason; Ison, Catherine A.; Chisholm, Stephanie; Butcher, Philip D.; Sadiq, Syed Tariq

    2016-01-01

    Introduction Increasing use of nucleic acid amplification tests (NAATs) as the primary means of diagnosing gonococcal infection has resulted in diminished availability of Neisseria gonorrhoeae antimicrobial susceptibility data. We conducted a prospective diagnostic assessment of a real-time PCR assay (NGSNP) enabling direct detection of gonococcal ciprofloxacin susceptibility from a range of clinical sample types. Methods NGSNP, designed to discriminate an SNP associated with ciprofloxacin resistance within the N. gonorrhoeae genome, was validated using a characterized panel of geographically diverse isolates (n = 90) and evaluated to predict ciprofloxacin susceptibility directly on N. gonorrhoeae-positive NAAT lysates derived from genital (n = 174) and non-genital (n = 116) samples (n = 290), from 222 culture-confirmed clinical episodes of gonococcal infection. Results NGSNP correctly genotyped all phenotypically susceptible (n = 49) and resistant (n = 41) panel isolates. Ciprofloxacin-resistant N. gonorrhoeae was responsible for infection in 29.7% (n = 66) of clinical episodes evaluated. Compared with phenotypic susceptibility testing, NGSNP demonstrated sensitivity and specificity of 95.8% (95% CI 91.5%–98.3%) and 100% (95% CI 94.7%–100%), respectively, for detecting ciprofloxacin-susceptible N. gonorrhoeae, with a positive predictive value of 100% (95% CI 97.7%–100%). Applied to urogenital (n = 164), rectal (n = 40) and pharyngeal samples alone (n = 30), positive predictive values were 100% (95% CI 96.8%–100%), 100% (95% CI 87.2%–100%) and 100% (95% CI 82.4%–100%), respectively. Conclusions Genotypic prediction of N. gonorrhoeae ciprofloxacin susceptibility directly from clinical samples was highly accurate and, in the absence of culture, will facilitate use of tailored therapy for gonococcal infection, sparing use of current empirical treatment regimens and enhancing acquisition of susceptibility data for

  3. Neisseria gonorrhoeae triggers the PGE2/IL-23 pathway and promotes IL-17 production by human memory T cells.

    PubMed

    Stefanelli, Paola; Teloni, Raffaela; Carannante, Anna; Mariotti, Sabrina; Nisini, Roberto; Gagliardi, Maria Cristina

    2012-10-01

    PGE2 is a potent modulator of the T helper (Th)17 immune response that plays a critical role in the host defense against bacterial, fungal and viral infections. We recently showed high serum levels of interleukin (IL)-17 in patients with gonococcal infection and we hypothesized that Neisseria gonorrhoeae could exploit a PGE2 mediated mechanism to promote IL-17 production. Here we show that N. gonorrhoeae induces human dendritic cell (DC) maturation, secretion of prostaglandin E2 and proinflammatory cytokines, including the pro-Th17 cytokine IL-23. Blocking PGE2 endogenous synthesis selectively reduces IL-23 production by DC in response to gonococcal stimulation, confirming recent data on PGE2/IL-23 crosstalk. N. gonorrhoeae stimulated DC induce a robust IL-17 production by memory CD4(+) T cells and this function correlates with PGE2 production. Our findings delineate a previously unknown role for PGE2 in the immune response to N. gonorrhoeae, suggesting its contribute via Th17 cell expansion. PMID:22542425

  4. New concepts in immunity to Neisseria gonorrhoeae: innate responses and suppression of adaptive immunity favor the pathogen, not the host.

    PubMed

    Liu, Yingru; Feinen, Brandon; Russell, Michael W

    2011-01-01

    It is well-known that gonorrhea can be acquired repeatedly with no apparent development of protective immunity arising from previous episodes of infection. Symptomatic infection is characterized by a purulent exudate, but the host response mechanisms are poorly understood. While the remarkable antigenic variability displayed by Neisseria gonorrhoeae and its capacity to inhibit complement activation allow it to evade destruction by the host's immune defenses, we propose that it also has the capacity to avoid inducing specific immune responses. In a mouse model of vaginal gonococcal infection, N. gonorrhoeae elicits Th17-driven inflammatory-immune responses, which recruit innate defense mechanisms including an influx of neutrophils. Concomitantly, N. gonorrhoeae suppresses Th1- and Th2-dependent adaptive immunity, including specific antibody responses, through a mechanism involving TGF-β and regulatory T cells. Blockade of TGF-β alleviates the suppression of specific anti-gonococcal responses and allows Th1 and Th2 responses to emerge with the generation of immune memory and protective immunity. Genital tract tissues are naturally rich in TGF-β, which fosters an immunosuppressive environment that is important in reproduction. In exploiting this niche, N. gonorrhoeae exemplifies a well-adapted pathogen that proactively elicits from its host innate responses that it can survive and concomitantly suppresses adaptive immunity. Comprehension of these mechanisms of gonococcal pathogenesis should allow the development of novel approaches to therapy and facilitate the development of an effective vaccine. PMID:21833308

  5. A Neisseria gonorrhoeae Immunoglobulin A1 Protease Mutant Is Infectious in the Human Challenge Model of Urethral Infection

    PubMed Central

    Johannsen, Diana B.; Johnston, David M.; Koymen, Hakan O.; Cohen, Myron S.; Cannon, Janne G.

    1999-01-01

    Many mucosal pathogens, including Neisseria gonorrhoeae, produce proteases that cleave immunoglobulin A (IgA), the predominant immunoglobulin class produced at mucosal surfaces. While considerable circumstantial evidence suggests that IgA1 protease contributes to gonococcal virulence, there is no direct evidence that N. gonorrhoeae requires IgA1 protease activity to infect a human host. We constructed a N. gonorrhoeae iga mutant without introducing new antibiotic resistance markers into the final mutant strain and used human experimental infection to test the ability of the mutant to colonize the male urethra and to cause gonococcal urethritis. Four of the five male volunteers inoculated with the Iga− mutant became infected. In every respect—clinical signs and symptoms, incubation period between inoculation and infection, and the proportion of volunteers infected—the outcome of human experimental infection with FA1090iga was indistinguishable from that previously reported for a variant of parent strain FA1090 matching the mutant in expression of Opa proteins, lipooligosaccharide, and pilin. These results indicate that N. gonorrhoeae does not require IgA1 protease production to cause experimental urethritis in males. PMID:10338512

  6. Mannose-binding lectin is present in human semen and modulates cellular adhesion of Neisseria gonorrhoeae in vitro

    PubMed Central

    Wing, J B; Jack, D L; Lee, M E; Pacey, A A; Kinghorn, G R; Read, R C

    2009-01-01

    Mannose-binding lectin (MBL) is an innate immune molecule present in blood and some mucosal tissues, which can influence microbial attachment and inflammatory responses of host cells during infection. In this study MBL was found to be present at a low concentration in semen samples in the range 1·2–24·9 ng/ml. Co-incubation of bacteria with semen resulted in the binding of MBL to the bacterial surface. Neisseria gonorrhoeae is a common cause of genitourinary infection. MBL bound to N. gonorrhoeae with strain-to-strain variation in the intensity of binding and nature of the bacterial receptor. Pretreatment with MBL concentrations similar to those found in human serum modulated the adhesion of N. gonorrhoeae strain FA1090 but not strain MS11 to epithelial cells. This effect was dose-dependent. This work demonstrates that MBL is present in human semen and modifies cellular responses to N. gonorrhoeae in a concentration-dependent manner. PMID:19664150

  7. Cloning of the recA gene of Neisseria gonorrhoeae and construction of gonococcal recA mutants.

    PubMed Central

    Koomey, J M; Falkow, S

    1987-01-01

    Interspecific complementation of an Escherichia coli recA mutant was used to identify recombinant plasmids within a genomic cosmid library derived from Neisseria gonorrhoeae that carry the gonococcal recA gene. These plasmids complement the E. coli recA mutation in both homologous recombination functions and resistance to DNA damaging agents. Subcloning, deletion mapping, and transposon Tn5 mutagenesis were used to localize the gonococcal gene responsible for suppression of the E. coli RecA- phenotype. Defined mutations in and near the cloned gonococcal recA gene were constructed in vitro and concurrently associated with a selectable genetic marker for N. gonorrhoeae and the mutated alleles were then reintroduced into the gonococcal chromosome by transformation-mediated marker rescue. This work resulted in the construction of two isogenic strains of N. gonorrhoeae, one of which expresses a reduced proficiency in homologous recombination activity and DNA repair function while the other displays an absolute deficiency in these capacities. These gonococcal mutants behaved similarly to recA mutants of other procaryotic species and displayed phenotypes consistent with the data obtained by heterospecific complementation in an E. coli recA host. The functional activities of the recA gene products of N. gonorrhoeae and E. coli appear to be highly conserved. Images PMID:3100504

  8. Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains.

    PubMed Central

    Dupuy, B; Pugsley, A P

    1994-01-01

    The assembly of type IV pili in Neisseria gonorrhoeae is a complex process likely to require the products of many genes. One of these is the enzyme prepilin peptidase, which cleaves and then N methylates the precursor pilin subunits prior to their assembly into pili. We have used a PCR amplification strategy to clone the N. gonorrhoeae prepilin peptidase gene, pilDNg. A single copy of the gene is shown to be present in the chromosome. Its product promotes correct cleavage of the gonococcal prepillin in Escherichia coli cells carrying both the prepilin peptidase gene and the pilin structural gene. PilDNg also cleaves prePulG, a type IV pilin-like protein of Klebsiella oxytoca. Moreover, PilDNg complements a mutation in the gene coding for the prepilin peptidase-like protein of K. oxytoca, pulO, partially restoring PulG-PulO-dependent extracellular secretion of the enzyme pullulanase. Finally, we show that genes homologous to pilDNg are present and expressed in a variety of species in the genus Neisseria, including some commensal strains. Images PMID:7906688

  9. New Ceftriaxone- and Multidrug-Resistant Neisseria gonorrhoeae Strain with a Novel Mosaic penA Gene Isolated in Japan.

    PubMed

    Nakayama, Shu-Ichi; Shimuta, Ken; Furubayashi, Kei-Ichi; Kawahata, Takuya; Unemo, Magnus; Ohnishi, Makoto

    2016-07-01

    We have characterized in detail a new ceftriaxone- and multidrug-resistant Neisseria gonorrhoeae strain (FC428) isolated in Japan in 2015. FC428 differed from previous ceftriaxone-resistant strains and contained a novel mosaic penA allele encoding a new mosaic penicillin-binding protein 2 (PBP 2). However, the resistance-determining 3'-terminal region of penA was almost identical to the regions of two previously reported ceftriaxone-resistant strains from Australia and Japan, indicating that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread. PMID:27067334

  10. Importance of multidrug efflux pumps in the antimicrobial resistance property of clinical multidrug-resistant isolates of Neisseria gonorrhoeae.

    PubMed

    Golparian, Daniel; Shafer, William M; Ohnishi, Makoto; Unemo, Magnus

    2014-06-01

    The contribution of drug efflux pumps in clinical isolates of Neisseria gonorrhoeae that express extensively drug-resistant or multidrug-resistant phenotypes has heretofore not been examined. Accordingly, we assessed the effect on antimicrobial resistance of loss of the three gonococcal efflux pumps associated with a known capacity to export antimicrobials (MtrC-MtrD-MtrE, MacA-MacB, and NorM) in such clinical isolates. We report that the MIC of several antimicrobials, including seven previously and currently recommended for treatment was significantly impacted. PMID:24733458

  11. Comparison of three real-time PCR assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in young pregnant women.

    PubMed

    Peuchant, Olivia; de Diego, Sabrina; Le Roy, Chloé; Frantz-Blancpain, Sandrine; Hocké, Claude; Bébéar, Cécile; de Barbeyrac, Bertille

    2015-12-01

    We compared 3 commercial real-time PCR assays, the Abbott RealTime CT/NG, the cobas® 4800 CT/NG, and the Cepheid Xpert® CT/NG, for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in vaginal swabs collected prospectively from pregnant women aged <25 years. The overall agreement among 2 assays ranged from 98.9% to 99.5% with a kappa score between 0.94 and 0.97 for C. trachomatis. For N. gonorrhoeae, the overall agreement was 100%. All kits allowed prompt and specific results for C. trachomatis and N. gonorrhoeae in young pregnant women. PMID:26423658

  12. Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.

    PubMed Central

    Makino, S; van Putten, J P; Meyer, T F

    1991-01-01

    Neisseria gonorrhoeae is a facultative intracellular bacterium capable of penetrating into certain human epithelial cell types. In order to identify gonococcal factors essential for invading Chang human conjunctiva cells, a gentamicin selection assay for the quantification of viable intracellular bacteria was used in conjunction with microscopy. The results demonstrate a correlation between the invasive behaviour of gonococci and the expression of Opa proteins, a family of variable outer membrane proteins present in all pathogenic Neisseria species. However, only particular Opa proteins supported invasion into Chang cells as indicated by the use of two unrelated gonococcal strains. Invasion was sensitive to cytochalasin D, and strong adherence mediated by the Opa proteins appeared to be essential for the internalization of gonococci. In contrast pili, which also conferred binding to Chang conjunctiva cells, did not support cellular invasion but rather were inhibitory. Images PMID:1673923

  13. The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. II. Use of hen antiserum to gonococcal lipopolysaccharide in a rapid slide test for the identification of N. gonorrhoeae from primary isolates and secondary cultures.

    PubMed

    Wallace, R; Ashton, F E; Ryan, A; Diena, B B

    1978-02-01

    An antiserum has been prepared in hens to R-type gonococcal lipopolysaccharide (LPS) and used in a simple slide-agglutination test for the identification of Neisseria gonorrhoeae. Anti-LPS serum agglutinated gonococcal cells representative of the four colony types of N. gonorrhoeae. Absorption of the antiserum with LPS removed the agglutinating activity. Secondary cultures (1120) were tested without observation of the colony type and all were agglutinated. No agglutination occurred with strains of Neisseria meningitidis, Neisseria lactamica, non-pathogenic Neisseria. Pseudomonas aeruginosa, Branhamella catarrhalis, or with species of lactobacilli and Acinetobacter. Cross-reactivity of the antiserum occurred with some streptococci. The anti-LPS serum was used to identify N. gonorrhoeae in primary isolates from the cervix, urethra, and pharynx. Of 251 gonococcal isolates tested, 249 were agglutinated by the antiserum, while all of the corresponding second cultures were agglutinated. The antiserum did not agglutinate N. meningitidis found in primary isolates from pharyngeal specimens. Anti-LPS hen serum should be useful for the rapid identification of N. gonorrhoeae in primary isolates or secondary cultures. PMID:417781

  14. Extragenital Infections Caused by Chlamydia trachomatis and Neisseria gonorrhoeae: A Review of the Literature.

    PubMed

    Chan, Philip A; Robinette, Ashley; Montgomery, Madeline; Almonte, Alexi; Cu-Uvin, Susan; Lonks, John R; Chapin, Kimberle C; Kojic, Erna M; Hardy, Erica J

    2016-01-01

    In the United States, sexually transmitted diseases due to Chlamydia trachomatis and Neisseria gonorrhoeae continue to be a major public health burden. Screening of extragenital sites including the oropharynx and rectum is an emerging practice based on recent studies highlighting the prevalence of infection at these sites. We reviewed studies reporting the prevalence of extragenital infections in women, men who have sex with men (MSM), and men who have sex only with women (MSW), including distribution by anatomical site. Among women, prevalence was found to be 0.6-35.8% for rectal gonorrhea (median reported prevalence 1.9%), 0-29.6% for pharyngeal gonorrhea (median 2.1%), 2.0-77.3% for rectal chlamydia (median 8.7%), and 0.2-3.2% for pharyngeal chlamydia (median 1.7%). Among MSM, prevalence was found to be 0.2-24.0% for rectal gonorrhea (median 5.9%), 0.5-16.5% for pharyngeal gonorrhea (median 4.6%), 2.1-23.0% for rectal chlamydia (median 8.9%), and 0-3.6% for pharyngeal chlamydia (median 1.7%). Among MSW, the prevalence was found to be 0-5.7% for rectal gonorrhea (median 3.4%), 0.4-15.5% for pharyngeal gonorrhea (median 2.2%), 0-11.8% for rectal chlamydia (median 7.7%), and 0-22.0% for pharyngeal chlamydia (median 1.6%). Extragenital infections are often asymptomatic and found in the absence of reported risk behaviors, such as receptive anal and oral intercourse. We discuss current clinical recommendations and future directions for research. PMID:27366021

  15. Identification of TbpA residues required for transferrin-iron utilization by Neisseria gonorrhoeae.

    PubMed

    Noto, Jennifer M; Cornelissen, Cynthia Nau

    2008-05-01

    Neisseria gonorrhoeae requires iron for survival in the human host and therefore expresses high-affinity receptors for iron acquisition from host iron-binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter critical for iron acquisition, while TbpB is a surface-exposed lipoprotein that increases the efficiency of iron uptake. The precise mechanism by which TbpA mediates iron acquisition has not been elucidated; however, the process is distinct from those of characterized siderophore transporters. Similar to these TonB-dependent transporters, TbpA is proposed to have two distinct domains, a beta-barrel and a plug domain. We hypothesize that the TbpA plug coordinates iron and therefore potentially functions in multiple steps of transferrin-mediated iron acquisition. To test this hypothesis, we targeted a conserved motif within the TbpA plug domain and generated single, double, and triple alanine substitution mutants. Mutagenized TbpAs were expressed on the gonococcal cell surface and maintained wild-type transferrin binding affinity. Single alanine substitution mutants internalized iron at wild-type levels, while the double and triple mutants showed a significant decrease in iron uptake. Moreover, the triple alanine substitution mutant was unable to grow on transferrin as a sole iron source; however, expression of TbpB compensated for this defect. These data indicate that the conserved motif between residues 120 and 122 of the TbpA plug domain is critical for transferrin-iron utilization, suggesting that this region plays a role in iron acquisition that is shared by both TbpA and TbpB. PMID:18347046

  16. Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

    PubMed

    Klotman, Mary E; Rapista, Aprille; Teleshova, Natalia; Micsenyi, Amanda; Jarvis, Gary A; Lu, Wuyuan; Porter, Edith; Chang, Theresa L

    2008-05-01

    Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection. PMID:18424739

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

    PubMed

    Rodríguez-Tirado, Carolina; Maisey, Kevin; Rodríguez, Felipe E; Reyes-Cerpa, Sebastián; Reyes-López, Felipe E; Imarai, Mónica

    2012-03-01

    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 β-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 β-catenin from the adherens junction complex, followed by a redistribution and reorganization of actin cytoskeleton and fibronectin within the extracellular matrix. PMID:22146107

  18. Instability of expression of lipooligosaccharides and their epitopes in Neisseria gonorrhoeae.

    PubMed Central

    Schneider, H; Hammack, C A; Apicella, M A; Griffiss, J M

    1988-01-01

    We assessed variation in the expression of lipooligosaccharide (LOS) components and their epitopes within populations of a strain of Neisseria gonorrhoeae by using the monoclonal antibodies (MAbs) O6B4 and 3F11 and immunoenzymatic, immuno-colloidal gold electron microscopic, and sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedures. Wild-type organisms varied in binding of both MAbs. We used the intensity of immunoenzymatic colony blot color to distinguish four binding variants for each MAb: red (R), pink (P), and colorless (nonreactive [N]) and an N back to R (N-R) revertant. R to P to R and R to N to R variation occurred at frequencies of 0.2% and 0.02%, respectively. The electrophoretic LOS profiles and MAb immunoblot patterns of the R, P, and N-R variants were the same as those of the wild type. LOSs of the N variants, in contrast, were of lower Mr, bound neither 3F11 nor O6B4 MAb, and contained as their major component the 3.6-kilodalton LOS that bears the L8LOS epitope of N. meningitidis. Results of immunoelectron microscopic studies were consistent with LOS binding patterns. Large number of colloidal gold particles were deposited about both R and P variants, distally from R organisms, but proximally from P organisms. N variant organisms, like their LOS, bound neither of the MAbs. N-R variant organisms were like the wild type in that they showed much variation in the amounts of MAb they bound. Images PMID:3126149

  19. Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae.

    PubMed Central

    Bos, M P; Grunert, F; Belland, R J

    1997-01-01

    Opacity (Opa) protein variation in Neisseria gonorrhoeae is implicated in the pathogenesis of gonorrhea, possibly by mediating adherence and entry of the bacteria into human tissues. One particular Opa protein mediates adherence to epithelial cells through cell surface proteoglycans. Recently, two other eukaryotic cell receptors for Opa proteins have been reported. These receptors are members of a subgroup of the carcinoembryonic (CEA) gene family that express CD66 antigens. CEA family members vary in their distribution in human tissues. In order to understand whether interactions between Opa and CEA-like molecules play any role in pathogenesis, we must investigate which CEA family members are able to serve as Opa receptors and which Opa proteins recognize CEA-like molecules. We therefore studied HeLa cells that were stably transfected with five different members of the CEA family, i.e., CEA, CEA gene family member 1a (CGM1a), CGM6, nonspecific cross-reacting antigen (NCA), and biliary glycoprotein a (BGPa). We infected these transfectants with all possible 11 Opa variants of gonococcal strain MS11 and determined the numbers of bacteria that were bound and internalized. To account for proteoglycan-mediated adherence, infection assays were also performed in the presence of heparin. Our results show that of the 11 Opa variants of MS11, the same 4 recognized CGM1a and NCA. CGM6, however, was not recognized by any Opa variant of MS11. CEA was recognized by at least 9 of 11 Opa variants, and the BGP transfectants specifically bound and internalized 10 of 11 Opa variants and also bound Opa-negative gonococci. Immunofluorescence experiments showed that clustering of CEA-like molecules occurred upon infection of HeLa transfectants with those Opa variants that interacted specifically with the CEA family member. Together these data show that CEA family members are differentially recognized by gonococcal Opa variants, suggesting that this phenomenon may contribute to cell

  20. Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

    PubMed

    Stein, Daniel C; LeVan, Adriana; Hardy, Britney; Wang, Liang-Chun; Zimmerman, Lindsey; Song, Wenxia

    2015-01-01

    Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions. PMID:26244560

  1. Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells

    PubMed Central

    Stein, Daniel C.; LeVan, Adriana; Hardy, Britney; Wang, Liang-Chun; Zimmerman, Lindsey; Song, Wenxia

    2015-01-01

    Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen–related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions. PMID:26244560

  2. Mismatch correction modulates mutation frequency and pilus phase and antigenic variation in Neisseria gonorrhoeae.

    PubMed

    Criss, Alison K; Bonney, Kevin M; Chang, Rhoda A; Duffin, Paul M; LeCuyer, Brian E; Seifert, H Steven

    2010-01-01

    The mismatch correction (MMC) system repairs DNA mismatches and single nucleotide insertions or deletions postreplication. To test the functions of MMC in the obligate human pathogen Neisseria gonorrhoeae, homologues of the core MMC genes mutS and mutL were inactivated in strain FA1090. No mutH homologue was found in the FA1090 genome, suggesting that gonococcal MMC is not methyl directed. MMC mutants were compared to a mutant in uvrD, the helicase that functions with MMC in Escherichia coli. Inactivation of MMC or uvrD increased spontaneous resistance to rifampin and nalidixic acid, and MMC/uvrD double mutants exhibited higher mutation frequencies than any single mutant. Loss of MMC marginally enhanced the transformation efficiency of DNA carrying a single nucleotide mismatch but not that of DNA with a 1-kb insertion. Unlike the exquisite UV sensitivity of the uvrD mutant, inactivating MMC did not affect survival after UV irradiation. MMC and uvrD mutants exhibited increased PilC-dependent pilus phase variation. mutS-deficient gonococci underwent an increased frequency of pilin antigenic variation, whereas uvrD had no effect. Recombination tracts in the mutS pilin variants were longer than in parental gonococci but utilized the same donor pilS loci. These results show that gonococcal MMC repairs mismatches and small insertion/deletions in DNA and also affects the recombination events underlying pilin antigenic variation. The differential effects of MMC and uvrD in gonococci unexpectedly reveal that MMC can function independently of uvrD in this human-specific pathogen. PMID:19854909

  3. Identification of Regulatory Elements That Control Expression of the tbpBA Operon in Neisseria gonorrhoeae

    PubMed Central

    Vélez Acevedo, Rosuany N.; Ronpirin, Chalinee; Kandler, Justin L.; Shafer, William M.

    2014-01-01

    Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5′ endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon. PMID:24837286

  4. Chemical characterization of binding properties of opacity-associated protein II from Neisseria gonorrhoeae.

    PubMed Central

    Bessen, D; Gotschlich, E C

    1987-01-01

    Binding of an opacity-associated protein II (PIIop) from Neisseria gonorrhoeae to eucaryotic macromolecules was studied. HeLa cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose, and purified PIIop bound to approximately 50 distinct molecular species. The binding of PIIop to HeLa cell components was stable in high salt and nonionic detergent and was not inhibited by a variety of monosaccharides and polyionic substances. PIIop binding behavior was compared with that of two model carbohydrate-binding proteins, wheat germ agglutinin (WGA) and concanavalin A (ConA). Model glycoproteins (ovomucoid, fetuin, mucin, ovalbumin) inhibited binding by PIIop, WGA, and ConA to various degrees. HeLa cell glycopeptides, generated by pronase digestion of chloroform-methanol-extracted cells, were tested for their ability to inhibit binding by PIIop to Western blots of HeLa cell macromolecules. HeLa cell extracts inhibited PIIop binding before pronase treatment, but inhibitory activity was lost as a result of pronase digestion. Direct binding to defined glycosylated and nonglycosylated proteins revealed that ConA and WGA bound only glycoproteins, whereas PIIop bound to proteins lacking carbohydrate as well. PIIop binding to human and bovine serum albumins was of high affinity and required partial unfolding of albumin; native albumin was not bound by PIIop; however, both the denatured, reduced form of albumin and the compact, nonreduced form of carboxymethylated albumin were bound strongly by PIIop. Albumin-PIIop interaction did not involve covalent bond formation through sulfhydryl groups. The predominant binding interactions of PIIop found in this study were with protein rather than carbohydrate, and the chemical nature of the interactions is more complex than involvement of purely ionic or hydrophobic forces. Images PMID:3098683

  5. Differential response of human monocytes to Neisseria gonorrhoeae variants expressing pili and opacity proteins.

    PubMed Central

    Knepper, B; Heuer, I; Meyer, T F; van Putten, J P

    1997-01-01

    Experiments in vitro suggest that Neisseria gonorrhoeae surface variation plays a key role in gonococcal pathogenesis by providing the appropriate bacterial phenotypes to go through different stages of the infection. Here we report on the effects of phase and antigen variation of two major gonococcal adhesins, pili and opacity (Opa) outer membrane proteins, on the interaction of the gonococci with human monocytes. Using a set of recombinants of gonococcus strain MS11 that each express 1 of 11 genetically defined Opa proteins or a defined type of pilus, we found that both Opa proteins and pili promote bacterial phagocytosis by monocytes in the absence of serum and that this feature largely depends on the type of protein that is expressed. One of the Opa proteins (Opa[50]) strongly promoted uptake by monocytes but had little effect on the interaction with polymorphonuclear leukocytes under the conditions employed. Similarly, the phagocytosis-promoting effect of the pili was much more pronounced in monocytes than in neutrophils (4-fold versus 22-fold stimulation of uptake, respectively). Only a subpopulation of both types of phagocytes actively ingested bacteria, as has been observed during natural infections. Measurements of luminol-enhanced chemiluminescence demonstrated that phagocytosis of opaque but not piliated gonococci was accompanied by an increase in oxygen-reactive metabolites. These findings demonstrate that the monocyte response towards gonococci is highly dependent on the bacterial phenotype and differs from the neutrophil response. This diversity in bacterial behavior towards various types of human phagocytic cells underlines the biological impact of gonococcal surface variation and may explain previous contradictory results on this subject. PMID:9317017

  6. [Laboratory practices: diagnostics and antibiotics resistance testing of Neisseria gonorrhoeae in Germany].

    PubMed

    Loenenbach, Anna; Dudareva-Vizule, S; Buder, S; Sailer, A; Kohl, P K; Bremer, V

    2015-08-01

    Recent years have seen a world-wide increase in antimicrobial resistance (AMR) in cases of infection with Neisseria gonorrhoeae (NG). NG infection is not notifiable in Germany and there is a lack of information available about the spread and AMR of NG infections. The objective of the study was to provide information on diagnostic methods and AMR testing in cases of NG infections in German laboratories. A cross-sectional survey was undertaken in Germany between June and August 2013 using an online questionnaire. Laboratories performing NG diagnostics were identified and described with regard to the diagnostic methods used, the number of tests performed, the antibiotics tested and the AMR observed, in addition to general laboratory information. In total, 188 of the 521 participating laboratories performed NG diagnostics; these were included in the further statistical analysis. 92.6 % of the 188 laboratories performed culture. A median of 60 (IQR 15-270) samples per quarter (SPQ) were tested, with an overall positivity rate of 4.1 and 6.9 % among men. Most (82.1 %) of the 151 laboratories performing NG culture tested for AMR as well. The most frequently tested antibiotics were ciprofloxacin (94.8 %), penicillin (93.1 %), doxycycline (70.0 %) and ceftriaxone (67.2 %). The most frequently observed AMR ever were those against ciprofloxacin (87.1 %), penicillin (78.3 %), doxycycline (56.6 %) and azithromycin (35.1 %; all percentages refer to laboratories). The laboratories used different standards regarding susceptibility criteria. The emergence and spread of AMR shows that it is crucial to assess and monitor the scope and trends of multidrug-resistant gonorrhea. The data collected on diagnostic methods and AMR testing in cases of NG infections in German laboratories constitute an important basis for future monitoring. PMID:26112875

  7. Extragenital Infections Caused by Chlamydia trachomatis and Neisseria gonorrhoeae: A Review of the Literature

    PubMed Central

    Chan, Philip A.; Montgomery, Madeline; Almonte, Alexi; Lonks, John R.; Chapin, Kimberle C.; Kojic, Erna M.; Hardy, Erica J.

    2016-01-01

    In the United States, sexually transmitted diseases due to Chlamydia trachomatis and Neisseria gonorrhoeae continue to be a major public health burden. Screening of extragenital sites including the oropharynx and rectum is an emerging practice based on recent studies highlighting the prevalence of infection at these sites. We reviewed studies reporting the prevalence of extragenital infections in women, men who have sex with men (MSM), and men who have sex only with women (MSW), including distribution by anatomical site. Among women, prevalence was found to be 0.6–35.8% for rectal gonorrhea (median reported prevalence 1.9%), 0–29.6% for pharyngeal gonorrhea (median 2.1%), 2.0–77.3% for rectal chlamydia (median 8.7%), and 0.2–3.2% for pharyngeal chlamydia (median 1.7%). Among MSM, prevalence was found to be 0.2–24.0% for rectal gonorrhea (median 5.9%), 0.5–16.5% for pharyngeal gonorrhea (median 4.6%), 2.1–23.0% for rectal chlamydia (median 8.9%), and 0–3.6% for pharyngeal chlamydia (median 1.7%). Among MSW, the prevalence was found to be 0–5.7% for rectal gonorrhea (median 3.4%), 0.4–15.5% for pharyngeal gonorrhea (median 2.2%), 0–11.8% for rectal chlamydia (median 7.7%), and 0–22.0% for pharyngeal chlamydia (median 1.6%). Extragenital infections are often asymptomatic and found in the absence of reported risk behaviors, such as receptive anal and oral intercourse. We discuss current clinical recommendations and future directions for research. PMID:27366021

  8. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure.

    PubMed

    Unemo, Magnus; Golparian, Daniel; Nicholas, Robert; Ohnishi, Makoto; Gallay, Anne; Sednaoui, Patrice

    2012-03-01

    Recently, the first Neisseria gonorrhoeae strain (H041) highly resistant to the expanded-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are the last remaining options for first-line gonorrhea treatment, was isolated in Japan. Here, we confirm and characterize a second strain (F89) with high-level cefixime and ceftriaxone resistance which was isolated in France and most likely caused a treatment failure with cefixime. F89 was examined using six species-confirmatory tests, antibiograms (33 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of known gonococcal resistance determinants (penA, mtrR, penB, ponA, and pilQ). F89 was assigned to MLST sequence type 1901 (ST1901) and NG-MAST ST1407, which is a successful gonococcal clone that has spread globally. F89 has high-level resistance to cefixime (MIC = 4 μg/ml) and ceftriaxone (MIC = 1 to 2 μg/ml) and resistance to most other antimicrobials examined. A novel penA mosaic allele (penA-CI), which was penA-XXXIV with an additional A501P alteration in penicillin-binding protein 2, was the primary determinant for high-level ESC resistance, as determined by transformation into a set of recipient strains. N. gonorrhoeae appears to be emerging as a superbug, and in certain circumstances and settings, gonorrhea may become untreatable. Investigations of the biological fitness and enhanced understanding and monitoring of the ESC-resistant clones and their international transmission are required. Enhanced disease control activities, antimicrobial resistance control and surveillance worldwide, and public health response plans for global (and national) perspectives are also crucial. Nevertheless, new treatment strategies and/or drugs and, ideally, a vaccine are essential to develop for efficacious gonorrhea management. PMID:22155830

  9. Global gene expression and the role of sigma factors in Neisseria gonorrhoeae in interactions with epithelial cells.

    PubMed

    Du, Ying; Lenz, Jonathan; Arvidson, Cindy Grove

    2005-08-01

    Like many bacterial pathogens, Neisseria gonorrhoeae must adapt to environmental changes in order to successfully colonize and proliferate in a new host. Modulation of gene expression in response to environmental signals is an efficient mechanism used by bacteria to achieve this goal. Using DNA microarrays and a tissue culture model for gonococcal infection, we examined global changes in gene expression in N. gonorrhoeae in response to adherence to host cells. Among those genes induced upon adherence to human epithelial cells in culture was rpoH, which encodes a homolog of the heat shock sigma factor, sigma(32) (RpoH), as well as genes of the RpoH regulon, groEL and groES. Attempts to construct an rpoH null mutant in N. gonorrhoeae were unsuccessful, suggesting that RpoH is essential for viability of N. gonorrhoeae. The extracytoplasmic sigma factor, RpoE (sigma(E)), while known to regulate rpoH in other bacteria, was found not to be necessary for the up-regulation of rpoH in gonococci upon adherence to host cells. To examine the role of RpoH in host cell interactions, an N. gonorrhoeae strain conditionally expressing rpoH was constructed. The results of our experiments showed that while induction of rpoH expression is not necessary for adherence of gonococci to epithelial cells, it is important for the subsequent invasion step, as gonococci depleted for rpoH invade cells two- to threefold less efficiently than a wild-type strain. Taken together, these results indicate that sigma(32), but not sigma(E), is important for the response of gonococci in the initial steps of an infection. PMID:16040997

  10. Novel cost-effective quality control approach for the Cepheid Xpert CT/NG assay for the detection of Chlamydia Trachomatis and Neisseria Gonorrhoeae.

    PubMed

    Chan, Maurice; Jiang, Boran; Ng, Siew Yong Lily; Tan, Thean Yen

    2016-06-01

    The Xpert CT/NG is a rapid assay for detection of Neisseria gonorrhoeae and Chlamydia trachomatis. QC materials must be formulated to emulate human specimens, and are prohibitively expensive. A creative, cost-effective QC approach is proposed. The acceptable sample types for the Xpert CT/NG assay were extended to include eye swabs. PMID:27091503

  11. Complete Genome Sequences of Three Neisseria gonorrhoeae Laboratory Reference Strains, Determined Using PacBio Single-Molecule Real-Time Technology

    PubMed Central

    Trees, David L.; Nicholas, Robert A.

    2015-01-01

    Neisseria gonorrhoeae, the etiological agent that causes the sexually transmitted infection gonorrhea, is a significant public health concern due to the emergence of antimicrobial resistance. We report the complete genome sequences of three reference isolates with varied antimicrobial susceptibility that will aid in elucidating the genetic mechanisms that confer resistance. PMID:26358608

  12. Genomic Epidemiology and Molecular Resistance Mechanisms of Azithromycin-Resistant Neisseria gonorrhoeae in Canada from 1997 to 2014.

    PubMed

    Demczuk, Walter; Martin, Irene; Peterson, Shelley; Bharat, Amrita; Van Domselaar, Gary; Graham, Morag; Lefebvre, Brigitte; Allen, Vanessa; Hoang, Linda; Tyrrell, Greg; Horsman, Greg; Wylie, John; Haldane, David; Archibald, Chris; Wong, Tom; Unemo, Magnus; Mulvey, Michael R

    2016-05-01

    The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZM(r)) represents a public health threat of untreatable gonorrhea infections. Genomic epidemiology through whole-genome sequencing was used to describe the emergence, dissemination, and spread of AZM(r) strains. The genomes of 213 AZM(r) and 23 AZM-susceptible N. gonorrhoeae isolates collected in Canada from 1989 to 2014 were sequenced. Core single nucleotide polymorphism (SNP) phylogenomic analysis resolved 246 isolates into 13 lineages. High-level AZM(r) (MICs ≥ 256 μg/ml) was found in 5 phylogenetically diverse isolates, all of which possessed the A2059G mutation (Escherichia coli numbering) in all four 23S rRNA alleles. One isolate with high-level AZM(r) collected in 2009 concurrently had decreased susceptibility to ceftriaxone (MIC = 0.125 μg/ml). An increase in the number of 23S rRNA alleles with the C2611T mutations (E. coli numbering) conferred low to moderate levels of AZM(r) (MICs = 2 to 4 and 8 to 32 μg/ml, respectively). Low-level AZM(r) was also associated with mtrR promoter mutations, including the -35A deletion and the presence of Neisseria meningitidis-like sequences. Geographic and temporal phylogenetic clustering indicates that emergent AZM(r) strains arise independently and can then rapidly expand clonally in a region through local sexual networks. PMID:26935729

  13. Antibodies directed to Neisseria gonorrhoeae impair nerve growth factor-dependent neurite outgrowth in Rat PC12 cells.

    PubMed

    Reuss, B

    2014-03-01

    In children born from mothers with prenatal infections with the Gram-negative bacterium Neisseria gonorrhoeae, schizophrenia risk is increased in later life. Since cortical neuropil formation is frequently impaired during this disease, actions of a rabbit polyclonal antiserum directed to N. gonorrhoeae on neurite outgrowth in nerve growth factor-stimulated PC12 cells were investigated here. It turned out that 10 μg/ml of the antiserum leads indeed to a significant reduction in neurite outgrowth, whereas an antiserum directed to Neisseria meningitidis had no such effect. Furthermore, reduction in neurite outgrowth could be reversed by the neuroleptic drugs haloperidol, clozapine, risperidone, and olanzapine. On the molecular level, the observed effects seem to include the known neuritogenic transcription factors FoxO3a and Stat3, since reduced neurite outgrowth caused by the antiserum was accompanied by a reduced phosphorylation of both factors. In contrast, restitution of neurite outgrowth by neuroleptic drugs revealed no correlation to the phosphorylation state of these factors. The present report gives a first hint that bacterial infections could indeed lead to impaired neuropil formation in vitro; however, the in vivo relevance of this finding for schizophrenia pathogenesis remains to be clarified in the future. PMID:24203572

  14. The second nationwide surveillance of the antimicrobial susceptibility of Neisseria gonorrhoeae from male urethritis in Japan, 2012-2013.

    PubMed

    Hamasuna, Ryoichi; Yasuda, Mitsuru; Ishikawa, Kiyohito; Uehara, Shinya; Hayami, Hiroshi; Takahashi, Satoshi; Matsumoto, Tetsuro; Yamamoto, Shingo; Minamitani, Shinichi; Watanabe, Akira; Iwata, Satoshi; Kaku, Mitsuo; Kadota, Junichi; Sunakawa, Keisuke; Sato, Junko; Hanaki, Hideaki; Tsukamoto, Taiji; Kiyota, Hiroshi; Egawa, Shin; Tanaka, Kazushi; Arakawa, Soichi; Fujisawa, Masato; Kumon, Hiromi; Kobayashi, Kanao; Matsubara, Akio; Naito, Seiji; Kuroiwa, Kentaro; Hirayama, Hideo; Narita, Harunori; Hosobe, Takahide; Ito, Shin; Ito, Kenji; Kawai, Shuichi; Ito, Masayasu; Chokyu, Hirofumi; Matsumura, Masaru; Yoshioka, Masaru; Uno, Satoshi; Monden, Koichi; Takayama, Kazuo; Kaji, Shinichi; Kawahara, Motoshi; Sumii, Toru; Kadena, Hitoshi; Yamaguchi, Takamasa; Maeda, Shinichi; Nishi, Shohei; Nishimura, Hirofumi; Shirane, Takeshi; Yoh, Mutsumasa; Akiyama, Kikuo; Imai, Toshio; Kano, Motonori

    2015-05-01

    Worldwide, the most important concern in the treatment of sexually transmitted infections is the increase in antimicrobial resistant Neisseria gonorrhoeae strains including resistance to cephalosporins, penicillins, fluoroquinolones or macrolides. To investigate the trends of antimicrobial susceptibility among N. gonorrhoeae strains isolated from male patients with urethritis, a Japanese surveillance committee conducted the second nationwide surveillance study. Urethral discharge was collected from male patients with urethritis at 26 medical facilities from March 2012 to January 2013. Of the 151 specimens, 103 N. gonorrhoeae strains were tested for susceptibility to 20 antimicrobial agents. None of the strains was resistant to ceftriaxone, but the minimum inhibitory concentration (MIC) 90% of ceftriaxone increased to 0.125 μg/ml, and 11 (10.7%) strains were considered less susceptible with an MIC of 0.125 μg/ml. There were 11 strains resistant to cefixime, and the MICs of these strains were 0.5 μg/ml. The distributions of the MICs of fluoroquinolones, such as ciprofloxacin, levofloxacin and tosufloxacin, were bimodal. Sitafloxacin, a fluoroquinolone, showed strong activity against all strains, including strains resistant to other three fluoroquinolones, such as ciprofloxacin, levofloxacin and tosufloxacin. The azithromycin MICs in 2 strains were 1 μg/ml. PMID:25727286

  15. Increasing Incidence of High-Level Tetracycline-Resistant Neisseria gonorrhoeae due to Clonal Spread and Foreign Import

    PubMed Central

    Lee, Hyukmin; Kim, Hyunsoo; Kim, Hyo Jin; Suh, Young Hee; Yong, Dongeun; Jeong, Seok Hoon; Chong, Yunsop

    2016-01-01

    Purpose The detection of high-level tetracycline-resistant strains of Neisseria gonorrhoeae (TRNG) can make important epidemiological contributions that are relevant to controlling infections from this pathogen. In this study, we aimed to determine the incidence of TRNG isolates over time and also to investigate the characteristics and genetic epidemiology of these TRNG isolates in Korea. Materials and Methods The antimicrobial susceptibilities of 601 isolates of N. gonorrhoeae from 2004 to 2011 were tested by standard Clinical and Laboratory Standards Institute methods. To determine the molecular epidemiological relatedness, N. gonorrhoeae multi-antigen sequence typing was performed. Results The incidence of TRNG increased from 2% in 2004 to 21% in 2011. The minimum inhibitory concentration distributions of ceftriaxone and susceptibility of ciprofloxacin in TRNG were different from non-TRNG and varied according to the year of isolation. Most of the TRNG isolates collected from 2004 to 2007 exhibited genetic relatedness, with sequence type (ST) 1798 being the most common. From 2008 to 2011, the STs of the isolates became more variable and introduction of genetically unrelated TRNG were noted. Conclusion The increased incidence of TRNG strains until 2007 appears to be due, at least in part, to clonal spread. However, we propose that the emergence of various STs since 2008 could be associated with foreign import. PMID:26847286

  16. Determination of in vitro synergy for dual antimicrobial therapy against resistant Neisseria gonorrhoeae using Etest and agar dilution.

    PubMed

    Wind, Carolien M; de Vries, Henry J C; van Dam, Alje P

    2015-03-01

    In response to antimicrobial resistance of Neisseria gonorrhoeae to last-resort extended-spectrum cephalosporins, combination therapy of azithromycin+ceftriaxone is now recommended. Dual therapy can be effective to treat monoresistant strains as well as multidrug-resistant strains, preferably employing the effect of in vitro synergy. As reports on in vitro synergy of azithromycin+ceftriaxone in N. gonorrhoeae are conflicting, in this study an evaluation of this combination was performed using a cross-wise Etest method and agar dilution. Synergy was defined as a fractional inhibitory concentration index (FICI) of ≤0.5. To identify other dual treatment options for gonorrhoea, in vitro synergy was evaluated for 65 dual antimicrobial combinations using Etest. Azithromycin, cefixime, ceftriaxone, colistin, ertapenem, fosfomycin, gentamicin, minocycline, moxifloxacin, rifampicin, spectinomycin and tigecycline were screened for synergy in all possible combinations. No synergy or antagonism was found for any of the 65 combinations. The geometric mean FICI ranged from 0.82 to 2.00. The mean FICI of azithromycin+ceftriaxone was 1.18 (Etest) and 0.55 (agar dilution). The difference between both methods did not result in a difference in interpretation of synergy. Ceftriaxone-resistant strain F89 was tested in all combinations and no synergy was found for any of them. Most importantly, the ceftriaxone minimum inhibitory concentration of F89 was not decreased below the breakpoint with any concentration of azithromycin. PMID:25532741

  17. Neisseria gonorrhoea, Chlamydia trachomatis, and Treponema pallidum infection in antenatal and gynecological patients at Korle-Bu Teaching Hospital, Ghana.

    PubMed

    Apea-Kubi, Kwasi Akyem; Yamaguchi, Shinya; Sakyi, Bright; Kishimoto, Toshio; Ofori-Adjei, David; Hagiwara, Toshikatsu

    2004-12-01

    Five hundred and seventeen women attending the gynecology and obstetrics clinics of the Korle-Bu Teaching Hospital were examined for sexually transmitted infections (STIs). Vaginal swabs were examined for Trichomonas vaginalis, Candida albicans, and Gardnerella vaginalis infection. Endocervical swabs were examined for Neisseria gonorrhoea and Chlamydia trachomatis using a recently developed RNA detection kit. Strain typing was performed to identify serovars of C. trachomatis. Sera were analyzed for Treponema pallidum with a passive-particle agglutination assay kit. The prevalence of infection with N. gonorrhoea was 0.6%, C. trachomatis 3.0%, and T. pallidum 5.6%. Eight samples were PCR-positive for C. trachomatis. Five of these were serovar G, and the rest were serovar E. All cases of mixed infections occurred in pregnant women. In conclusion, a high transmissible risk of T. pallidum infection was observed among our study population and in particular among our pregnant women. The absence of association between the presenting symptoms, clinical findings, and specific pathogens has implications for the syndromic approach to STI case management. The low prevalence of C. trachomatis and N. gonorrhoea may be due to self medication and requires further research in primary health institutions in rural areas to compare rates. PMID:15623949

  18. Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro.

    PubMed

    Gong, Zijian; Lai, Wei; Liu, Min; Hua, Zhengshuang; Sun, Yayin; Xu, Qingfang; Xia, Yue; Zhao, Yue; Xie, Xiaoyuan

    2016-04-01

    The emergence of ceftriaxone-resistantNeisseria gonorrhoeaeis currently a global public health concern. However, the mechanism of ceftriaxone resistance is not yet fully understood. To investigate the potential genes related to ceftriaxone resistance inNeisseria gonorrhoeae, we subcultured six gonococcal strains with increasing concentrations of ceftriaxone and isolated the strains that became resistant. After analyzing several frequently reported genes involved in ceftriaxone resistance, we found only a single mutation inpenA(A501V). However, differential analysis of the genomes and transcriptomes between pre- and postselection strains revealed many other mutated genes as well as up- and downregulated genes. Transformation of the mutatedpenAgene into nonresistant strains increased the MIC between 2.0- and 5.3-fold, and transformation of mutatedftsXincreased the MIC between 3.3- and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, whilepilM,pilN, andpilQwere downregulated. Furthermore, the resistant strain developed cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and downregulated PilMNQ. In conclusion, antimicrobial pressure could result in the emergence of ceftriaxone resistance, and the evolution of resistance ofNeisseria gonorrhoeaeto ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry. PMID:26787702

  19. Control of pili and sialyltransferase expression in Neisseria gonorrhoeae is mediated by the transcriptional regulator CrgA.

    PubMed

    Matthias, Kathryn A; Rest, Richard F

    2014-03-01

    Contact-regulated gene A (CrgA) is a transcriptional regulator present in the pathogenic Neisseria that functions as both an activator and a repressor of transcription following contact with host cells. While its mechanism of action has been studied extensively in Neisseria meningitidis, the specific subset of genes that CrgA targets has been debated. Although the majority of these constitute virulence genes, suggesting that CrgA is important in pathogenesis, no study to date has examined the effects of CrgA in Neisseria gonorrhoeae. In this report, we generated a knockout mutant of crgA (ΔcrgA) in the serum-sensitive gonococcal strain F62. crgA deletion resulted in a reduction in the transcript and protein levels of the primary pilin component pilE via mechanisms that were both contact-dependent and -independent. In contrast, ΔcrgA overexpressed the main determinant of serum resistance in F62, lipooligosaccharide sialyltransferase (Lst). CrgA-mediated lst repression was direct as both recombinant and native CrgA bound to the lst promoter at multiple locations in EMSA and ChIP assays respectively. The increase in Lst levels associated with crgA deletion correlated with enhanced protection against killing by normal human serum. These data suggest a role for CrgA in virulence regulation during both cell adherence and planktonic growth. PMID:24433334

  20. Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific and South East Asian Regions, 2010.

    PubMed

    Lahra, Monica M

    2012-03-01

    The World Health Organization (WHO) Gonococcal Antimicrobial Surveillance Programme (GASP) has conducted continuous surveillance of antimicrobial resistance in Neisseria gonorrhoeae in the WHO Western Pacific Region (WPR) to optimise antibiotic treatment and control of gonococcal disease since 1992. From 2007, this has been enhanced by the inclusion of data from the WHO South East Asian Region (SEAR). Over time, there has been recruitment of additional centres in both regions. This report provides an analysis of antimicrobial resistance in N. gonorrhoeae in the WHO WPR and SEAR derived from results of the 2010 GASP surveillance. In 2010 there were 9,744 N. gonorrhoeae isolates examined for their susceptibility to one or more of the antibiotics used for the treatment of gonorrhoea, incorporating External Quality Assurance controlled methods, from reporting centres in 19 countries and/or jurisdictions. A high proportion of penicillin and quinolone resistance was again detected amongst isolates tested in the 'Asian' countries of WHO WPR and SEAR. In contrast, lower levels of penicillin and quinolone resistance were reported from the Pacific Islands of Fiji and New Caledonia. The proportion of gonococci reported as having 'decreased susceptibility' to the third-generation cephalosporin antibiotic ceftriaxone varied widely, ranging from 1.3% to 55.8%. There is a continued need for revision and clarification of some of the in vitro criteria that are currently used to categorise the clinical importance of gonococci with different ceftriaxone and oral cephalosporin MIC levels, and to relate these to treatment outcome. Azithromycin resistance was very low in most countries reporting, except in Mongolia where it was 34%. The number of instances of spectinomycin resistance remained low. A high proportion of strains tested continued to exhibit high-level plasmid mediated resistance to tetracyclines. The continuing emergence and spread of antibiotic resistant gonococci in and

  1. Neisseria gonorrhoeae bacterioferritin: structural heterogeneity, involvement in iron storage and protection against oxidative stress.

    PubMed

    Chen, C Y; Morse, S A

    1999-10-01

    The iron-storage protein bacterioferritin (Bfr) from Neisseria gonorrhoeae strain F62 was identified in cell-free extracts and subsequently purified by column chromatography. Gonococcal Bfr had an estimated molecular mass of 400 kDa by gel filtration; however, analysis by SDS-PAGE revealed that it was composed of 18 kDa (BfrA) and 22 kDa (BfrB) subunits. DNA encoding BfrB was amplified by PCR using degenerate primers derived from the N-terminal amino acid sequence of BfrB and from a C-terminal amino acid sequence of Escherichia coli Bfr. The DNA sequence of bfrA was subsequently obtained by genome walking using single-specific-primer PCR. The two Bfr genes were located in tandem with an intervening gap of 27 bp. A potential Fur-binding sequence (12 of 19 bp identical to the consensus neisserial fur sequence) was located within the 5' flanking region of bfrA in front of a putative -35 hexamer. The homology between the DNA sequences of bfrA and bfrB was 55.7%; the deduced amino acid sequences of BfrA (154 residues) and BfrB (157 residues) showed 39.7% identity, and showed 41.3% and 56.1% identity, respectively, to E. coli Bfr. Expression of recombinant BfrA and BfrB in E. coli strain DH5alpha was detected on Western blots probed with polyclonal anti-E. coli Bfr antiserum. Most Bfrs are homopolymers with identical subunits; however, the evidence presented here suggests that gonococcal Bfr was composed of two similar but not identical subunits, both of which appear to be required for the formation of a functional Bfr. A Bfr-deficient mutant was constructed by inserting the omega fragment into the BfrB gene. The growth of the BfrB-deficient mutant in complex medium was reduced under iron-limited conditions. The BfrB-deficient mutant was also more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These results demonstrate that gonococcal Bfr plays an important role in iron storage and protection from iron-mediated oxidative stress. PMID:10537219

  2. Azithromycin resistance is coevolving with reduced susceptibility to cephalosporins in Neisseria gonorrhoeae in Ontario, Canada.

    PubMed

    Allen, Vanessa G; Seah, Christine; Martin, Irene; Melano, Roberto G

    2014-05-01

    Azithromycin (AZM) is routinely recommended as a component of dual therapy for gonorrhea in combination with third-generation cephalosporins (3GC). In this study, we examined the prevalence of AZM-resistant (AZM(r)) Neisseria gonorrhoeae from July 2010 to February 2013, assessed the rate of concurrent cephalosporin resistance under the current treatment recommendations, and analyzed the clonal distribution of AZM(r) isolates in Ontario, Canada. Nineteen AZM(r) clinical isolates (one per patient; MIC, ≥2 μg/ml) were included in the study. Susceptibility profiles of these isolates to 11 antibiotics, molecular typing, characterization of macrolide resistance mechanisms, and penicillin-binding protein 2 (PBP2) patterns were determined for all the isolates. Two groups were defined based on AZM(r) level; group A isolates displayed high-level resistance (MIC, ≥2,048 μg/ml) due to mutations (A2143G) in the four copies of the 23S rRNA rrl gene, and group B isolates had moderate resistance to AZM (MICs, 2 to 8 μg/ml, C2599T mutation in the rrl gene), with a subgroup belonging to sequence type 3158 (ST3158) (n = 8), which also showed reduced susceptibility to 3GC (MICs, 0.12 to 0.25 μg/ml, PBP2 pattern XXXIV). This AZM(r) phenotype was not observed in previous provincial surveillance in 2008 (the ST3158 clone was found, with AZM MICs of 0.25 to 0.5 μg/ml associated with mtrR mutations). We hypothesized that the AZM mutant prevention concentration (MPC) in the ST3158 subpopulation we found in 2008 was higher than the MPC in wild-type isolates (AZM MIC, ≤0.031 μg/ml), increasing the chances of additional selection of AZM(r) mutations. Full AZM resistance is now emerging in this clone together with reduced susceptibility to 3GC, threatening the future efficacy of these antibiotics as therapeutic options for treatment of gonorrhea. PMID:24514092

  3. In vitro activity of fosfomycin alone and in combination with ceftriaxone or azithromycin against clinical Neisseria gonorrhoeae isolates.

    PubMed

    Hauser, Christoph; Hirzberger, Lea; Unemo, Magnus; Furrer, Hansjakob; Endimiani, Andrea

    2015-03-01

    New therapeutic strategies are needed to combat the emergence of infections due to multidrug-resistant Neisseria gonorrhoeae. In this study, fosfomycin (FOS) was tested against 89 N. gonorrhoeae isolates using the Etest method, showing MIC50/MIC90s of only 8/16 μg/ml (range, ≤1 to 32 μg/ml). FOS in combination with ceftriaxone (CRO) or azithromycin (AZT) was then evaluated using the checkerboard method for eight strains, including N. gonorrhoeae F89 (CRO-resistant) and AZT-HLR (high-level AZT-resistant). All combinations that included FOS gave indifferent effects (fractional inhibitory concentration [FIC] index values, 1.2 to 2.3 for FOS plus CRO, 1.8 to 3.2 for FOS plus AZT). Time-kill experiments for FOS, CRO, AZT, and their combinations (at 0.5×, 1×, 2×, and 4× the MIC) were performed against N. gonorrhoeae strain ATCC 49226, one N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence type 1407 (ST1407) strain, F89, and AZT-HLR. For all strains, at 24 h, the results indicated that (i) FOS was bactericidal at 2× the MIC, but after >24 h, there was regrowth of bacteria; (ii) CRO was bactericidal at 0.5× the MIC; (iii) AZT was bactericidal at 4× the MIC; (iv) CRO plus AZT was less bactericidal than was CRO alone; (v) FOS plus AZT was bactericidal at 2× the MIC; and (vi) CRO plus AZT and FOS plus CRO were both bactericidal at 0.5× the MIC, but FOS plus CRO had more rapid effects. FOS is appealing for use in the management of N. gonorrhoeae infections because of its single and oral formulation. However, our results suggest it be used in combination with CRO. After the appropriate clinical trials are conducted, this strategy could be implemented for the treatment of infections due to isolates possessing resistance to CRO and/or AZT. PMID:25547354

  4. In Vitro Activity of Fosfomycin Alone and in Combination with Ceftriaxone or Azithromycin against Clinical Neisseria gonorrhoeae Isolates

    PubMed Central

    Hauser, Christoph; Hirzberger, Lea; Unemo, Magnus; Furrer, Hansjakob

    2014-01-01

    New therapeutic strategies are needed to combat the emergence of infections due to multidrug-resistant Neisseria gonorrhoeae. In this study, fosfomycin (FOS) was tested against 89 N. gonorrhoeae isolates using the Etest method, showing MIC50/MIC90s of only 8/16 μg/ml (range, ≤1 to 32 μg/ml). FOS in combination with ceftriaxone (CRO) or azithromycin (AZT) was then evaluated using the checkerboard method for eight strains, including N. gonorrhoeae F89 (CRO-resistant) and AZT-HLR (high-level AZT-resistant). All combinations that included FOS gave indifferent effects (fractional inhibitory concentration [FIC] index values, 1.2 to 2.3 for FOS plus CRO, 1.8 to 3.2 for FOS plus AZT). Time-kill experiments for FOS, CRO, AZT, and their combinations (at 0.5×, 1×, 2×, and 4× the MIC) were performed against N. gonorrhoeae strain ATCC 49226, one N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence type 1407 (ST1407) strain, F89, and AZT-HLR. For all strains, at 24 h, the results indicated that (i) FOS was bactericidal at 2× the MIC, but after >24 h, there was regrowth of bacteria; (ii) CRO was bactericidal at 0.5× the MIC; (iii) AZT was bactericidal at 4× the MIC; (iv) CRO plus AZT was less bactericidal than was CRO alone; (v) FOS plus AZT was bactericidal at 2× the MIC; and (vi) CRO plus AZT and FOS plus CRO were both bactericidal at 0.5× the MIC, but FOS plus CRO had more rapid effects. FOS is appealing for use in the management of N. gonorrhoeae infections because of its single and oral formulation. However, our results suggest it be used in combination with CRO. After the appropriate clinical trials are conducted, this strategy could be implemented for the treatment of infections due to isolates possessing resistance to CRO and/or AZT. PMID:25547354

  5. α-2,3-Sialyltransferase Expression Level Impacts the Kinetics of Lipooligosaccharide Sialylation, Complement Resistance, and the Ability of Neisseria gonorrhoeae to Colonize the Murine Genital Tract

    PubMed Central

    Gulati, Sunita; Burrowes, Elizabeth; Zheng, Bo; Ram, Sanjay; Rice, Peter A.

    2015-01-01

    ABSTRACT  Neisseria meningitidis and Neisseria gonorrhoeae modify the terminal lacto-N-neotetraose moiety of their lipooligosaccharide (LOS) with sialic acid. N. gonorrhoeae LOS sialylation blocks killing by complement, which is mediated at least in part by enhanced binding of the complement inhibitor factor H (FH). The role of LOS sialylation in resistance of N. meningitidis to serum killing is less well defined. Sialylation in each species is catalyzed by the enzyme LOS α-2,3-sialyltransferase (Lst). Previous studies have shown increased Lst activity in N. gonorrhoeae compared to N. meningitidis due to an ~5-fold increase in lst transcription. Using isogenic N. gonorrhoeae strains engineered to express gonococcal lst from either the N. gonorrhoeae or N. meningitidis lst promoter, we show that decreased expression of lst (driven by the N. meningitidis promoter) reduced LOS sialylation as determined by less incorporation of tritium-labeled cytidine monophospho-N-acetylneuraminic acid (CMP-NANA; the donor molecule for sialic acid). Diminished LOS sialylation resulted in reduced rates of FH binding and increased pathway activation compared to N. gonorrhoeae promoter-driven lst expression. The N. meningitidis lst promoter generated sufficient Lst to sialylate N. gonorrhoeae LOS in vivo, and the level of sialylation after 24 h in the mouse genital tract was sufficient to mediate resistance to human serum ex vivo. Despite demonstrable LOS sialylation in vivo, gonococci harboring the N. meningitidis lst promoter were outcompeted by those with the N. gonorrhoeae lst promoter during coinfection of the vaginal tract of estradiol-treated mice. These data highlight the importance of high lst expression levels for gonococcal pathogenesis. PMID:25650401

  6. Potential impact of vaccination against Neisseria meningitidis on Neisseria gonorrhoeae in the United States: results from a decision-analysis model.

    PubMed

    Régnier, Stéphane A; Huels, Jasper

    2014-01-01

    Components in 4CMenB vaccine against Neisseria meningitidis serogroup B have shown to potentially cross-react with Neisseria gonorrhoeae. We modeled the theoretical impact of a US 4CMenB vaccination program on gonorrhea outcomes. A decision-analysis model was populated using published healthcare utilization and cost data. A two-dose adolescent vaccination campaign was assumed, with protective immunity starting at age 15 years and a base-case efficacy against gonorrhea of 20%. The 20%-efficacy level is an assumption since no clinical data have yet quantified the efficacy of 4CMenB against Neisseria gonorrhoea. Key outcome measures were reductions in gonorrhea and HIV infections, reduction in quality-adjusted life-years (QALYs) lost, and the economically justifiable price assuming a willingness-to-pay threshold of $75,000 per QALY gained. Adolescent vaccination with 4CMenB would prevent 83,167 (95% credible interval [CrI], 44,600-134,600) gonorrhea infections and decrease the number of HIV infections by 55 (95% CrI, 2-129) per vaccinated birth cohort in the USA. Excluding vaccination costs, direct medical costs for gonorrhea would reduce by $28.7 million (95% CrI, $6.8-$70.0 million), and income and productivity losses would reduce by $40.0 million (95% CrI, $8.2-$91.7 million). Approximately 83% of the reduction in lost productivity is generated by avoiding HIV infections. At a cost of $75,000 per QALY gained, and incremental to the vaccine's effect on meningococcal disease, a price of $26.10 (95% CrI, $9.10-$57.20) per dose, incremental to the price of the meningococcal vaccine, would be justified from the societal perspective. At this price, the net cost per infection averted would be $1,677 (95% CrI, $404-$2,564). Even if the cross-immunity of 4CMenB vaccine and gonorrhea is only 20%, the reduction in gonorrhea infections and associated costs would be substantial. PMID:25483706

  7. Draft Genome Sequence of a Dominant, Multidrug-Resistant Neisseria gonorrhoeae Strain, TCDC-NG08107, from a Sexual Group at High Risk of Acquiring Human Immunodeficiency Virus Infection and Syphilis▿

    PubMed Central

    Chen, Chun-Chen; Hsia, Ko-Chiang; Huang, Chung-Ter; Wong, Wing-Wai; Yen, Muh-Yong; Li, Lan-Hui; Lin, Kun-Yen; Chen, Kuo-Wei; Li, Shu-Ying

    2011-01-01

    Neisseria gonorrhoeae infection is the second major cause of sexually transmitted diseases worldwide. Development of resistance to multiple classes of antimicrobials in N. gonorrhoeae has compromised treatment and disease control. Herein, we report the availability of the draft genome sequence of a multidrug-resistant N. gonorrhoeae isolate, TCDC-NG08107, which spread in groups of men who have sex with men (MSM) in Taiwan. PMID:21257765

  8. Building a web-based tool to support clinical decisions in the control of Chlamydia trachomatis and Neisseria gonorrhoeae infections.

    PubMed

    Zhao, Kun; Qiu, Fasheng; Chen, Guantao

    2013-12-20

    Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are the agents of two common, sexually transmitted diseases afflicting women in the United States (http://www.cdc.gov). We designed a novel web-based application that offers simple recommendations to help optimize medical outcomes with CT and GC prevention and control programs. This application takes population groups, prevalence rates, parameters for available screening assays and treatment regimens (costs, sensitivity, and specificity), as well as budget limits as inputs. Its output suggests optimal screening and treatment strategies for selected at-risk groups, commensurate with the clinic's budget allocation. Development of this tool illustrates how a clinical informatics application based on rigorous mathematics might have a significant impact on real-world clinical issues. PMID:24564848

  9. Analysis of antibodies in local and disseminated Neisseria gonorrhoeae infections by means of gel electrophoresis-derived ELISA.

    PubMed

    Hadfield, S G; Glynn, A A

    1982-10-01

    Major antigens in Neisseria gonorrhoeae were identified by surface labelling the organisms with 125I and electrophoresing extracts in polyacrylamide with sodium dodecyl sulphate. Horizontal slices of the gels were cut out and tested in individual wells against patients' sera using ELISA. Serum from local gonococcal infections reacted with Protein II and, probably, lipopolysaccharide, but not with Protein I in deoxycholate (DOC) extracts and gave no reaction with Triton X-100 extracts. Serum from disseminated gonococcal infections reacted with Protein I in the DOC extract and with pili and a number of undefined possibly cytoplasmic membrane antigens in the Triton X-100 extract. The significance of the results and the potential of the method are discussed. PMID:6811421

  10. Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae.

    PubMed

    Hemarajata, P; Yang, S; Soge, O O; Humphries, R M; Klausner, J D

    2016-03-01

    In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions. PMID:26739156