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Sample records for neisseria gonorrhoeae isolated

  1. 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

  2. [Neisseria gonorrhoeae infections].

    PubMed

    Furuya, Ryusaburo; Tanaka, Masatoshi

    2009-01-01

    Neisseria gonorrhoeae infections are common bacterial sexually transmitted diseases. Men will usually experience lower urinary tract symptons attributed to urethritis, epididymitis, proctitis, or prostatitis, with associated mucopurulent urethral discharge. Many women are asymptomatic. But, occasionally, they have symptons of vaginal and pelvic discomfort of dysuria, and these infections can lead to pelvic inflammatory disease. Recentry, high prevalence of Neisseria gonorrhoeae isolates resistant to antimicrobial agents is a serious problem in the treatment of gonorrhea. For example, in Fukuoka city, Japan, the proportion of the isolates resistant to ciprofloxacin (CPFX) were 73.4% in 2006 and it was still so high. The proportion of the isolates resistant to tetracycline (TC) was 38.5% in 2006 and that of isolates resistant to penicillin G (PCG) was 17.5%. Owing to this high prevalence of antimicrobial-resistant Neisseria gonorrhoeae in Japan, the clinical efficacy rates of oral antimicrobial agents have become lower. So, as first-line therapy for gonococcal infections, only three parenteral regimens of single doses of ceftriaxone, cefodizime or spectinomycin are recommended by the Japanese Society for Sexually Transmitted Diseases. In the circumstances, we studied in vitro activity of combinations of oral agents such as, beta-lactam and azithromycin, fluoroquinolone and azithromycin, or beta-lactam and fluoroquinolone against Neisseria gonorrhoeae. The cefixime+azithromycin combination demonstrated greater synergy than other combinations.

  3. Epidemiological characterisation of Neisseria gonorrhoeae isolates from the Far East.

    PubMed Central

    Odugbemi, T O; Whittington, W L; DeWitt, W; Perkins, G; Johnson, S; Biddle, J; Piziak, M; Albritton, W L

    1983-01-01

    One hundred strains of Neisseria gonorrhoeae (including 30 penicillinase producing (PPNG) strains) originating from Korea were characterised by plasmid analysis, auxotyping, and serogrouping. Eighty per cent of the isolates possessed the conjugative 24.5 megadalton (Mdal) plasmid. A novel 7.8 Mdal plasmid was present in four isolates (one PPNG and three non-PPNG strains). Seventy five per cent of all the strains tested were wild type and belonged to serogroup WII, while 20% were proline requiring and belonged to serogroup WII. Two of the remaining strains were tyrosine auxotrophs, while another strain was arginine requiring; these three strains carried the conjugative plasmid and belonged to serogroup WII. Images PMID:6412959

  4. High-level tetracycline resistant Neisseria gonorrhoeae isolated in Portugal.

    PubMed

    Ferreira, E; Louro, D; Gomes, J P; Catry, M A; Pato, M V

    1997-05-01

    The first high-level tetracycline resistance (MIC > or = 16 mg/l) isolates of Neisseria gonorrhoeae (TRNG) were reported in 1990 from patients attending a Sexual Transmitted Disease (STD) Center in Lisbon. The TRNG prevalence was 4% in 1991, 5.3% in 1992 and 10,8% in 1994, exploding to 52.2% in 1995. The tet M determinant was evaluated by PCR. The digests of PCRP using HpaII produced the restriction pattern 2 for all the strains, except one (pattern 3). 78.3% of the TRNG strains were beta-lactamase producers and the 4.5 MDa penicillinase plasmid was the dominant (83%), 90% and 93.3% of the TRNG strains belonged to the auxotype NR and to the serogroup IA, respectively. The IA-8/NR class represented 58.3% of the TRNG isolates, suggesting a clonal spreading.

  5. Characterization of multiresistant strains of Neisseria gonorrhoeae isolated in Nicaragua.

    PubMed

    Castro, I; Bergeron, M G; Chamberland, S

    1993-01-01

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

  6. Identification of Drug Resistance Loci in Various Clinical Isolates of Neisseria gonorrhoeae

    PubMed Central

    Maier, Thomas W.; Warner, Patricia; Zubryzycki, Leonard; Chila, Maria

    1977-01-01

    Extending previous results obtained for three strains of Neisseria gonorrhoeae, four naturally occurring, multiply resistant isolates were used in transformation studies. Six drug resistance loci corresponding to those found in the previously studied strains were identified. In addition, the mtr and ery-2 loci were found to be allelic. PMID:410364

  7. Conjunctivitis Caused by Neisseria gonorrhoeae Isolates with Reduced Cephalosporin Susceptibility and Multidrug Resistance

    PubMed Central

    Kitagawa, Yutaka; Maruyama, Yosuke; Yamaguchi, Satoshi; Sakane, Yuri; Miyamoto, Hitoshi; Ohashi, Yuichi

    2013-01-01

    We report two cases of conjunctivitis caused by Neisseria gonorrhoeae with reduced cephalosporin susceptibility. Patients showed no response to cefmenoxime eye drops and intravenous ceftriaxone administration. The patients' condition improved after the addition of oral minocycline. The isolates contained the mosaic penA for reduction of β-lactam susceptibility. PMID:24025911

  8. Changing Antimicrobial Resistance Profiles among Neisseria gonorrhoeae Isolates in Italy, 2003 to 2012

    PubMed Central

    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

    2014-01-01

    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

  9. In vitro inhibition of growth of Neisseria gonorrhoeae by Neisseria meningitidis isolated from the pharynx of homosexual men.

    PubMed

    Bisaillon, J G; Turgeon, P; Dubreuil, D; Beaudet, R; Sylvestre, M; Ashton, F E

    1984-01-01

    Despite the high prevalence of pharyngeal gonorrhea and of meningococcal carriage among homosexual men, Neisseria gonorrhoeae and Neisseria meningitidis are rarely co-isolated from the throat. Forty-seven meningococcal isolates from the pharynx of homosexual men were examined, by a lawn-spotting method, for their ability to inhibit N. gonorrhoeae in vitro. Eight (17%) of the meningococcal isolates were inhibitory when tested against gonococci from the same patient, while 31 (66%) were inhibitory when tested against N. gonorrhoeae strain 650 (T1). The colonial type T1 of a given strain was, in all cases tested, more sensitive to the inhibitory activities than the corresponding T4 type. Since the meningococci co-isolated from the throat with gonococci were at least as inhibitory in vitro as those isolated without gonococci, the natural resistance to gonococcal pharyngitis cannot be explained on the basis of the inhibitory activities produced by the meningococci in vitro. The inhibitory strains of N. meningitidis were identified in decreasing importance as: nonserogroupable, W135, C, B, 29E, and X. The addition of trypsin to the solid medium removed the inhibition produced by the meningococci, an observation suggesting the involvement of protein inhibitors.

  10. Description of an Unusual Neisseria meningitidis Isolate Containing and Expressing Neisseria gonorrhoeae-Specific 16S rRNA Gene Sequences

    PubMed Central

    Skvoretz, Rhonda; Montgomery-Fullerton, Megan; Jonas, Vivian; Brentano, Steve

    2013-01-01

    An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination. PMID:23863567

  11. In vitro activity of tigecycline alone and antimicrobial combinations against clinical Neisseria gonorrhoeae isolates.

    PubMed

    Lee, Hyukmin; Kim, Hyunsoo; Seo, Young Hee; Yong, Dongeun; Jeong, Seok Hoon; Lee, Kyungwon; Chong, Younsop

    2017-02-01

    In this study, we determined the in vitro activity of various combinations of antimicrobial agents against 54 Neisseria gonorrhoeae isolates. The combined activity of ceftriaxone (CRO) and azithromycin (AZM), CRO and doxycycline (DOX), CRO and spectinomycin (SPT), cefixime (CFX) and AZM, CFX and DOX, and CFX and SPT was determined using a checkerboard method. The fractional inhibitory concentration index (FICI) values for all combinations were either additive or indifferent, and no synergistic or antagonistic effects were found. The FICI comparison in each combination did not show any difference according to the N.gonorrhoeae-resistant phenotypes and genotypic characteristics, including penicillinase-producing N. gonorrhoeae, tetracycline-resistant N. gonorrhoeae, stratified MIC of all antibiotics, and N. gonorrhoeae multiantigen sequence typing. MIC50 and MIC90 of tigecycline by agar dilution were 0.5 mg/L and 0.5 mg/L, respectively, which were lower than that of tetracycline and DOX. Additive/indifference results could suggest that combinations that include CRO may be used safely without a significant likelihood of generating resistance.

  12. [Resistance to ciprofloxacin of Neisseria gonorrhoeae strains isolated in Poland in 2012-2013].

    PubMed

    Młynarczyk-Bonikowska, Beata; Kujawa, Marlena; Młynarczyk, Grażyna; Malejczyk, Magdalena; Majewski, Sławomir

    2014-01-01

    Ciprofloxacin is commonly used in Poland specially for the treatment of urinary tract infections including urethritis. Patients are often treated without pathogen identification and antimicrobial resistance tests. Neisseria gonorrhoeae infection is one of the most common causes of urethritis in Poland. The resistance of bacteria to a wide range of antibiotics including ciprofloxacine makes the therapy of gonorrhoea more difficult. The mechanism of ciprofloxacine action depends on inactivation of bacterial topoisomerase II (gyrase) and topoisomerase IV. A resistance to ciprofloxacine occurring in Neisseria gonorrhoeae is mainly due to mutations in bacterial gyrA (encoding topoisomerase II) and/or parC (encoding topoisomerase IV ) genes. High level resistance is an effect of combination of three or four mutations. Another, less important mechanism of ciprofloxacin resistance, that can coexist with mutations in gyrA and parC genes related to the overproduction of membrane pumps proteins. 65 Neisseria gonorrhoeae strains isolated from patients of Department of Dermatology and Wenereology in Warsaw in the second half of 2012 and first of 2013 was investigated. The strains were cultured on chocolate agar plates in a 5% CO2 atmosphere at 37 degrees C and identified by colony morphology, Gram stain and oxidase reaction, followed by carbohydrate utilization test. Ciprofloxacin susceptibility was determined by E-Tests (bioMerieux). Bacteria were incubated at 35 degrees C in 5% CO2 for 24 h on chocolate agar plates. Tests were performed according to producers recommendations. The results (sensitive or resistant) were interpreted according to EUCAST recommendations. The MIC (Minimal inhibitory concentration) of Ciprofloxacin in investigated strains ranged from 0,002 to > 32 mg/L, MIC50 = 8 mg/L, MIC90 = > 32 mg/L. It was shown that only 38.5% of the strains were sensitive to ciprofloxacin according to EUCAST criteria from 2013 year. Due to the high percentage of ciprofloxacin

  13. Comparative evaluation of New York City and modified Thayer-Martin media for isolation of Neisseria gonorrhoeae.

    PubMed

    Greenwood, J R; Voss, J; Smith, R F; Wallace, H; Peter, C; Nachtigall, M; Maier, T; Wilber, J; Butsumyo, A

    1986-12-01

    Commercially manufactured New York City (NYC) medium and modified Thayer-Martin (MTM) medium were compared for their ability to isolate Neisseria gonorrhoeae from clinical specimens. Twenty-seven public health laboratories throughout California evaluated 4,802 specimens collected from patients attending either sexually transmitted disease or family planning clinics. Total of 726 and 737 N. gonorrhoeae isolates were recovered from NYC and MTM medium, respectively. Although less contamination was noted on NYC medium, MTM medium was equivalent to commercially prepared NYC medium for the isolation of N. gonorrhoeae from clinical specimens.

  14. Analysis of penicillinase-producing Neisseria gonorrhoeae isolates in Madrid (Spain) from 1983-85.

    PubMed Central

    Fenoll, A.; Berrón, S.; Vázquez, J. A.

    1987-01-01

    Between April 1983 and December 1985, 576 strains of Neisseria gonorrhoeae were isolated in our laboratory from patients attending Sexually Transmitted Diseases (STD) clinics. Of these, 61 (10.6%) were penicillinase-producing. Studies on these strains by plasmid analysis, auxotyping and serogrouping showed that the predominant type strains harboured the Asian resistance plasmid, were prototrophic, and were of serogroup W II/W III. About half of the strains, both of the African and Asian type, harboured the transfer plasmid. Strains of serogroup W II/W III were less sensitive to tetracycline and cefoxitin than serogroup W I strains. Images Fig. 2 PMID:2960555

  15. 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

  16. Neisseria gonorrhoeae isolates from four centres in Papua New Guinea remain susceptible to amoxycillin-clavulanate therapy.

    PubMed

    Toliman, Pamela J; Lupiwa, Tony; Law, Gregory J; Reeder, John C; Siba, Peter M

    2010-01-01

    Antibiotic-resistant strains of Neisseria gonorrhoeae have the potential to undermine treatment and control of gonorrhoea, which remains a highly prevalent sexually transmitted infection (STI) in Papua New Guinea (PNG). The standard treatment regimen for gonorrhoea in PNG based on amoxycillin and clavulanic acid (amoxycillin-clavulanate) was introduced about 15 years ago and there is some concern that over time circulating strains may have developed resistance to this therapy. To investigate this, N. gonorrhoeae isolates (n = 52) were collected from STI clinics in geographically representative centres in PNG and tested for their in vitro susceptibility to a range of antibiotics. All 52 isolates tested were found susceptible to amoxycillin-clavulanate, despite 40% (n = 21) being penicillinase producers and thus resistant to penicillin. These findings indicate that amoxycillin-clavulanate therapy remains an effective treatment for gonococcal infections in PNG, and support the maintenance of the present standard treatment for gonorrhoea in PNG.

  17. Comparison of Virulence Markers of Peritoneal and Fallopian Tube Isolates with Endocervical Neisseria gonorrhoeae Isolates from Women with Acute Salpingitis

    PubMed Central

    Draper, D. L.; James, J. F.; Brooks, G. F.; Sweet, R. L.

    1980-01-01

    Neisseria gonorrhoeae strains which cause acute salpingitis are presumed to ascend the genital tract from the cervix. Previous studies utilized isolates obtained from endocervical canal cultures, although it was not known if the isolates truly represented the organisms present in the fallopian tubes. In this study, we compared N. gonorrhoeae isolates from endocervical canal cultures with fallopian tube or peritoneal cul-de-sac isolates or isolates from both sites obtained at laparoscopy. Potential virulence markers were studied, including colony phenotype, auxotype, antimicrobial agent susceptibility, protein patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and susceptibility to normal human serum. Six of seven cervical isolates had the same antibiograms and molecular weight for major outer membrane proteins as those of the corresponding peritoneal isolates. Auxotypes also were the same and included prototrophic, proline-requiring, and proline-and-arginine-requiring isolates. The isolates as a group appeared to be very susceptible to the bactericidal action of pooled serum from normal women. Colony phenotypes varied between sites; the fallopian tubecul-de-sac isolates were predominantly of transparent phenotype and piliated. The cervical isolates were either mixtures of equal quantities of opaque and transparent phenotypes or predominantly opaque phenotype. By these markers, patients' N. gonorrhoeae cervical isolates appeared to be the same as their isolates from fallopian tubes except for a difference or shift in colony phenotype. Images Fig. 1 PMID:6769811

  18. [Antimicrobial resistance of Neisseria gonorrhoeae strains isolated from sex workers in Ankara].

    PubMed

    Zarakolu, Pinar; Sakizligil, Bülent; Unal, Serhat

    2006-01-01

    The prevalence of gonococcal infections among sexually transmitted infections is decreasing particularly in developed countries, but the increase in antimicrobial resistance of Neisseria gonorrhoeae is an emerging issue. There is lack of data about the epidemiology and the resistance pattern of the pathogen in our country. Gonococcal infections are recently included among the reportable diseases in Turkey. The specific laboratory tests are difficult, expensive and seldomly used for diagnosis in our country. The infection is usuallly treated empirically. In this study, 30 N. gonorrhoeae strains isolated from clinical samples (endocervical, rectal and urethral swabs) obtained from registered/unregistered sex workers admitted to Ankara Municipiality Hospital of Dermatology and Venereal Diseases were tested for beta-lactamase production and the susceptibility to various antimicrobials. The susceptibility testing was performed by agar dilution method, and the results were evaluated according to Clinical and Laboratory Standards Institute (CLSI) recommendations. Of the isolates, 70% was found resistant to penicilin and beta-lactamase production was observed in 48% of them. The susceptibility rates of the isolates to ceftriaxone, cefixime, ciprofloxacin, and tetracycline were found as 100%, 100%, 97%, and 40%, respectively.

  19. Isolation of Neisseria gonorrhoeae mutants that show enhanced trafficking across polarized T84 epithelial monolayers.

    PubMed

    Hopper, S; Wilbur, J S; Vasquez, B L; Larson, J; Clary, S; Mehr, I J; Seifert, H S; So, M

    2000-02-01

    Initiation of a gonococcal infection involves attachment of Neisseria gonorrhoeae to the plasma membrane of an epithelial cell in the mucosal epithelium and its internalization, transepithelial trafficking, and exocytosis from the basal membrane. Piliation and expression of certain Opa proteins and the immunoglobulin A1 protease influence the transcytosis process. We are interested in identifying other genetic determinants of N. gonorrhoeae that play a role in transcellular trafficking. Using polarized T84 monolayers as a model epithelial barrier, we have assayed an N. gonorrhoeae FA1090 minitransposon (mTn) mutant bank for isolates that traverse the monolayer more quickly than the isogenic wild-type (WT) strain. From an initial screen, we isolated four mutants, defining three genetic loci, that traverse monolayers significantly more quickly than their WT parent strain. These mutants adhere to and invade cells normally and do not affect the integrity of the monolayer barrier. Backcrosses of the mutations into the WT FA1090 strain yielded mutants with a similar fast-trafficking phenotype. In two mutants, the mTns had inserted 370 bp apart into the same locus, which we have named fit, for fast intracellular trafficker. Backcrosses of one of these mutants into the MS11A genetic background also yielded a fast-trafficking mutant. The fit locus contains two overlapping open reading frames, fitA and fitB, whose deduced amino acid sequences have predicted molecular weights of 8.6 and 15.3, respectively. Neither protein contains a signal sequence. FitA has a potential helix-turn-helix motif, while the deduced sequence of FitB offers no clues to its function. fitA or fitB homologues are present in the genomes of Pseudomonas syringae and Rhizobium meliloti, but not Neisseria meningitidis. Replication of the MS11A fitA mutant in A431 and T84 cells is significantly accelerated compared to that of the isogenic WT strain. In contrast, growth of this mutant in liquid media is

  20. 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.

  1. Comparison of Antimicrobial Susceptibility of Urogenital Neisseria gonorrhoeae Isolates Obtained From Women and Men.

    PubMed

    Kidd, Sarah; Moore, Page C; Kirkcaldy, Robert D; Philip, Susan S; Wiesenfeld, Harold C; Papp, John R; Kerndt, Peter R; Venkatasubramanian, Lalitha; Ghanem, Khalil G; Hook, Edward W

    2015-08-01

    The US system for gonococcal antimicrobial susceptibility surveillance monitors trends exclusively among men with urethral infection, the population from whom the yield of gonococcal culture is highest. Little is known about the susceptibility of female urogenital isolates, and it is unclear whether gonococcal susceptibility among men who report sex exclusively with women (MSW) is representative of susceptibility among women. Using isolates collected during a recent treatment trial in 5 US cities, we performed a secondary analysis to compare antimicrobial susceptibilities of Neisseria gonorrhoeae urogenital isolates obtained from women, MSW, and men who have sex with men (MSM). Pretreatment isolates were collected from trial participants; minimum inhibitory concentrations (MICs) were determined by agar dilution. Geometric mean MICs were adjusted for geographic location using general linear models. Susceptibility data for urogenital isolates from 56 women, 252 MSW, and 170 MSM were studied. The adjusted geometric mean ceftriaxone MIC was similar among women (0.0067 μg/mL; 95% confidence interval [CI], 0.0049-0.0092 μg/mL) and MSW (0.0060 μg/mL; 95% CI, 0.0053-0.0066 μg/mL). In contrast, the adjusted geometric mean ceftriaxone MIC was higher among MSM (0.0098 μg/mL; 95% CI, 0.0082-0.0119 μg/mL) than among MSW. This same pattern was observed for other antimicrobials, including cefixime and azithromycin Ceftriaxone, cefixime, and azithromycin MICs were higher among MSM than among MSW, but were similar among women and MSW. These findings suggest that gonococcal antimicrobial susceptibility surveillance based on urethral isolates from MSW may adequately represent susceptibility of urogenital N. gonorrhoeae in women.

  2. 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

  3. [Resistance to azithromycin of Neisseria gonorrhoeae strains isolated in Poland in 2012-2013 years].

    PubMed

    Młynarczyk-Bonikowska, Beata; Kujawa, Marlena; Młynarczyk, Grażyna; Malejczyk, Magdalena; Majewski, Sławomir

    2014-01-01

    Azithromycin is one of the most commonly used macrolide antibiotics. As other macrolides it inhibits bacterial proteins synthesis by binding with V domene of bacterial 23S rRNA. Resistance to azithromycin can be related to: 1. Mutations in gene encoding 23S rRNA. Significant effect on azithromycin MIC (minimal inhibitory concentration) is not observed when the mutation occurs only in 1 allele. In case of mutations occurring in 4 alleles, more common mutation C2611T is associated with MIC 2-16 mg/L and the second mutation A2059T results in high level resistance to azithromycin MIC > 256 mg/l 2. Over- production of membrane pumps proteins MtrCDE and MacAB, that remove antibiotics from bacterial cells. The mechanism is not able to cause azithromycin resistance itself but coexisting with other mechanisms of resistance can additionally increase MIC. 3. Synthesis of 23S rRNA methylases ErmB, ErmF, ErmC, ErmA. These enzymes cause demethylation of adenine (A2058) in V domain of 23S rRNA. The mechanism was common in the past, but it has been replaced by mutations in in V domain of 23S rRNA. Nowadays 23S rRNA methylases are very rare in N. gonorhoeae. Sixty five Neisseria gonorrhoeae strains isolated from patients of the Department of Dermatology and Venereology in Warsaw in the second half of 2012 and first of 2013 were investigated. The strains were cultured on chocolate agar plates in a 5% CO2 atmosphere at 37 °C and identified by colony morphology, Gram staining and oxidase reaction, followed by carbohydrate utilization test. Azithromycin susceptibility was determined by E-Tests (bioMerieux). Bacteria were incubated at 37°C in 5% CO2 for 24 h on chocolate agar plates. Tests were performed according to producers recommendations. The results (sensitive or resistant) were interpreted according to EUCAST recommendations. The MIC of azithromycin in investigated strains ranged from 0,064 to 4 mg/L, MIC50 = 0.5 mg/L, MIC90 = 2 mg/L. It was shown that only 38.5% of the strains

  4. Molecular analyses of TEM genes and their corresponding penicillinase-producing Neisseria gonorrhoeae isolates in Bangkok, Thailand.

    PubMed

    Nakayama, Shu-Ichi; Tribuddharat, Chanwit; Prombhul, Sasiprapa; Shimuta, Ken; Srifuengfung, Somporn; Unemo, Magnus; Ohnishi, Makoto

    2012-02-01

    Neisseria gonorrhoeae is a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials introduced for first-line treatment of gonorrhea. In the present study, 96 N. gonorrhoeae isolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A β-lactamase (TEM) gene variant, bla(TEM-135), that may be a precursor in the transitional stage of a traditional bla(TEM-1) gene into an extended-spectrum β-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins in N. gonorrhoeae, was identified. Clonal analysis using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand. Molecular analysis of the bla(TEM-135) gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of the bla(TEM-135) allele in the penicillinase-producing N. gonorrhoeae population may call for monitoring for the possible emergence of ESBL-producing N. gonorrhoeae in the future. This study identified a bla(TEM) variant (bla(TEM-135)) that is a possible intermediate precursor for an ESBL, which warrants international awareness.

  5. Molecular Analyses of TEM Genes and Their Corresponding Penicillinase-Producing Neisseria gonorrhoeae Isolates in Bangkok, Thailand

    PubMed Central

    Nakayama, Shu-ichi; Tribuddharat, Chanwit; Prombhul, Sasiprapa; Shimuta, Ken; Srifuengfung, Somporn; Unemo, Magnus

    2012-01-01

    Neisseria gonorrhoeae is a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials introduced for first-line treatment of gonorrhea. In the present study, 96 N. gonorrhoeae isolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A β-lactamase (TEM) gene variant, blaTEM-135, that may be a precursor in the transitional stage of a traditional blaTEM-1 gene into an extended-spectrum β-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins in N. gonorrhoeae, was identified. Clonal analysis using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand. Molecular analysis of the blaTEM-135 gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of the blaTEM-135 allele in the penicillinase-producing N. gonorrhoeae population may call for monitoring for the possible emergence of ESBL-producing N. gonorrhoeae in the future. This study identified a blaTEM variant (blaTEM-135) that is a possible intermediate precursor for an ESBL, which warrants international awareness. PMID:22143532

  6. 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

  7. 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

  8. Activity of gemifloxacin tested against Neisseria gonorrhoeae isolates including antimicrobial-resistant phenotypes.

    PubMed

    Pottumarthy, Sudha; Fritsche, Thomas R; Jones, Ronald N

    2006-02-01

    The antigonococcal potency of gemifloxacin and 5 reference comparator antimicrobials was determined for a selected collection of gonococcal isolates. The 250 Neisseria gonorrhoeae strains were inclusive of (1) 50 historic strains from Japan with elevated fluoroquinolone minimal inhibitory concentration values (QRNG) and (2) 200 contemporary strains from clinical specimens in 2004 (176 from 6 sentinel sites in the United States and 24 bacteremic isolates from the SENTRY Antimicrobial Surveillance Program). The rank order of potency of the tested antimicrobials for the entire collection was: ceftriaxone (MIC(90) = 0.06 microg/mL) > gemifloxacin (1 microg/mL) > tetracycline (2 microg/mL) > ciprofloxacin = levofloxacin = penicillin (4 microg/mL). The activity of gemifloxacin was not affected by penicillinase production; however, its activity was decreased for penicillin-resistant strains. Cross-resistance between gemifloxacin and the older fluoroquinolones was present, and the gemifloxacin MIC90 value was higher for the ciprofloxacin-resistant strains compared with the ciprofloxacin-susceptible strains (2 versus 0.016 mug/mL, respectively). More than 20.0% of the recent clinical strains were resistant to penicillin, tetracycline, and ciprofloxacin with >30.0% of gonococci resistant to ciprofloxacin in Washington State and Hawaii. The historic QRNG strains from the Far East were predominantly of intermediate susceptibility (88.0%) to ciprofloxacin, with a gemifloxacin MIC90 value of only 0.25 microg/mL. The bacteremic gonococcal strains were exquisitely susceptible to the 3 quinolones tested (ciprofloxacin, levofloxacin, and gemifloxacin, MIC90 at < or = 0.03 microg/mL) and ceftriaxone (MIC90 = 0.06 microg/mL). In summary, the potency of gemifloxacin was competitive and positioned between that of ceftriaxone and older quinolones or penicillin or tetracycline, irrespective of the gonococcal resistance phenotype. Gemifloxacin should be considered for further development

  9. Neisseria gonorrhoeae Antimicrobial Susceptibility Surveillance - The Gonococcal Isolate Surveillance Project, 27 Sites, United States, 2014.

    PubMed

    Kirkcaldy, Robert D; Harvey, Alesia; Papp, John R; Del Rio, Carlos; Soge, Olusegun O; Holmes, King K; Hook, Edward W; Kubin, Grace; Riedel, Stefan; Zenilman, Jonathan; Pettus, Kevin; Sanders, Tremeka; Sharpe, Samera; Torrone, Elizabeth

    2016-07-15

    Gonorrhea is the second most commonly reported notifiable disease in the United States; 350,062 gonorrhea cases were reported in 2014. Sexually transmitted infections caused by Neisseria gonorrhoeae are a cause of pelvic inflammatory disease in women, which can lead to serious reproductive complications including tubal infertility, ectopic pregnancy, and chronic pelvic pain. Prevention of sequelae and of transmission to sexual partners relies largely on prompt detection and effective antimicrobial treatment. However, treatment has been compromised by the absence of routine antimicrobial susceptibility testing in clinical care and evolution of antimicrobial resistance to the antibiotics used to treat gonorrhea. 2014. The Gonococcal Isolate Surveillance Project (GISP) was established in 1986 as a sentinel surveillance system to monitor trends in antimicrobial susceptibilities of N. gonorrhoeae strains in the United States. Each month, N. gonorrhoeae isolates are collected from up to the first 25 men with gonococcal urethritis attending each of the participating sexually transmitted disease (STD) clinics at 27 sites. The number of participating sites has varied over time (21-30 per year). Selected demographic and clinical data are abstracted from medical records. Isolates are tested for antimicrobial susceptibility using agar dilution at one of five regional laboratories. A total of 5,093 isolates were collected in 2014. Of these, 25.3% were resistant to tetracycline, 19.2% to ciprofloxacin, and 16.2% to penicillin (plasmid-based, chromosomal, or both). Reduced azithromycin susceptibility (Azi-RS) (defined as minimum inhibitory concentration [MIC] ≥2.0 µg/mL) increased from 0.6% in 2013 to 2.5% in 2014. The increase occurred in all geographic regions, but was greatest in the Midwest, and among all categories of sex of sex partners (men who have sex with men [MSM], men who have sex with men and women [MSMW], and men who have sex with women [MSW]). No Azi-RS isolates

  10. In Vitro Susceptibilities of 400 Spanish Isolates of Neisseria gonorrhoeae to Gemifloxacin and 11 Other Antimicrobial Agents

    PubMed Central

    Berrón, S.; Vázquez, J. A.; Giménez, M. J.; de la Fuente, L.; Aguilar, L.

    2000-01-01

    The in vitro activity of gemifloxacin versus those of 11 other antimicrobial agents against 400 strains of Neisseria gonorrhoeae was determined by microdilution with supplemented GC agar. A total of 37.5% of the strains were β-lactamase positive. A total of 70 and 6.4% of the β-lactamase-negative strains exhibited intermediate and high-level penicillin resistance, respectively. Ceftriaxone and gemifloxacin were the most active drugs (MICs at which 90% of isolates are inhibited, 0.01 versus 0.007 μg/ml, respectively), with 100% of strains inhibited by 0.12 μg/ml. PMID:10952612

  11. Clonally related Neisseria gonorrhoeae isolates with decreased susceptibility to the extended-spectrum cephalosporin cefotaxime in Amsterdam, the Netherlands.

    PubMed

    Heymans, Raymond; Bruisten, Sylvia M; Golparian, Daniel; Unemo, Magnus; de Vries, Henry J C; van Dam, Alje P

    2012-03-01

    From 2006 to 2008, Neisseria gonorrhoeae isolates were identified with decreased susceptibility to the extended-spectrum cephalosporin (ESC) cefotaxime among visitors of the Amsterdam sexually transmitted infections (STI) clinic, the Netherlands. Spread, clonality, and characteristics of 202 isolates were examined using antibiograms, conventional penA mosaic gene PCR, and N. gonorrhoeae multiple-locus variable-number tandem repeat analysis (NG-MLVA). A strictly defined subset was further characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and sequencing of ESC resistance determinants (penA, mtrR, and porB1b). Seventy-four N. gonorrhoeae isolates with a cefotaxime MIC of >0.125 μg/ml (group A), 54 with a cefotaxime MIC of 0.125 μg/ml (group B), and a control group of 74 with a cefotaxime MIC of <0.125 μg/ml (group C) were included. Fifty-three clonally related penA mosaic-positive isolates (penicillin-binding protein 2 type XXXIV) were identified in group A (n = 47 isolates; 64%) and B (n = 6 isolates; 11%). The 53 penA mosaic-positive isolates were predominantly NG-MAST ST1407 (87%) and contained an mtrR promoter A deletion (98%) and porB1b alterations G101K/A102N. All were assigned to the same NG-MLVA cluster that comprised in total 56 isolates. A correlation was found between decreased cefotaxime susceptibility and ST1407 that was highly prevalent among visitors of the Amsterdam STI clinic. The rapid spread of this strain, which also has been identified in many other countries, might be facilitated by high-risk sexual behavior and should be monitored closely to identify potential treatment failure. Quality-assured surveillance of ESC susceptibility on the national and international levels and exploration of new drugs and/or strategies for treatment of gonorrhea are crucial.

  12. Clonally Related Neisseria gonorrhoeae Isolates with Decreased Susceptibility to the Extended-Spectrum Cephalosporin Cefotaxime in Amsterdam, the Netherlands

    PubMed Central

    Heymans, Raymond; Bruisten, Sylvia M.; Golparian, Daniel; Unemo, Magnus; de Vries, Henry J. C.

    2012-01-01

    From 2006 to 2008, Neisseria gonorrhoeae isolates were identified with decreased susceptibility to the extended-spectrum cephalosporin (ESC) cefotaxime among visitors of the Amsterdam sexually transmitted infections (STI) clinic, the Netherlands. Spread, clonality, and characteristics of 202 isolates were examined using antibiograms, conventional penA mosaic gene PCR, and N. gonorrhoeae multiple-locus variable-number tandem repeat analysis (NG-MLVA). A strictly defined subset was further characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and sequencing of ESC resistance determinants (penA, mtrR, and porB1b). Seventy-four N. gonorrhoeae isolates with a cefotaxime MIC of >0.125 μg/ml (group A), 54 with a cefotaxime MIC of 0.125 μg/ml (group B), and a control group of 74 with a cefotaxime MIC of <0.125 μg/ml (group C) were included. Fifty-three clonally related penA mosaic-positive isolates (penicillin-binding protein 2 type XXXIV) were identified in group A (n = 47 isolates; 64%) and B (n = 6 isolates; 11%). The 53 penA mosaic-positive isolates were predominantly NG-MAST ST1407 (87%) and contained an mtrR promoter A deletion (98%) and porB1b alterations G101K/A102N. All were assigned to the same NG-MLVA cluster that comprised in total 56 isolates. A correlation was found between decreased cefotaxime susceptibility and ST1407 that was highly prevalent among visitors of the Amsterdam STI clinic. The rapid spread of this strain, which also has been identified in many other countries, might be facilitated by high-risk sexual behavior and should be monitored closely to identify potential treatment failure. Quality-assured surveillance of ESC susceptibility on the national and international levels and exploration of new drugs and/or strategies for treatment of gonorrhea are crucial. PMID:22214779

  13. Homology of cryptic plasmid of Neisseria gonorrhoeae with plasmids from Neisseria meningitidis and Neisseria lactamica.

    PubMed

    Ison, C A; Bellinger, C M; Walker, J

    1986-10-01

    DNA probe hybridisation was used to examine the relation between the cryptic plasmid from Neisseria gonorrhoeae and plasmids carried by pharyngeal isolates of Neisseria meningitidis and Neisseria lactamica. The complete gonococcal cryptic plasmid and HinfI derived digestion fragments subcloned into Escherichia coli were used to probe Southern blots of plasmid extracts. Homology was found to a plasmid of approximate molecular weight 4.5 kilobase pairs (Kb) but not to plasmids of less than 3.2 Kb or 6.5 Kb. Eleven of 16 strains of N meningitidis and two of six strains of N lactamica carried plasmids that showed strong hybridisation with the 4.2 Kb gonococcal plasmid. Hybridisation of plasmids from non-gonococcal species of neisseria with the gonococcal cryptic plasmid indicates that caution should be taken when using the cryptic plasmid as a diagnostic probe for gonorrhoea.

  14. Mosaic-like structure of penicillin-binding protein 2 Gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime.

    PubMed

    Ameyama, Satoshi; Onodera, Shoichi; Takahata, Masahiro; Minami, Shinzaburo; Maki, Nobuko; Endo, Katsuhisa; Goto, Hirokazu; Suzuki, Hiroo; Oishi, Yukihiko

    2002-12-01

    Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 micro g/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 micro g/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae.

  15. Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from symptomatic men attending the Nanjing sexually transmitted diseases clinic (2011-2012): genetic characteristics of isolates with reduced sensitivity to ceftriaxone.

    PubMed

    Li, Sai; Su, Xiao-Hong; Le, Wen-Jing; Jiang, Fa-Xing; Wang, Bao-Xi; Rice, Peter A

    2014-11-27

    Evolving gonococcal antimicrobial resistance (AMR) poses a serious threat to public health. The aim of this study was to: update antimicrobial susceptibility data of Neisseria gonorrhoeae recently isolated in Nanjing, China and identify specific deteminants of antimicrobial resistance and gentoypes of isolates with decreased sensitivity to ceftriaxone. 334 N. gonorrhoeae isolates were collected consecutively from symptomatic men attending the Nanjing STD Clinic between April 2011 and December 2012. The minimum inhibitory concentrations (MICs) for penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone were determined by agar plate dilution for each isolate. Penicillinase-producing N. gonorrhoeae (PPNG) and tetracycline-resistant N. gonorrhoeae (TRNG) were examined and typed for β-lactamase and tetM encoding plasmids respectively. Isolates that displayed elevated MICs to ceftriaxone (MIC ≥0.125 mg/L) were also tested for mutations in penA, mtrR, porB1b, ponA and pilQ genes and characterized by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). 98.8% (330/334) of N. gonorrhoeae isolates were resistant to ciprofloxacin; 97.9% (327/334) to tetracycline and 67.7% (226/334) to penicillin. All isolates were susceptible to ceftriaxone (MIC ≤0.25 mg/L) and spectinomycin (MIC ≤32 mg/L). Plasmid mediated resistance was exhibited by 175/334 (52%) of isolates: 120/334 (36%) of isolates were PPNG and 104/334 (31%) were TRNG. 90.0% (108/120) of PPNG isolates carried the Asia type β-lactamase encoding plasmid and 96% (100/104) of TRNG isolates carried the Dutch type tetM containing plasmid. Elevated MICs for ceftriaxone were present in 15 (4.5%) isolates; multiple mutations were found in penA, mtrR, porB1b and ponA genes. The 15 isolates were distributed into diverse NG-MAST sequence types; four different non-mosaic penA alleles were identified, including one new type. N. gonorrhoeae isolates in Nanjing generally retained similar antimicrobial

  16. Antimicrobial resistance, genetic resistance determinants for ceftriaxone and molecular epidemiology of Neisseria gonorrhoeae isolates in Nanjing, China.

    PubMed

    Chen, Shao-Chun; Yin, Yue-Ping; Dai, Xiu-Qin; Unemo, Magnus; Chen, Xiang-Sheng

    2014-11-01

    Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major problem worldwide. This study investigated the AMR, genetic ceftriaxone resistance determinants and molecular epidemiology of N. gonorrhoeae in Nanjing, China. N. gonorrhoeae isolates were collected in 2007 (n = 198) and 2012 (n = 80). The susceptibility to ceftriaxone, spectinomycin, ciprofloxacin and tetracycline was determined using an agar-dilution method. The ceftriaxone resistance determinants penA, mtrR and penB were examined using sequencing. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was performed for molecular epidemiology. All isolates were resistant to ciprofloxacin, 42.4% produced β-lactamase and 34.9% showed high-level resistance to tetracycline (MIC ≥16 mg/L). In total, 5.4% of isolates were resistant to ceftriaxone; however, all of these isolates were obtained in 2007 and the susceptibility to ceftriaxone appeared to have increased. All isolates were susceptible to spectinomycin. No penA mosaic alleles were found. Non-mosaic penA alleles with A501T and G542S alterations, an H105Y alteration in mtrR and an A102D/N alteration in porB1b were statistically associated with decreased susceptibility or resistance to ceftriaxone. The most prevalent NG-MAST sequence types (STs) were ST568 (n = 13), ST270 (n = 9) and ST421 (n = 7). ST270 was the most common ST in isolates with decreased susceptibility or resistance to ceftriaxone. Ceftriaxone, ideally 500 mg and together with azithromycin (1-2 g), should be recommended for treatment of gonorrhoea in Nanjing, China. However, N. gonorrhoeae strains with resistance to ceftriaxone have been found in Nanjing. NG-MAST and ceftriaxone resistance determinant analysis can be valuable to supplement the antimicrobial resistance surveillance in China, which needs to be further strengthened. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights

  17. Neisseria gonorrhoeae isolated from disseminated and localised infections in pre-penicillin era. Auxotypes and antibacterial drug resistances.

    PubMed Central

    Catlin, B W; Reyn, A

    1982-01-01

    Interest in the evolution of gonococcal auxotrophy led to a study of 72 strains isolated between 1935 and 1948 from the urogenital tract (57 patients), the eye (two patients), and from disseminated gonococcal infections (11 patients and probably two others). Two cervical isolates with nutritional requirements for proline, arginine, histidine, and biotin were oxidase-positive, Gram-negative diplococci, but their identity as Neisseria gonorrhoeae was uncertain because they were atypically susceptible to colistin and did not produce acid in glucose media. The N gonorrhoeae strains were highly susceptible to 11 other antibacterial drugs but not to sulphadiazine. Defects of one or more pathways for the biosynthesis of methionine, proline, arginine, threonine, lysine, the branched-chain amino acids, hypoxanthine, and thiamine pyrophosphate were found in 39 of the 70 strains, including four isolated in the presulphanilamide era. Unexpectedly, methionine was required for the growth of 11 (21%) of the 52 Danish strains and for 13 (72%) of 18 strains isolated in the USA. The Danish strains included 28 (54%) that did not require any of the compounds used for differentiating auxotypes, whereas this type was represented by only three (17%) of the USA strains. None of the gonococci required uracil or other pyrimidines. This suggests that the requirements for arginine, hypoxanthine, and uracil commonly found in recent isolates from disseminated gonococcal infections probably evolved treatment with sulphonamide was replaced by penicillin. PMID:6805848

  18. 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

  19. Long-term antibiotic susceptibility pattern of strains of Neisseria gonorrhoeae isolated in Czechoslovakia in the years 1957-1982.

    PubMed

    Hejzlar, H; Drazdáková, M; Podaná, B; Kvicera, J; Kittnar, E; Hajková, L; Povolná, A; Sedmidubský, V; Nedvídková, J; Janatová, E; Pellar, T; Stastná, L

    1982-01-01

    Over the past 25 years a total of 7492 strains of Neisseria gonorrhoeae have been isolated in Czechoslovakia, mainly in Prague (64%). All these strains have been tested for susceptibility to the following antibiotics: penicillin G, ampicillin, tetracycline, spectinomycin, erythromycin, doxycycline, kanamycin, rifampin, chloramphenicol, gentamicin, cephalothin, cephaloridine, lincomycin and clindamycin. In addition, seven derivatives of newer antibiotics of penicillin and cephalosporin series were tested in 1981. The study showed that in 1957 the MIC of 0.03 units of penicillin per ml was effective against 95% of strains, but in 1981 only 37% of isolates were sensitive to this concentration. The first gonococcal strains with the MIC value of 4.0 units/ml to penicillin were detected in 1981. This tendency towards decreased gonococcal susceptibility to benzylpenicillin is alarming. Over the last eight years there have been described sporadic isolations of strains relatively resistant to tetracycline (MIC = 8.0 mg/l). The susceptibility to spectinomycin has been tested in over 4000 gonococcal strains, since 1967. The test showed that this antibiotic remained highly effective against the gonococcal infection with over 95% of gonococci with the MIC value of 16.0 mg/l. No fully spectinomycin resistant strains have been found. Penicillin G as well as spectinomycin and cefotaxim are still considered the antibiotics of the first choice in the treatment of gonorrhoea. The alternative antibiotics may include cefuroxim, chloramphenicol and, in cases of sensitive strains, tetracyclines.

  20. Resistance to Ceftriaxone and Azithromycin in Neisseria gonorrhoeae Isolates From 7 Countries of South America and the Caribbean: 2010-2011.

    PubMed

    Thakur, Sidharath Dev; Araya, Pamela; Borthagaray, Graciela; Galarza, Patricia; Hernandez, Alina Llop; Payares, Daisy; Sanabria Cruz, Olga Marina; Carvallo, Maria Elena Trigoso; Corredor, Aura Helena; Dillon, Jo-Anne R

    2017-03-01

    Seven countries in Latin America and the Caribbean report on (2010 and 2011) the susceptibility of 2235 isolates of Neisseria gonorrhoeae to 6 antibiotics. Thirteen isolates had ceftriaxone minimum inhibitory concentrations (MICs) of 0.125 to ≥ 0.25 mg/L. The percentage of resistant isolates to the following antibiotics was: azithromycin, 1.0% to 1.7%; ciprofloxacin, 42.1% to 36.2%; penicillin, 31% to 35%; tetracycline, 21.8% to 22.6%.

  1. Auxotypes and Penicillin Susceptibilities of Neisseria gonorrhoeae Isolated from Patients with Gonorrhea Involving Two or More Sites

    PubMed Central

    Catlin, B. Wesley; Pace, Paul J.

    1977-01-01

    A system of auxotyping described in 1973 is based on the differing nutritional requirement patterns of Neisseria gonorrhoeae strains. Our ongoing evaluation of the reliability of auxotyping has involved a study of the constancy of characteristics of gonococci isolated at one time from two or more sites of a given subject. The auxotypes and minimal inhibitory concentration (MIC) of penicillin G were determined for 181 isolates obtained from 84 patients with uncomplicated gonorrhea, for 16 isolates from 8 couples with uncomplicated gonorrhea, and for 21 isolates from 12 other patients, 9 with disseminated gonococcal infection and three consorts. The penicillin MIC served to distinguish between many members of auxotypes 1, 2, and 3, which are commonly involved in uncomplicated gonorrhea. Thus, for proline-requiring gonococci (auxotype 2) the MIC ranged from 0.01 to 1.2 IU of penicillin per ml. The profile of gonococcal responses to seven other antibacterial drugs provided useful additional information where the extent of phenotypic similarity was in doubt. In all but seven instances, the gonococci isolated from different sites of the same patient, or from a consort, had the same nutritional requirements and penicillin MIC. The gonococci isolated from one patient with disseminated gonococcal infection and from one of her two sexual contacts had nutritional requirements for arginine, hypoxanthine, uracil, and thiamine pyrophosphate, whereas the strain isolated from her second contact differed in having no requirement for thiamine pyrophosphate. The paired cervical and rectal isolates from one patient with uncomplicated gonorrhea differed only with respect to a requirement for hypoxanthine. Pairs of isolates from three patients differed slightly in degree of susceptibility to penicillin. In the remaining two instances, however, numerous differences between the isolates from the endocervix and the anal canal of a given patient indicated the presence of concomitant

  2. Trends in antimicrobial susceptibility for azithromycin and ceftriaxone in Neisseria gonorrhoeae isolates in Amsterdam, the Netherlands, between 2012 and 2015

    PubMed Central

    Wind, Carolien M; Schim van der Loeff, Maarten F; van Dam, Alje P; de Vries, Henry JC; van der Helm, Jannie J

    2017-01-01

    Resistance of Neisseria gonorrhoeae to azithromycin and ceftriaxone has been increasing in the past years. This is of concern since the combination of these antimicrobials is recommended as the first-line treatment option in most guidelines. To analyse trends in antimicrobial resistance, we retrospectively selected all consultations with a positive N. gonorrhoeae culture at the sexually transmitted infection clinic, Amsterdam, the Netherlands, from January 2012 through September 2015. Minimum inhibitory concentrations (MICs) for azithromycin and ceftriaxone were analysed per year, and determinants associated with decreased susceptibility to azithromycin (MIC > 0.25 mg/L) or ceftriaxone (MIC > 0.032 mg/L) were assessed. Between 2012 and 2015 azithromycin resistance (MIC > 0.5 mg/L) was around 1.2%, the percentage of isolates with intermediate MICs (> 0.25 and ≤ 0.5 mg/L) increased from 3.7% in 2012, to 8.6% in 2015. Determinants associated with decreased azithromycin susceptibility were, for men who have sex with men (MSM), infections diagnosed in the year 2014, two infected sites, and HIV status (HIV; associated with less decreased susceptibility); for heterosexuals this was having ≥ 10 sex partners (in previous six months). Although no ceftriaxone resistance (MIC > 0.125 mg/L) was observed during the study period, the proportion of isolates with decreased ceftriaxone susceptibility increased from 3.6% in 2012, to 8.4% in 2015. Determinants associated with decreased ceftriaxone susceptibility were, for MSM, infections diagnosed in 2014, and pharyngeal infections; and for heterosexuals, infections diagnosed in 2014 or 2015, being of female sex, and having ≥ 10 sex partners. Continued decrease of azithromycin and ceftriaxone susceptibility will threaten future treatment of gonorrhoea. Therefore, new treatment strategies are warranted. PMID:28079519

  3. Trends in antimicrobial susceptibility for azithromycin and ceftriaxone in Neisseria gonorrhoeae isolates in Amsterdam, the Netherlands, between 2012 and 2015.

    PubMed

    Wind, Carolien M; Schim van der Loeff, Maarten F; van Dam, Alje P; de Vries, Henry Jc; van der Helm, Jannie J

    2017-01-05

    Resistance of Neisseria gonorrhoeae to azithromycin and ceftriaxone has been increasing in the past years. This is of concern since the combination of these antimicrobials is recommended as the first-line treatment option in most guidelines. To analyse trends in antimicrobial resistance, we retrospectively selected all consultations with a positive N. gonorrhoeae culture at the sexually transmitted infection clinic, Amsterdam, the Netherlands, from January 2012 through September 2015. Minimum inhibitory concentrations (MICs) for azithromycin and ceftriaxone were analysed per year, and determinants associated with decreased susceptibility to azithromycin (MIC > 0.25 mg/L) or ceftriaxone (MIC > 0.032 mg/L) were assessed. Between 2012 and 2015 azithromycin resistance (MIC > 0.5 mg/L) was around 1.2%, the percentage of isolates with intermediate MICs (> 0.25 and ≤ 0.5 mg/L) increased from 3.7% in 2012, to 8.6% in 2015. Determinants associated with decreased azithromycin susceptibility were, for men who have sex with men (MSM), infections diagnosed in the year 2014, two infected sites, and HIV status (HIV; associated with less decreased susceptibility); for heterosexuals this was having ≥ 10 sex partners (in previous six months). Although no ceftriaxone resistance (MIC > 0.125 mg/L) was observed during the study period, the proportion of isolates with decreased ceftriaxone susceptibility increased from 3.6% in 2012, to 8.4% in 2015. Determinants associated with decreased ceftriaxone susceptibility were, for MSM, infections diagnosed in 2014, and pharyngeal infections; and for heterosexuals, infections diagnosed in 2014 or 2015, being of female sex, and having ≥ 10 sex partners. Continued decrease of azithromycin and ceftriaxone susceptibility will threaten future treatment of gonorrhoea. Therefore, new treatment strategies are warranted. This article is copyright of The Authors, 2017.

  4. Phenotypic and genotypic properties of Neisseria gonorrhoeae isolates in Norway in 2009: antimicrobial resistance warrants an immediate change in national management guidelines.

    PubMed

    Hjelmevoll, S O; Golparian, D; Dedi, L; Skutlaberg, D H; Haarr, E; Christensen, A; Jørgensen, S; Nilsen, Ø J; Unemo, M; Skogen, V

    2012-06-01

    Despite rapidly diminishing treatment options for Neisseria gonorrhoeae and high levels of ciprofloxacin resistance worldwide, Norwegian guidelines still recommend ciprofloxacin as empirical treatment for gonorrhea. The present study aimed to characterize phenotypical and genotypical properties of N. gonorrhoeae isolates in Norway in 2009. All viable N. gonorrhoeae isolates (n = 114) from six university hospitals in Norway (2009) were collected, representing 42% of all notified gonorrhea cases. Epidemiological data were collected from the Norwegian Surveillance System for Communicable Diseases and linked to phenotypical and genotypical characteristics for each N. gonorrhoeae isolate. Resistance levels to the antimicrobials examined were: ciprofloxacin 78%, azithromycin 11%, cefixime 3.5%, ceftriaxone 1.8%, and spectinomycin 0%. The minimum inhibitory concentrations of gentamicin varied from 1.5 to 8 mg/L. Forty-one (36%) of the isolates were β-lactamase-producing, 17 displayed penA mosaic alleles, and 72 different N. gonorrhoeae multiantigen sequence types (ST; 37 novel) were identified. The most common ST was ST1407 (n = 11), containing penA mosaic allele. Four of these isolates displayed intermediate susceptibility/resistance to cefixime. The N. gonorrhoeae strains circulating in Norway were highly diverse. The level of ciprofloxacin resistance was high and the Norwegian management guidelines should promptly exclude ciprofloxacin as an empirical treatment option for gonorrhea.

  5. Evaluation of an rRNA-derived oligonucleotide probe for culture confirmation of Neisseria gonorrhoeae.

    PubMed Central

    Rossau, R; Duhamel, M; Van Dyck, E; Piot, P; Van Heuverswyn, H

    1990-01-01

    The reliability of an rRNA-derived oligonucleotide probe for Neisseria gonorrhoeae was tested with 187 N. gonorrhoeae isolates, 81 Neisseria meningitidis isolates, and several strains of other bacterial species. The probe proved to be 100% specific and 100% sensitive. N. gonorrhoeae cells could also be reliably identified in contaminated cultures with the oligonucleotide probe. The 2.6-megadalton cryptic plasmid used as a probe for N. gonorrhoeae was shown to be less sensitive, detecting 179 of 181 N. gonorrhoeae isolates. Images PMID:1693630

  6. Multidrug-Resistant Neisseria gonorrhoeae Isolates from Nanjing, China, Are Sensitive to Killing by a Novel DNA Gyrase Inhibitor, ETX0914 (AZD0914).

    PubMed

    Su, Xiao-Hong; Wang, Bao-Xi; Le, Wen-Jing; Liu, Yu-Rong; Wan, Chuan; Li, Sai; Alm, Richard A; Mueller, John P; Rice, Peter A

    2015-10-19

    We tested the activity of ETX0914 against 187 Neisseria gonorrhoeae isolates from men with urethritis in Nanjing, China, in 2013. The MIC50, MIC90, and MIC range for ETX0914 were 0.03 μg/ml, 0.06 μg/ml, and ≤0.002 to 0.125 μg/ml, respectively. All isolates were resistant to ciprofloxacin, and 36.9% (69/187) were resistant to azithromycin. Of the isolates, 46.5% were penicillinase-producing N. gonorrhoeae (PPNG), 36% were tetracycline-resistant N. gonorrhoeae (TRNG), and 13% (24 isolates) had an MIC of 0.125 μg/ml for ceftriaxone. ETX0914 may be an effective treatment option for gonorrhea.

  7. Multidrug-Resistant Neisseria gonorrhoeae Isolates from Nanjing, China, Are Sensitive to Killing by a Novel DNA Gyrase Inhibitor, ETX0914 (AZD0914)

    PubMed Central

    Su, Xiao-Hong; Le, Wen-Jing; Liu, Yu-Rong; Wan, Chuan; Li, Sai; Alm, Richard A.; Mueller, John P.; Rice, Peter A.

    2015-01-01

    We tested the activity of ETX0914 against 187 Neisseria gonorrhoeae isolates from men with urethritis in Nanjing, China, in 2013. The MIC50, MIC90, and MIC range for ETX0914 were 0.03 μg/ml, 0.06 μg/ml, and ≤0.002 to 0.125 μg/ml, respectively. All isolates were resistant to ciprofloxacin, and 36.9% (69/187) were resistant to azithromycin. Of the isolates, 46.5% were penicillinase-producing N. gonorrhoeae (PPNG), 36% were tetracycline-resistant N. gonorrhoeae (TRNG), and 13% (24 isolates) had an MIC of 0.125 μg/ml for ceftriaxone. ETX0914 may be an effective treatment option for gonorrhea. PMID:26482313

  8. Susceptibilities of Neisseria gonorrhoeae Isolates Containing Amino Acid Substitutions in GyrA, with or without Substitutions in ParC, to Newer Fluoroquinolones and Other Antibiotics

    PubMed Central

    Tanaka, Masatoshi; Nakayama, Hiroshi; Haraoka, Masashi; Saika, Takeshi; Kobayashi, Intetsu; Naito, Seiji

    2000-01-01

    We examined the antimicrobial susceptibilities of 85 Neisseria gonorrhoeae isolates, classified according to the presence of amino acid substitutions in the GyrA and ParC proteins, to 12 fluoroquinolones and 7 other antibiotics. Sitafloxacin and HSR-903 showed excellent activity against N. gonorrhoeae, including strains with both GyrA and ParC substitutions. Among the strains with various GyrA substitutions, strains with a serine-91-to-phenylalanine mutation required the highest MICs of all of the fluoroquinolones tested and were cross-resistant to structurally unrelated β-lactams. PMID:10602748

  9. Comparison of Antimicrobial Susceptibility of Urogenital Neisseria gonorrhoeae Isolates Obtained from Women and Men

    PubMed Central

    Kidd, Sarah; Moore, Page C.; Kirkcaldy, Robert D.; Philip, Susan S.; Wiesenfeld, Harold C.; Papp, John R.; Kerndt, Peter R.; Venkatasubramanian, Lalitha; Ghanem, Khalil G.; Hook, Edward W.

    2015-01-01

    Background The United States’ (US) system for gonococcal antimicrobial susceptibility surveillance monitors trends exclusively among men with urethral infection, the population from whom the yield of gonococcal culture is highest. Little is known about the susceptibility of female urogenital isolates, and it is unclear whether gonococcal susceptibility among men who report sex exclusively with women (MSW) is representative of susceptibility among women. Methods Using isolates collected during a recent treatment trial in five US cities, we performed a secondary analysis to compare antimicrobial susceptibilities of N. gonorrhoeae urogenital isolates obtained from women, MSW, and men who have sex with men (MSM). Pre-treatment isolates were collected from trial participants; minimum inhibitory concentrations (MICs) were determined by agar dilution. Geometric mean MICs were adjusted for geographic location using general linear models. Results Susceptibility data for urogenital isolates from 56 women, 252 MSW, and 170 MSM were studied. The adjusted geometric mean ceftriaxone MIC was similar among women (0.0067 μg/ml, 95% CI 0.0049–0.0092 μg/ml) and MSW (0.0060 μg/ml, 95% CI 0.0053–0.0066 μg/ml). In contrast, the adjusted geometric mean ceftriaxone MIC was higher among MSM (0.0098 μg/ml, 95% CI 0.0082–0.0119 μg/ml) than MSW. This same pattern was observed for other antimicrobials, including cefixime and azithromycin Conclusions Ceftriaxone, cefixime, and azithromycin MICs were higher among MSM than MSW, but were similar among women and MSW. These findings suggest that gonococcal antimicrobial susceptibility surveillance based on urethral isolates from MSW may adequately represent susceptibility of urogenital N. gonorrhoeae in women. PMID:26165435

  10. Nucleic Acid Amplification Testing for Neisseria gonorrhoeae

    PubMed Central

    Whiley, David M.; Tapsall, John W.; Sloots, Theo P.

    2006-01-01

    Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review. PMID:16436629

  11. Incidence of Neisseria gonorrhoeae isolates negative by Syva direct fluorescent-antibody test but positive by Gen-Probe accuprobe test in a sexually transmitted disease clinic population.

    PubMed Central

    Beebe, J L; Rau, M P; Flageolle, S; Calhoon, B; Knapp, J S

    1993-01-01

    To determine the accuracy of the Syva (Palo Alto, Calif.) direct fluorescent-antibody (DFA) test in comparison with the Gen-Probe (San Diego, Calif.) Accuprobe culture confirmation test, we tested 395 isolates of Neisseria gonorrhoeae from cultures obtained from patients attending a sexually transmitted disease clinic from 1 July 1991 through 30 June 1992. All isolates were tested for DFA reactivity with a polyclonal reagent (Difco Laboratories, Detroit, Mich.) and a monoclonal reagent (Syva, Inc., direct specimen test) and for specific molecular probe reactivity by the Gen-Probe Accuprobe culture confirmation test for N. gonorrhoeae. The 395 isolates gave positive results for the Gen-Probe culture confirmation test and the Difco polyclonal direct specimen test. However, 18 (4.6%) of the isolates were negative for N. gonorrhoeae by the Syva DFA test. With the exception of six beta-lactamase-positive isolates, all isolates that were negative by Syva DFA were sensitive to penicillin, tetracycline, spectinomycin, and ceftriaxone by disk-diffusion susceptibility testing. Auxotyping and serotyping studies indicated that strains negative by Syva DFA consisted of several variants. The frequency of N. gonorrhoeae isolates showing negative results by Syva DFA in this patient population ranged from 0 to 11.5%/month. Laboratories using only the Syva DFA test for confirmation of N. gonorrhoeae may incur a significant risk of misidentification. PMID:8408585

  12. Proctitis associated with Neisseria cinerea misidentified as Neisseria gonorrhoeae in a child.

    PubMed

    Dossett, J H; Appelbaum, P C; Knapp, J S; Totten, P A

    1985-04-01

    An 8-year-old boy developed proctitis. Rectal swabs yielded a Neisseria sp. that was repeatedly identified by API (Analytab Products, Plainview, N.Y.), Minitek (BBL Microbiology Systems, Cockeysville, Md.), and Bactec (Johnston Laboratories, Towson, Md.) methods as Neisseria gonorrhoeae. Subsequent testing in a reference laboratory yielded an identification of Neisseria cinerea. It is suggested that identification of a Neisseria sp. isolated from genital or rectal sites in a child be confirmed by additional serological, growth, and antibiotic susceptibility tests and, if necessary, by a reference laboratory. The implications of such misidentifications are discussed.

  13. Interference by Neisseria gonorrhoeae growth by other bacterial species.

    PubMed Central

    Kraus, S J; Geller, R C; Perkins, G H; Rhoden, D L

    1976-01-01

    Growth of Neisseria gonorrhoeae from clinical specimens has been enhanced by the use of selective media that inhibit the simultaneous growth of other microorganisms. One explanation for this enhancement could be that certain other bacteria inhibit gonococcal growth. This hypothesis was examined by testing 167 bacterial isolates for in vitro gonococcal inhibition; 34.1% of the isolates failed to inhibit the gonococcus, but 12.0% produced weak inhibition and 53.9% strongly inhibited N. gonorrhoeae. The pattern of in vitro gonococcal inhibition was consistently the same for all the individual isolates within some species, but individual isolates within other bacterial species varied in their ability to inhibit the gonococcus. Consistently strong in vitro N. gonorrhoeae inhibitors were Citrobacter diversus, Enterobacter cloacae, Serratia marcescens, and Pseudomonas. The in vivo significance of gonococcal interference was demonstrated in the subcutaneous chamber model of N. gonorrhoeae infection. Images PMID:823175

  14. Molecular Typing of Neisseria gonorrhoeae Isolates by Opa-Typing and Ribotyping in New Delhi, India.

    PubMed

    Khaki, Pejvak; Bhalla, Preena; Fayaz, Ahmad Mir; Moradi Bidhendi, Sohiela; Esmailzadeh, Majid; Sharma, Pawan

    2009-01-01

    Control and preventive measures for gonococcal infections are based on precise epidemiological characteristics of N. gonorrhoeae isolates. In the present study the potential utility of opa-typing and ribotyping for molecular epidemiological study of consecutive gonococcal strains was determined. Sixty gonococcal isolates were subjected to ribotyping with two restriction enzymes, AvaII and HincII, and opa-typing with TaqI and HpaII for epidemiological characterization of gonococcal population. Ribotyping with AvaII yielded 6 ribotype patterns while twelve RFLP patterns were observed with HincII. Opa-typing of the 60 isolates revealed a total 54 opa-types, which 48 were unique and 6 formed clusters. Fifty-two opa-types were observed with TaqI-digested PCR product while opa-typing with HpaII demonstrated 54 opa-types. The opa-types from isolates that were epidemiologically unrelated were distinct, whereas those from the sexual contacts were identical. The results showed that opa-typing is highly useful for characterizing gonococcal strains from sexual contacts and has more discriminatory than ribotyping that could differentiate between gonococci of the same ribotype. The technique even with a single restriction enzyme has a high level of discrimination (99.9%) between epidemiologically unrelated isolates. In conclusion, the molecular methods such as opa-typing and ribotyping can be used for epidemiological characterization of gonococcal strains.

  15. A national quality assurance survey of Neisseria gonorrhoeae testing.

    PubMed

    Trembizki, Ella; Lahra, Monica; Stevens, Kerrie; Freeman, Kevin; Hogan, Tiffany; Hogg, Geoff; Lawrence, Andrew; Limnios, Athena; Pearson, Julie; Smith, Helen; Nissen, Michael; Sloots, Theo; Whiley, David

    2014-01-01

    The aims of this study were to (1) conduct a national survey of Neisseria gonorrhoeae identification by National Neisseria Network (NNN) reference laboratories contributing data to the Australian Gonococcal Surveillance Programme and (2) determine the prevalence in Australia of strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays for confirmation of gonococcal nucleic acid amplification tests. Gonococcal clinical isolates referred to NNN laboratories for the first half of 2012 were screened using in-house real-time PCR assays targeting multicopy opa, porA pseudogene and cppB genes. There were 2455 clinical gonococcal isolates received in the study period; 98.6 % (2420/2455) of isolates harboured all three gene targets, 0.12 % (3/2455) were porA-negative, 0.04 % (1/2455) opa-negative and 1.14 % (28/2455) cppB-negative by PCR. Notably, no isolates were simultaneously negative for two targets. However, three isolates failed to be amplified by all three PCR methods, one isolate of which was shown to be a commensal Neisseria strain by 16S rRNA sequencing. Using PCR as the reference standard the results showed that (1) identification of N. gonorrhoeae isolates by NNN laboratories was highly specific (99.96 %) and (2) strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays are present but not widespread throughout Australia at this point in time.

  16. Whole-Genome Phylogenomic Heterogeneity of Neisseria gonorrhoeae Isolates with Decreased Cephalosporin Susceptibility Collected in Canada between 1989 and 2013

    PubMed Central

    Lynch, Tarah; Martin, Irene; Van Domselaar, Gary; Graham, Morag; Bharat, Amrita; Allen, Vanessa; Hoang, Linda; Lefebvre, Brigitte; Tyrrell, Greg; Horsman, Greg; Haldane, David; Garceau, Richard; Wylie, John; Wong, Tom; Mulvey, Michael R.

    2014-01-01

    A large-scale, whole-genome comparison of Canadian Neisseria gonorrhoeae isolates with high-level cephalosporin MICs was used to demonstrate a genomic epidemiology approach to investigate strain relatedness and dynamics. Although current typing methods have been very successful in tracing short-chain transmission of gonorrheal disease, investigating the temporal evolutionary relationships and geographical dissemination of highly clonal lineages requires enhanced resolution only available through whole-genome sequencing (WGS). Phylogenomic cluster analysis grouped 169 Canadian strains into 12 distinct clades. While some N. gonorrhoeae multiantigen sequence types (NG-MAST) agreed with specific phylogenomic clades or subclades, other sequence types (ST) and closely related groups of ST were widely distributed among clades. Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) emerged among a group of diverse strains in Canada during the 1990s with a variety of nonmosaic penA alleles, followed in 2000/2001 with the penA mosaic X allele and then in 2007 with ST1407 strains with the penA mosaic XXXIV allele. Five genetically distinct ESC-DS lineages were associated with penA mosaic X, XXXV, and XXXIV alleles and nonmosaic XII and XIII alleles. ESC-DS with coresistance to azithromycin was observed in 5 strains with 23S rRNA C2599T or A2143G mutations. As the costs associated with WGS decline and analysis tools are streamlined, WGS can provide a more thorough understanding of strain dynamics, facilitate epidemiological studies to better resolve social networks, and improve surveillance to optimize treatment for gonorrheal infections. PMID:25378573

  17. Plasmid-mediated resistance to tetracyclines among Neisseria gonorrhoeae strains isolated in Poland between 2012 and 2013

    PubMed Central

    Młynarczyk-Bonikowska, Beata; Kujawa, Marlena; Malejczyk, Magdalena; Młynarczyk, Grażyna

    2016-01-01

    Introduction One of two main mechanisms of resistance in tetracycline-resistant Neisseria gonorrhoeae (TRNG) is associated with the presence of TetM protein responsible for actively blocking of the tetracycline target site in the 30S ribosomal subunit. This mechanism is encoded by conjugative plasmids. The second mechanism is chromosomal in nature and due to mutations in specific genes. Aim To determine the incidence and type of tetM determinants in TRNG strains isolated from patients presenting with gonorrhea infection to the Dermatology and Venereology Clinic in Warsaw in 2012–2013. Material and methods Tetracycline and doxycycline susceptibility was determined by E-Tests. The presence and type of the tetM gene were determined by polymerase chain reaction. Results Tetracycline resistance was detected in 50.8% of the evaluated strains. The TRNG strains containing the tetM plasmid constituted 13.8% of all the evaluated strains. Dutch type tetM constituted 12.3% and American type tetM 1.5% of all the evaluated strains. In the remaining TRNG strains, resistance to tetracyclines was presumably chromosome-encoded. The minimal inhibitory concentration (MIC) of tetracycline ranged from 0.25 to 32.0 mg/l, MIC50 = 2.0 mg/l, MIC90 = 32.0 mg/l. The MIC of doxycycline ranged from 0.25 to 32.0 mg/l, MIC50 = 4.0 mg/l, MIC90 = 16.0 mg/l. Conclusions Unlike most of European countries, in 2012–2013 in Poland, the Dutch type tetM was found to be much more common than the American type. Minimal inhibitory concentration values of tetracycline and doxycycline were similar, with doxycycline exhibiting a somewhat lower effectiveness in vitro than tetracycline towards chromosome-mediated tetracycline resistant strains of N. gonorrhoeae. PMID:28035227

  18. Conjugation of plasmids of Neisseria gonorrhoeae to other Neisseria species: potential reservoirs for the beta-lactamase plasmid.

    PubMed

    Genco, C A; Knapp, J S; Clark, V L

    1984-09-01

    The discovery that penicillinase production in Neisseria gonorrhoeae was plasmid mediated and the spread of the beta-lactamase encoding plasmids in gonococcal isolates since 1976, raise the possibility that a nonpathogenic indigenous bacterium could serve as a reservoir for these plasmids. We initiated studies to define the ability of commensal Neisseria species and Branhamella catarrhalis strains, as well as strains of the pathogen Neisseria meningitidis, to serve as recipients in conjugation with Neisseria gonorrhoeae. We found that with N. gonorrhoeae as the donor, 3 of 5 Neisseria cinerea, 2 of 5 Neisseria flava, 0 of 1 Neisseria flavescens, 1 of 3 Neisseria subflava, 0 of 6 B. catarrhalis, 0 of 7 Neisseria lactamica, 1 of 5 Neisseria mucosa, 1 of 7 Neisseria perflava/sicca, and 0 of 13 N. meningitidis strains gave detectable conjugation frequencies (greater than 10(-8). N. cinerea was the only species found to maintain the gonococcal conjugal plasmid (pLE2451). A N. cinerea transconjugant containing pLE2451 was observed to transfer both the beta-lactamase plasmid and pLE2451 to N. gonorrhoeae at high frequency.

  19. Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone.

    PubMed

    Osaka, Kazuyoshi; Takakura, Tadakazu; Narukawa, Kayo; Takahata, Masahiro; Endo, Katsuhisa; Kiyota, Hiroshi; Onodera, Shoichi

    2008-06-01

    Neisseria gonorrhoeae strains with reduced susceptibility to cefixime and ceftriaxone, with minimum inhibitory concentrations (MICs) of cefixime of 0.125-0.25 microg/ml and ceftriaxone of 0.031-0.125 microg/ml, were isolated from male urethritis patients in Tokyo, Japan, in 2006. The amino acid sequences of PenA, penicillin-binding protein 2, in these strains were of two types: PenA mosaic and nonmosaic strains. In the PenA mosaic strain, some regions in the transpeptidase-encoding domain in PenA were similar to those of Neisseria perflava/sicca, Neisseria cinerea, Neisseria flavescens, Neisseria polysaccharea, and Neisseria meningitidis. In the PenA nonmosaic strain, there was a mutation of Ala-501 to Val in PenA. In addition, we performed homology modeling of PenA wild-type and mosaic strains and compared them. The results of the modeling studies suggested that reduced susceptibility to cephems such as cefixime and ceftriaxone is due to a conformational alteration of the beta-lactam-binding pocket. These results also indicated that the mosaic structures and the above point mutation in PenA make a major contribution to the reduced susceptibility to cephem antibiotics.

  20. [Ciprofloxacin resistance of Neisseria gonorrhoeae according to sexual habits].

    PubMed

    García, Susana; Casco, Ricardo; Perazzi, Beatriz; De Mier, Carmen; Vay, Carlos; Famiglietti, Angela

    2008-01-01

    The first isolates of Neisseria gonorrhoeae resistant to fluoroquinolones in Argentina were reported in 2000. Since January 2005 to June 2007 Neisseria gonorrhoeae was studied in 595 men who have sex with men (MSM) and 571 heterosexual men. The gonorrhea prevalence in MSM and heterosexual men was 0.091(91/1000) and the Neisseria gonorrhoeae ciprofloxacin resistant (CRNG) was 20% in MSM and 3.8% in heterosexual men (p: 0.0416). Thirteen out of 106 isolates from 11 MSM and 2 heterosexual men were CRNG. Six out of eleven MSM had urethritis, one also carried Neisseria gonorrhoeae in rectum and 5 patients were asymptomatic carriers (rectum 2, pharynx 2, urethra 1). No epidemiological relation was found among the patients. Two heterosexual men had urethritis. The 8 symptomatic men were treated with ciprofloxacin but treatment failed in all of them. These patients and the asymptomatic ones were treated with ceftriaxone, 500 mg IM. The post treatment microbiological controls were negative. The CRNG isolates had ciprofloxacin MIC between 2 and 32 (microg/ml), all were negative to penicillinase, 4 out of 13 were chromosomally resistant to penicillin (MIC: 1 microg/ml). The MICs (microg/ml) ranges for several antimicrobial agents were: penicillin: 0.016-1; tetracycline: 0.125-2; ceftriaxone: 0.004-0.008; erythromycin: 0.032-2; azithromycin: 0.032-0.5; spectinomycin: 8-32. Due to the high level of ciprofloxacin-resistant N. gonorrhoeae isolated from MSM in our hospital, another antimicrobial agent for empirical therapy should be used in these patients.

  1. Gonococcal Antimicrobial Susceptibility and the Prevalence of blaTEM-1 and blaTEM-135 Genes in Neisseria gonorrhoeae Isolates from Thailand.

    PubMed

    Tribuddharat, Chanwit; Pongpech, Pintip; Charoenwatanachokchai, Angkana; Lokpichart, Somchai; Srifuengfung, Somporn; Sonprasert, Suthatta

    2017-03-24

    We studied the antimicrobial susceptibility and prevalence of the blaTEM-1 and blaTEM-135 genes in Neisseria gonorrhoeae isolates obtained in Thailand. The isolates were tested using the disk diffusion method, and 100% of 370 isolates were found susceptible to cefixime, ceftriaxone, cefotaxime, ceftazidime, cefepime, spectinomycin, and azithromycin. Some of the isolates were resistant to penicillin (85.7%), ciprofloxacin (88.0%), ofloxacin (97.4%), or tetracycline (89.1%). Penicillinase-producing N. gonorrhoeae accounted for 83.8% of isolates, with 70.0% of these further identified as penicillinase-producing plus tetracycline resistant N. gonorrhoeae. Penicillin, tetracycline, and ciprofloxacin are not recommended for treatment because of the high prevalence (89.7%) of multidrug resistant gonococci. A study of genes controlling enzyme of beta-lactamase production (blaTEM-1 and blaTEM-135) was performed using mismatch amplification mutation assay PCR method and DNA sequencing. Beta-lactamase positive N. gonorrhoeae carried blaTEM-1 (69.6%) and blaTEM-135 (30.4%), indicating that there is a significant increase and spread of blaTEM-135 among gonococci in Thailand.

  2. 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

  3. The prevalence and epidemiology of plasmid-mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates in Guangzhou, China, 2002-2012.

    PubMed

    Zheng, Heping; Wu, Xingzhong; Huang, Jinmei; Qin, Xiaolin; Xue, Yaohua; Zeng, Weiying; Lan, Yinyuan; Ou, Jiangli; Tang, Sanmei; Fang, Mingheng

    2015-10-09

    Gonococcal antimicrobial resistance is a global problem. Different resistance plasmids have emerged and spread among the isolates of Neisseria gonorrhoeae worldwide and in China. We conducted this study to monitor the plasmid-mediated penicillin and tetracycline resistance among N. gonorrhoeae isolates in Guangzhou from 2002 to 2012. Consecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending the STD clinic in Guangdong Provincial Centre for Skin Diseases and STIs Control and Prevention. Penicillinase-producing N. gonorrhoeae (PPNG) isolates were analyzed by the paper acidometric method. Plasmid-mediated resistance to tetracycline in N. gonorrhoeae (TRNG) isolates was screened by the agar plate dilution method. Plasmid types were determined for TRNG and PPNG isolates using polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) to penicillin and tetracycline were detected by the agar plate dilution. Of 1378 consecutive N. gonorrhoeae isolates, 429 PPNG and 639 TRNG isolates were identified. The prevalence of PPNG, TRNG, and PPNG/TRNG increased from 18.3 to 47.1 % (χ (2) = 31.57, p < 0.001), from 29.4 to 52.1 % (χ (2) = 16.28, p < 0.001) and from 10.0 to 26.2 % (χ (2) = 10.46, p < 0.001) between 2002 and 2012, respectively. Genotyping of plasmids among PPNGs showed that the majority (93.7 %) of the isolates were the Asian type plasmids, while the African type plasmid emerged in 2008 and rapidly increased to 14.0 % in 2012 (χ (2) = 25.03, p < 0.001). For TRNGs, all 639 isolates carried the Dutch type plasmid. MICs of penicillin G and tetracycline persisted at high levels and the MIC90s were 32-fold higher than the resistant cutoff point over 11 years. The prevalence rates of penicillin- and tetracycline-resistant N. gonorrhoeae varied from 90.9 to 91.1 % and from 88.3 to 89.3 % during 2002 to 2012, respectively. Resistance to penicillin and tetracycline among N. gonorrhoeae

  4. Worldwide Susceptibility Rates of Neisseria gonorrhoeae Isolates to Cefixime and Cefpodoxime: A Systematic Review and Meta-Analysis

    PubMed Central

    Yu, Rui-xing; Yin, Yueping; Wang, Guan-qun; Chen, Shao-chun; Zheng, Bing-jie; Dai, Xiu-qin; Han, Yan; Li, Qi; Zhang, Guo-yi; Chen, Xiangsheng

    2014-01-01

    Background Neisseria gonorrhoeae (NG) infection is a serious public health problem. The third-generation extended-spectrum cephalosporins (ESCs) have been used as the first-line treatment for NG infection for almost three decades. However, in recent years, treatment failures with the oral third-generation ESCs have been reported worldwide. This study aimed to estimate worldwide susceptibility rates of NG to cefixime and cefpodoxime by analyzing data from all relevant published studies. Methodology/principal findings Two researchers independently searched five databases to identify studies on susceptibilities of NG to cefixime and cefpodoxime published between January 1, 1984 and October 15, 2012. A fixed-effect model was used to perform group analysis, and a χ2 test was employed to make subgroup comparison. Publication bias was assessed with the Begg rank correlation test. The pooled susceptibility rate of NG isolates to cefixime was 99.8% (95% CI: 99.7%–99.8%). The cefixime susceptibility rate of NG isolates from men was significantly lower than that from patients without information of gender or from men and women; the susceptibility rate of NG isolates from Asia was significantly lower than that from other continents; and the susceptibility rate of NG isolates collected before or during 2003 was significantly higher than that after 2003. The pooled susceptibility rate of NG isolates to cefpodoxime was 92.8% (95% CI: 89.0%–95.3%), which was lower than that to cefixime (92.8% vs. 99.8%, χ2 = 951.809, P<0.01). Conclusions The susceptibility rate of NG isolates to cefixime varied with the gender of patients and geographical location from which NG isolates were collected, and declined with time. The reported lower susceptibility rate of NG isolates to cefixime and associated treatment failures, as well as the emergence of NG strains with cephalosporin resistance call for the more effective control of NG infection and the development of new antibiotics. PMID

  5. Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany.

    PubMed

    Horn, Nicole Nari; Kresken, Michael; Körber-Irrgang, Barbara; Göttig, Stephan; Wichelhaus, Cornelia; Wichelhaus, Thomas A

    2014-07-01

    Antimicrobial drug resistance in Neisseria gonorrhoeae has become an increasing public health problem. Hence, surveillance of resistance development is of crucial importance to implement adequate treatment guidelines. Data on the spread of antibiotic resistance among gonococcal isolates in Germany, however, is scarce. In a resistance surveillance study conducted by the Paul Ehrlich Society for Chemotherapy between October 2010 and December 2011, 23 laboratories all over Germany were requested to send N. gonorrhoeae isolates to the study laboratory in Frankfurt am Main. Species verification was performed biochemically using ApiNH and with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Etest method. For molecular epidemiological analysis, N. gonorrhoeae strains were genotyped by means of N. gonorrhoeae multi-antigen sequence typing. A total of 213 consecutive gonococcal isolates were analyzed in this nationwide study. Applying EUCAST breakpoints, high resistance rates were found for ciprofloxacin (74%) and tetracycline (41%). Penicillin non-susceptibility was detected in 80% of isolates. The rate of azithromycin resistance was 6%, while all strains were susceptible to spectinomycin, cefixime, and ceftriaxone. Molecular typing of gonococcal isolates revealed a great heterogeneity of 99 different sequence types (ST), but ST1407 predominated (n=39). This is the first comprehensive German multi-centre surveillance study on antibiotic susceptibility and molecular epidemiology of N. gonorrhoeae with implications for antibiotic choice for treatment of gonorrhoea. The World Health Organization supports the concept that an efficacious treatment of gonorrhoea results in at least 95% of infections being cured. Accordingly, as spectinomycin is not available on the German market, only the third generation cephalosporins cefixime and ceftriaxone are regarded as valuable drugs

  6. Antimicrobial resistance of Neisseria gonorrhoeae isolates in south-west Germany, 2004 to 2015: increasing minimal inhibitory concentrations of tetracycline but no resistance to third-generation cephalosporins

    PubMed Central

    Regnath, Thomas; Mertes, Thomas; Ignatius, Ralf

    2016-01-01

    Increasing antimicrobial resistance of Neisseria gonorrhoeae, particularly to third-generation cephalosporins, has been reported in many countries. We examined the susceptibility (determined by Etest and evaluated using the breakpoints of the European Committee on Antimicrobial Susceptibility Testing) of 434 N. gonorrhoeae isolates collected from 107 female and 327 male patients in Stuttgart, south-west Germany, between 2004 and 2015. During the study period, high proportions of isolates were resistant to ciprofloxacin (70.3%), tetracycline (48.4%; increasing from 27.5% in 2004/2005 to 57.7% in 2014/2015; p = 0.0002) and penicillin (25.6%). The proportion of isolates resistant to azithromycin was low (5.5%) but tended to increase (p = 0.08). No resistance and stable minimum inhibitory concentrations were found for cefixime, ceftriaxone, and spectinomycin. High-level resistance was found for ciprofloxacin (39.6%) and tetracycline (20.0%) but not for azithromycin; 16.3% of the isolates produced betalactamase. Thus, cephalosporins can still be used for the treatment of gonorrhoea in the study area. To avoid further increasing resistance to azithromycin, its usage should be limited to patients allergic to cephalosporins, or (in combination with cephalosporins) to patients for whom no susceptibility testing could be performed or those co-infected with chlamydiae. PMID:27632642

  7. Lipids of Branhamella catarrhalis and Neisseria gonorrhoeae.

    PubMed

    Beebe, J L; Wlodkowski, T J

    1976-07-01

    Three strains of Branhamella catarrhalis and three strains of Neisseria gonorrhoeae were analyzed with regard to their phospholipid and neutral lipid composition. B. catarrhalis (ATCC 23246) contained 5.12 +/- 0.34% lipid, determined gravimetrically, compared to 8.56 +/- 0.15% and 9.73 +/- 0.06% for two strains of N. gonorrhoeae. Cardiolipin, phosphatidylglycerol, and phosphatidyl-ethanolamine were identified in extracts of both species. In addition, B. catarrhalis contained small amounts of phosphatidylcholine, and N. gonorrhoeae contained small amounts of lyso-phosphatidylethanolamine, which accumulated with autolysis accompanying late cell culture growth. The kinetics of change of relative amounts of phospholipids in both species were measured and found to differ substantially. Neutral lipid accounted for 30.4% of the total lipid of B. catarrhalis (ATCC 23246) and 7.6% of the total lipid of N. gonorrhoeae NYH 002. Hydrocarbons, triglycerides, free fatty acids, coenzyme Q, diglycerides, and free hydroxy fatty acids were identified in the neutral lipid fraction of both species. The three strains of N. gonorrhoeae, sensitive, intermediate, and resistant to penicillin, exhibited no significant difference in the composition or metabolism of phospholipid.

  8. Lipids of Branhamella catarrhalis and Neisseria gonorrhoeae.

    PubMed Central

    Beebe, J L; Wlodkowski, T J

    1976-01-01

    Three strains of Branhamella catarrhalis and three strains of Neisseria gonorrhoeae were analyzed with regard to their phospholipid and neutral lipid composition. B. catarrhalis (ATCC 23246) contained 5.12 +/- 0.34% lipid, determined gravimetrically, compared to 8.56 +/- 0.15% and 9.73 +/- 0.06% for two strains of N. gonorrhoeae. Cardiolipin, phosphatidylglycerol, and phosphatidyl-ethanolamine were identified in extracts of both species. In addition, B. catarrhalis contained small amounts of phosphatidylcholine, and N. gonorrhoeae contained small amounts of lyso-phosphatidylethanolamine, which accumulated with autolysis accompanying late cell culture growth. The kinetics of change of relative amounts of phospholipids in both species were measured and found to differ substantially. Neutral lipid accounted for 30.4% of the total lipid of B. catarrhalis (ATCC 23246) and 7.6% of the total lipid of N. gonorrhoeae NYH 002. Hydrocarbons, triglycerides, free fatty acids, coenzyme Q, diglycerides, and free hydroxy fatty acids were identified in the neutral lipid fraction of both species. The three strains of N. gonorrhoeae, sensitive, intermediate, and resistant to penicillin, exhibited no significant difference in the composition or metabolism of phospholipid. Images PMID:819418

  9. Antimicrobial Susceptibilities and Plasmid Contents of Neisseria gonorrhoeae Isolates from Commercial Sex Workers in Dhaka, Bangladesh: Emergence of High-Level Resistance to Ciprofloxacin

    PubMed Central

    Bhuiyan, Bahar Uddin; Rahman, Motiur; Miah, Mohammed Ruhul Amin; Nahar, Shamsun; Islam, Nazrul; Ahmed, Monira; Rahman, Kazi Masihur; Albert, M. John

    1999-01-01

    Commercial sex workers (CSWs) serve as the most important reservoir of sexually transmitted diseases (STD), including gonorrhea. Periodic monitoring of the antimicrobial susceptibility profile of Neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. A study concerning the prevalence of gonococcal infection among CSWs was conducted in Bangladesh. The isolates were examined with regards to their antimicrobial susceptibility to, and the MICs of, penicillin, tetracycline, ciprofloxacin, cefuroxime, ceftriaxone, and spectinomycin by disk diffusion and agar dilution methods. The total plasmid profile of the isolates was also analyzed. Of the 224 CSWs, 94 (42%) were culture positive for N. gonorrhoeae. There was a good correlation between the results of the disk diffusion and agar dilution methods. Some 66% of the isolates were resistant to penicillin, and 34% were moderately susceptible to penicillin. Among the resistant isolates, 23.4% were penicillinase-producing N. gonorrhoeae (PPNG). 60.6% of the isolates were resistant and 38.3% were moderately susceptible to tetracycline, 17.5% were tetracycline-resistant N. gonorrhoeae, 11.7% were resistant and 26.6% had reduced susceptibility to ciprofloxacin, 2.1% were resistant and 11.7% had reduced susceptibility to cefuroxime, and 1% were resistant to ceftriaxone. All PPNG isolates contained a 3.2-MDa African type of plasmid, and a 24.2-MDa conjugative plasmid was present in 34.1% of the isolates. Since quinolones such as ciprofloxacin are recommended as the first line of therapy for gonorrhea, the emergence of significant resistance to ciprofloxacin will limit the usefulness of this drug for treatment of gonorrhea in Bangladesh. PMID:10074537

  10. Nucleotide sequence composition and method for detection of neisseria gonorrhoeae

    SciTech Connect

    Lo, A.; Yang, H.L.

    1990-02-13

    This patent describes a composition of matter that is specific for {ital Neisseria gonorrhoeae}. It comprises: at least one nucleotide sequence for which the ratio of the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria gonorrhoeae} to the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria meningitidis} is greater than about five. The ratio being obtained by a method described.

  11. Drug-resistant Neisseria gonorrhoeae: latest developments.

    PubMed

    Suay-García, B; Pérez-Gracia, M T

    2017-07-01

    Gonorrhea is the second most frequently reported notifiable disease in the United States and is becoming increasingly common in Europe. The purpose of this review was to assess the current state of drug-resistant Neisseria gonorrhoeae in order to evaluate future prospects for its treatment. An exhaustive literature search was conducted to include the latest research regarding drug resistance and treatment guidelines for gonorrhea. Gonococci have acquired all known resistance mechanisms to all antimicrobials used for treatment. Currently, the European Union, the United States, and the United Kingdom have established surveillance programs to assess, on a yearly basis, the development of gonococcal resistance. Current treatment guidelines are being threatened by the increasing number of ceftriaxone-, cefixime-, and azithromycin-resistant N. gonorrhoeae strains being detected worldwide. This has led the scientific community to develop new treatment options with new molecules in order to persevere in the battle against this "superbug".

  12. A Case-Control Study of Molecular Epidemiology in Relation to Azithromycin Resistance in Neisseria gonorrhoeae Isolates Collected in Amsterdam, the Netherlands, between 2008 and 2015.

    PubMed

    Wind, Carolien M; Bruisten, Sylvia M; Schim van der Loeff, Maarten F; Dierdorp, Mirjam; de Vries, Henry J C; van Dam, Alje P

    2017-06-01

    Neisseria gonorrhoeae resistance to ceftriaxone and azithromycin is increasing, which threatens the recommended dual therapy. We used molecular epidemiology to identify N. gonorrhoeae clusters and associations with azithromycin resistance in Amsterdam, the Netherlands. N. gonorrhoeae isolates (n = 143) were selected from patients visiting the Amsterdam STI Outpatient Clinic from January 2008 through September 2015. We included all 69 azithromycin-resistant isolates (MIC ≥ 2.0 mg/liter) and 74 frequency-matched susceptible controls (MIC ≤ 0.25 mg/liter). The methods used were 23S rRNA and mtrR sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), N. gonorrhoeae multilocus variable-number tandem-repeat analysis (NG-MLVA), and a specific PCR to detect mosaic penA genes. A hierarchical cluster analysis of NG-MLVA related to resistance and epidemiological characteristics was performed. Azithromycin-resistant isolates had C2611T mutations in 23S rRNA (n = 62, 89.9%, P < 0.001) and were NG-MAST genogroup G2992 (P < 0.001), G5108 (P < 0.001), or G359 (P = 0.02) significantly more often than susceptible isolates and were more often part of NG-MLVA clusters (P < 0.001). Two resistant isolates (2.9%) had A2059G mutations, and five (7.3%) had wild-type 23S rRNA. No association between mtrR mutations and azithromycin resistance was found. Twenty-four isolates, including 10 azithromycin-resistant isolates, showed reduced susceptibility to extended-spectrum cephalosporins. Of these, five contained a penA mosaic gene. Four of the five NG-MLVA clusters contained resistant and susceptible isolates. Two clusters consisting mainly of resistant isolates included strains from men who have sex with men and from heterosexual males and females. The co-occurrence of resistant and susceptible strains in NG-MLVA clusters and the frequent occurrence of resistant strains outside of clusters suggest that azithromycin resistance develops independently from the background genome

  13. In Vitro Activity of Gepotidacin (GSK2140944) against Neisseria gonorrhoeae

    PubMed Central

    Farrell, D. J.; Sader, H. S.; Rhomberg, P. R.; Scangarella-Oman, N. E.

    2017-01-01

    ABSTRACT Gepotidacin (formerly GSK2140944) is a novel, first-in-class, triazaacenaphthylene antibacterial that inhibits bacterial DNA gyrase and topoisomerase IV via a unique mechanism and has demonstrated in vitro activity against Neisseria gonorrhoeae, including drug-resistant strains, and also targets pathogens associated with other conventional and biothreat infections. Broth microdilution was used to evaluate the MIC and minimum bactericidal concentration (MBC) activity of gepotidacin and comparators against 25 N. gonorrhoeae strains (including five ciprofloxacin-nonsusceptible strains). Gepotidacin activity was also evaluated against three N. gonorrhoeae strains (including a ciprofloxacin-nonsusceptible strain) for resistance development, against three N. gonorrhoeae strains (including two tetracycline- and azithromycin-nonsusceptible strains) using time-kill kinetics and checkerboard methods, and against two N. gonorrhoeae strains for the investigation of postantibiotic (PAE) and subinhibitory (PAE-SME) effects. The MIC50 and MIC90 for gepotidacin against the 25 N. gonorrhoeae isolates tested were 0.12 and 0.25 μg/ml, respectively. The MBC50 and MBC90 for gepotidacin were 0.25 and 0.5 μg/ml, respectively. Gepotidacin was bactericidal, and single-step resistance selection studies did not recover any mutants, indicating a low rate of spontaneous single-step resistance. For combinations of gepotidacin and comparators tested using checkerboard methods, there were no instances where antagonism occurred and only one instance of synergy (with moxifloxacin; fractional inhibitory concentration, 0.375). This was not confirmed by in vitro time-kill studies. The PAE for gepotidacin against the wild-type strain ranged from 0.5 to >2.5 h, and the PAE-SME was >2.5 h. These in vitro data indicate that further study of gepotidacin is warranted for potential use in treating infections caused by N. gonorrhoeae. PMID:28069643

  14. Molecular analysis of antimicrobial resistance mechanisms in Neisseria gonorrhoeae isolates from Ontario, Canada.

    PubMed

    Allen, Vanessa G; Farrell, David J; Rebbapragada, Anuradha; Tan, Jingyuan; Tijet, Nathalie; Perusini, Stephen J; Towns, Lynn; Lo, Stephen; Low, Donald E; Melano, Roberto G

    2011-02-01

    Surveillance of gonococcal antimicrobial resistance and the molecular characterization of the mechanisms underlying these resistance phenotypes are essential in order to establish correct empirical therapies, as well as to describe the emergence of new mechanisms in local bacterial populations. To address these goals, 149 isolates were collected over a 1-month period (October-November 2008) at the Ontario Public Health Laboratory, Toronto, Canada, and susceptibility profiles (8 antibiotics) were examined. Mutations in previously identified targets or the presence of some enzymes related to resistance (r), nonsusceptibility (ns) (resistant plus intermediate categories), or reduced susceptibility (rs) to the antibiotics tested were also studied. A significant proportion of nonsusceptibility to penicillin (PEN) (89.2%), tetracycline (TET) (72.3%), ciprofloxacin (CIP) (29%), and macrolides (erythromycin [ERY] and azithromycin; 22.3%) was found in these strains. Multidrug resistance was observed in 18.8% of the collection. Although all the strains were susceptible to spectinomycin and extended-spectrum cephalosporins (ESC) (ceftriaxone and cefixime), 9.4% of them displayed reduced susceptibility to extended-spectrum cephalosporins. PBP 2 mosaic structures were found in all of these ESC(rs) isolates. Alterations in the mtrR promoter, MtrR repressor (TET(r), PEN(ns), ESC(rs), and ERY(ns)), porin PIB (TET(r) and PEN(ns)), and ribosomal protein S10 (TET(r)) and double mutations in gyrA and parC quinolone resistance-determining regions (QRDRs) (CIP(r)) were associated with and presumably responsible for the resistance phenotypes observed. This is the first description of ESC(rs) in Canada. The detection of this phenotype indicates a change in the epidemiology of this resistance and highlights the importance of continued surveillance to preserve the last antimicrobial options available.

  15. Purification and characterization of DNA methyltransferases from Neisseria gonorrhoeae.

    PubMed Central

    Piekarowicz, A; Yuan, R; Stein, D C

    1988-01-01

    Three DNA methyltransferases, M.NgoAI, and M.NgoBI and M.NgoBII, free of any nuclease activities were isolated from Neisseria gonorrhoeae strains WR220 and MUG116 respectively. M.NgoAI recognizes the sequence 5' GGCC 3' and methylates the first 5' cytosine on both strands. M.NgoBI and M.NgoBII recognize 5' TCACC 3' and 5' GTAN5CTC 3' respectively. M.NgoBII methylates cytosine on only one strand to produce 5' GTAN5mCTC 3'. Images PMID:3135534

  16. [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.

  17. High in vitro susceptibility to the novel spiropyrimidinetrione ETX0914 (AZD0914) among 873 contemporary clinical Neisseria gonorrhoeae isolates from 21 European countries from 2012 to 2014.

    PubMed

    Unemo, Magnus; Ringlander, Johan; Wiggins, Catherine; Fredlund, Hans; Jacobsson, Susanne; Cole, Michelle

    2015-09-01

    Resistance in Neisseria gonorrhoeae against all antimicrobials available for the treatment of gonorrhea has emerged. The first gonococcal strains with high-level resistance to ceftriaxone, the last option for first-line empirical antimicrobial monotherapy, were recently described. Consequently, new treatment options are essential. In this study, the in vitro activity of the novel spiropyrimidinetrione ETX0914 (AZD0914), a DNA topoisomerase II inhibitor, was investigated among contemporary consecutive clinical N. gonorrhoeae isolates obtained in 21 European countries and compared to the activities of antimicrobials currently or previously recommended for treatment. Consecutive clinical N. gonorrhoeae isolates (n = 873) cultured in 21 European countries from 2012 to 2014 were examined for their susceptibility to ETX0914. The MICs of ETX0914 were determined using the agar dilution method. For comparison, the MICs of ceftriaxone, cefixime, azithromycin, and ciprofloxacin were determined using Etest or the agar dilution method. For ETX0914, the MIC range, modal MIC, MIC50, and MIC90 were ≤0.002 to 0.25 mg/liter, 0.125 mg/liter, 0.064 mg/liter, and 0.125 mg/liter, respectively. The MIC values were substantially lower than those of the fluoroquinolone ciprofloxacin and most other antimicrobials examined. No cross-resistance with any other examined antimicrobial was observed. In conclusion, the in vitro susceptibility to the novel spiropyrimidinetrione ETX0914 (AZD0914) among 873 contemporary clinical isolates from 21 European countries was high, and no cross-resistance to antimicrobials currently or previously used for gonorrhea treatment was indicated. Additional studies investigating the in vitro and in vivo induction and mechanisms of ETX0914 resistance in gonococci, pharmacokinetics/pharmacodynamics in modeling/simulations and in humans, and performance in randomized controlled gonorrhea treatment trials are essential.

  18. High In Vitro Susceptibility to the Novel Spiropyrimidinetrione ETX0914 (AZD0914) among 873 Contemporary Clinical Neisseria gonorrhoeae Isolates from 21 European Countries from 2012 to 2014

    PubMed Central

    Ringlander, Johan; Wiggins, Catherine; Fredlund, Hans; Jacobsson, Susanne; Cole, Michelle

    2015-01-01

    Resistance in Neisseria gonorrhoeae against all antimicrobials available for the treatment of gonorrhea has emerged. The first gonococcal strains with high-level resistance to ceftriaxone, the last option for first-line empirical antimicrobial monotherapy, were recently described. Consequently, new treatment options are essential. In this study, the in vitro activity of the novel spiropyrimidinetrione ETX0914 (AZD0914), a DNA topoisomerase II inhibitor, was investigated among contemporary consecutive clinical N. gonorrhoeae isolates obtained in 21 European countries and compared to the activities of antimicrobials currently or previously recommended for treatment. Consecutive clinical N. gonorrhoeae isolates (n = 873) cultured in 21 European countries from 2012 to 2014 were examined for their susceptibility to ETX0914. The MICs of ETX0914 were determined using the agar dilution method. For comparison, the MICs of ceftriaxone, cefixime, azithromycin, and ciprofloxacin were determined using Etest or the agar dilution method. For ETX0914, the MIC range, modal MIC, MIC50, and MIC90 were ≤0.002 to 0.25 mg/liter, 0.125 mg/liter, 0.064 mg/liter, and 0.125 mg/liter, respectively. The MIC values were substantially lower than those of the fluoroquinolone ciprofloxacin and most other antimicrobials examined. No cross-resistance with any other examined antimicrobial was observed. In conclusion, the in vitro susceptibility to the novel spiropyrimidinetrione ETX0914 (AZD0914) among 873 contemporary clinical isolates from 21 European countries was high, and no cross-resistance to antimicrobials currently or previously used for gonorrhea treatment was indicated. Additional studies investigating the in vitro and in vivo induction and mechanisms of ETX0914 resistance in gonococci, pharmacokinetics/pharmacodynamics in modeling/simulations and in humans, and performance in randomized controlled gonorrhea treatment trials are essential. PMID:26077246

  19. Susceptibility to ceftriaxone and occurrence of penicillinase plasmids in Neisseria gonorrhoeae strains isolated in Poland in 2012-2013.

    PubMed

    Mlynarczyk-Bonikowska, Beata; Kujawa, Marlena; Mlynarczyk, Grazyna; Malejczyk, Magdalena; Majewski, Slawomir

    2016-07-01

    Recent years have seen rising concerns over increasing antibiotic resistance of the gonorrhea-causing bacterium, Neisseria gonorrhoeae. This is especially true for third-generation cephalosporins, which are currently recommended for the treatment of such infections. Therefore, susceptibility to these antibiotics should be monitored internationally to the greatest extent possible. The susceptibility of N. gonorrhoeae strains to ceftriaxone and penicillin, as well as production of beta-lactamase by the Cefinase test was determined. Moreover, the presence and type of penicillinase plasmids were determined by PCR. All strains were susceptible to ceftriaxone, the minimal inhibitory concentration (MIC) values ranged from 0.002 to 0.125 mg/L; MIC50 was =0.016 mg/L and MIC90 was =0.064 mg/L. As much as 7.7 % of the strains demonstrated ceftriaxone MIC of 0.125 mg/L. For penicillin, the MICs ranged from 0.064 to 32 mg/L; MIC50 was =0.5 mg/L and MIC90 was =4 mg/L. It was shown that only 1.5 % of the strains were sensitive to penicillin according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Among the penicillin-resistant strains, six (30.0 %) produced penicillinase. The MICs of penicillin were substantially higher for penicillinase-producing than for penicillin-resistant, penicillinase-negative strains. MICs of ceftriaxone for penicillinase-producing strains were low (0.002-0.016 mg/L). Three of the penicillinase-producing strains possessed plasmids of African type (50 %) and three Toronto/Rio type (50 %). An increase of the proportion of beta-lactamase-positive strains in the last years as well as emergence of strains with elevated MIC of ceftriaxone indicate a need to constantly monitor N. gonorrhoeae strains for their susceptibility to beta-lactam antibiotics, as well as for their ability to produce beta-lactamases.

  20. [E-test quantitative determination for evaluating Neisseria gonorrhoeae resistance].

    PubMed

    Filipiuc, Silvia; Iancu, Luminiţa Smaranda

    2010-01-01

    Thanks of underreported and of difficulties of isolation, antibiotic susceptibility profile of N. gonorrhoeae strains circulating is not sufficiently known in our country as well in the Suceava county. In addition, WHO' experts recommended the establishment of MIC (minimum inhibitory concentration) values using E-test strips, completing the database at the European level. To determine the type of resistance of N. gonorrhoeae strains by E-test in patients with gonorrhoea in Suceava county, in the 2009 -2010 period. We tested the sensitivity of 32 strains of N. gonorrhoeae isolated using classical algorithm and E-test strips according with CLSI 2008 (Clinical and Laboratory Standard Institute) standard. We tested the sensitivity for penicillin, amoxicillin, augmentin, clarytromycin, tetracycline, ceftriaxone, ciprofloxacin, and spectinomycin. Production of beta-lactamases was performed using API-NH test (Neisseria-Haemophylus-Biomerieux). 96.9% strains were sensitive for ceftriaxone and spectinomycin, each 10 strains (31.2%) were resistant for penicillin and tetracycline, 34.5% strains were sensitive for amoxicillin, 37.5% sensitive for ciprofloxacin, and 13/32 strains (40.6%) were sensitive for augmentine. 7 strains were beta-lactamases positive and sensitive to all antibiotics, excepting penicillin and tetracyclin. Our results, especially the low rate of sensitivity for penicillin and tetracycline (68.8%) were similar with other from Asia, America or Africa, including Iaşi region. Our results demonstrated for first time in the studied aria, using E-test strips, the level of resistance of N. gonorrhoeae offering useful informations for clinicians in order to treat the patients with ceftrixone and spectinomycine as empirical treatment, and for other antibiotics, according with antibiogram results.

  1. Evaluation of Six Commercial Nucleic Acid Amplification Tests for Detection of Neisseria gonorrhoeae and Other Neisseria Species▿

    PubMed Central

    Tabrizi, Sepehr N.; Unemo, Magnus; Limnios, Athena E.; Hogan, Tiffany R.; Hjelmevoll, Stig-Ove; Garland, Susanne M.; Tapsall, John

    2011-01-01

    Molecular detection of Neisseria gonorrhoeae in extragenital samples may result in false-positive results due to cross-reaction with commensal Neisseria species or Neisseria meningitidis. This study examined 450 characterized clinical culture isolates, comprising 216 N. gonorrhoeae isolates and 234 isolates of nongonococcal Neisseria species (n = 218) and 16 isolates of other closely related bacteria, with six commercial nucleic acid amplification tests (NAATs). The six NAATs tested were Gen-Probe APTIMA COMBO 2 and APTIMA GC, Roche COBAS Amplicor CT/NG and COBAS 4800 CT/NG tests, BD ProbeTec GC Qx amplified DNA assay, and Abbott RealTime CT/NG test. All assays except COBAS Amplicor CT/NG test where four (1.9%) isolates were not detected showed a positive result with all N. gonorrhoeae isolates (n = 216). Among the 234 nongonococcal isolates examined, initial results from all assays displayed some false-positive results due to cross-reactions. Specifically, the COBAS Amplicor and ProbeTec tests showed the highest number of false-positive results, detecting 33 (14.1%) and 26 (11%) nongonococcal Neisseria isolates, respectively. On the first testing, APTIMA COMBO 2, APTIMA GC, Abbott RealTime, and Roche COBAS 4800 showed lower level of cross-reactions with five (2.1%), four (1.7%), two (1%), and two (1%) of the isolates showing low-level positivity, respectively. Upon retesting of these nine nongonococcal isolates using freshly cultured colonies, none were positive by the APTIMA COMBO 2, Abbott RealTime, or COBAS 4800 test. In conclusion, the COBAS Amplicor and ProbeTec tests displayed high number of false-positive results, while the remaining NAATs showed only sporadic low-level false-positive results. Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites. PMID:21813721

  2. Role of outer-membrane proteins and lipopolysaccharide in conjugation between Neisseria gonorrhoeae and Neisseria cinerea.

    PubMed

    Genco, C A; Clark, V L

    1988-12-01

    Little is known concerning the mechanism involved in cell contact between the donor and recipient during conjugation in Neisseria gonorrhoeae. The formation of stable mating pairs during conjugation in Escherichia coli appears to require a specific protein as well as LPS in the outer membrane of the recipient cell. To attempt to identify the cell surface components necessary for conjugation in the neisseriae, we began a comparison of the outer membrane of Neisseria cinerea strains that can (Con+) and cannot (Con-) serve as recipients in conjugation with N. gonorrhoeae. There were no differences in outer-membrane protein profiles on SDS-polyacrylamide gel electrophoresis between Con+ and Con- strains that could be correlated with the ability to conjugate. However, whole outer membrane isolated from Con+ strains specifically inhibited conjugation while those from Con- strains did not. Proteolytic cleavage of outer-membrane proteins by trypsin, pronase or alpha-chymotrypsin abolished the inhibitory effect of Con+ outer membranes, suggesting that these outer membranes contained a protease-sensitive protein(s) involved in conjugation. Although periodate oxidation of Con+ outer-membrane carbohydrates did not abolish the inhibitory action of these membranes, purified LPS from both Con+ and Con- strains inhibited conjugation when added at low concentrations. These results suggest that conjugation requires the presence of a specific conjugal receptor that consists of both LPS and one or more outer-membrane proteins. Both Con+ and Con- strains contain the necessary LPS, but only Con+ strains contain the required protein(s).

  3. [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.

  4. Isolation and nucleotide sequence of the gene (aniA) encoding the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae.

    PubMed Central

    Hoehn, G T; Clark, V L

    1992-01-01

    When grown under anaerobic conditions, Neisseria gonorrhoeae, the etiologic agent of the sexually transmitted disease gonorrhea, expresses several novel outer membrane proteins. One of these, Pan 1, has an apparent molecular mass of 54 kDa in electrophoresis and is recognized by serum samples from patients with gonococcal infection. The presence of antibodies to this protein in patient sera suggests that Pan 1 is expressed during gonococcal infection and, more importantly, that N. gonorrhoeae grows anaerobically in vivo. We have cloned the Pan 1 structural gene, aniA, by screening a gonococcal lambda gt11 expression library with monospecific, polyclonal anti-Pan 1 antiserum. Three distinct immunoreactive recombinants, containing overlapping fragments of DNA, were isolated and confirmed to be coding for Pan 1 protein sequences. Northern (RNA blot) hybridization of an insert from an aniA recombinant to total gonococcal cellular RNA revealed the presence of a 1.5-kb transcript that was specific to RNA from anaerobically grown gonococci, indicating that the aniA gene is regulated at the transcriptional level and is monocistronic. To characterize the aniA gene, we have sequenced the entire 2-kb region spanned by the overlapping recombinants. We have also performed primer extension analysis on RNA isolated from aerobically and anaerobically grown gonococci in order to define the aniA promoter region. Two putative primer extension products specific to organisms grown anaerobically were identified by homology to known Escherichia coli promoter sequences, suggesting that the regulation of aniA expression involves multiple promoter regions. Images PMID:1383156

  5. 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. Published by Elsevier B.V.

  6. Species status of Neisseria gonorrhoeae: evolutionary and epidemiological inferences from multilocus sequence typing

    PubMed Central

    Bennett, Julia S; Jolley, Keith A; Sparling, P Frederick; Saunders, Nigel J; Hart, C Anthony; Feavers, Ian M; Maiden, Martin CJ

    2007-01-01

    Background Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species. Results A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters. Conclusion The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three

  7. Antimicrobial Resistance and Molecular Typing of Neisseria gonorrhoeae Isolates in Kyoto and Osaka, Japan, 2010 to 2012: Intensified Surveillance after Identification of the First Strain (H041) with High-Level Ceftriaxone Resistance

    PubMed Central

    Shimuta, Ken; Unemo, Magnus; Nakayama, Shu-ichi; Morita-Ishihara, Tomoko; Dorin, Misato; Kawahata, Takuya

    2013-01-01

    In 2009, the first high-level ceftriaxone-resistant Neisseria gonorrhoeae strain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) of Neisseria gonorrhoeae isolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193 N. gonorrhoeae isolates were collected and the MICs (μg/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, <0.5 μg/ml), and spectinomycin. The rates of resistance (intermediate susceptibility) to azithromycin, penicillin G, and ciprofloxacin were 3.6% (19.7%), 24.4% (71.0%), and 78.2% (0.5%), respectively. Multilocus sequence typing (MLST) showed that 40.9%, 19.2%, and 17.1% of isolates belonged to ST1901, ST7359, and ST7363, respectively. Furthermore, N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 μg/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic type penA alleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of the penA mosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the high-level ceftriaxone-resistant N. gonorrhoeae strain H041, suggesting that H041 might have caused only a sporadic case and has not spread further. PMID:23939890

  8. Azithromycin Resistance and Decreased Ceftriaxone Susceptibility in Neisseria gonorrhoeae, Hawaii, USA

    PubMed Central

    Papp, John R.; Abrams, A. Jeanine; Nash, Evelyn; Katz, Alan R.; Kirkcaldy, Robert D.; O’Connor, Norman P.; O’Brien, Pamela S.; Harauchi, Derek H.; Maningas, Eloisa V.; Soge, Olusegun O.; Kersh, Ellen N.; Komeya, Alan; Tomas, Juval E.; Wasserman, Glenn M.; Kunimoto, Gail Y.; Trees, David L.

    2017-01-01

    During 2016, eight Neisseria gonorrhoeae isolates from 7 patients in Hawaii were resistant to azithromycin; 5 had decreased in vitro susceptibility to ceftriaxone. Genomic analysis demonstrated a distinct phylogenetic clade when compared with local contemporary strains. Continued evolution and widespread transmission of these strains might challenge the effectiveness of current therapeutic options. PMID:28418303

  9. Azithromycin Resistance and Decreased Ceftriaxone Susceptibility in Neisseria gonorrhoeae, Hawaii, USA.

    PubMed

    Papp, John R; Abrams, A Jeanine; Nash, Evelyn; Katz, Alan R; Kirkcaldy, Robert D; O'Connor, Norman P; O'Brien, Pamela S; Harauchi, Derek H; Maningas, Eloisa V; Soge, Olusegun O; Kersh, Ellen N; Komeya, Alan; Tomas, Juval E; Wasserman, Glenn M; Kunimoto, Gail Y; Trees, David L; Whelen, A Christian

    2017-05-01

    During 2016, eight Neisseria gonorrhoeae isolates from 7 patients in Hawaii were resistant to azithromycin; 5 had decreased in vitro susceptibility to ceftriaxone. Genomic analysis demonstrated a distinct phylogenetic clade when compared with local contemporary strains. Continued evolution and widespread transmission of these strains might challenge the effectiveness of current therapeutic options.

  10. 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

  11. Clinical Neisseria gonorrhoeae isolate with a N. meningitidis porA gene and no prolyliminopeptidase activity, Sweden, 2011: danger of false-negative genetic and culture diagnostic results.

    PubMed

    Golparian, D; Johansson, E; Unemo, M

    2012-03-01

    We describe a Neisseria gonorrhoeae strain, found in Sweden in 2011, that harbours a N. meningitidis porA gene causing false-negative results in PCRs targeting the gonococcal porA pseudogene. Furthermore, the strain had no prolyliminopeptidase (PIP) activity that many commercial biochemical kits for species verification in culture rely on. Enhanced awareness of the spread of such strains and screening for them can be crucial.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed

    Unemo, Magnus

    2015-08-21

    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

  14. Evaluation of the coagglutination test for the identification of Neisseria gonorrhoeae in primary cultures.

    PubMed Central

    Johnston, N A

    1981-01-01

    The coagglutination (COA) test for the identification of Neisseria gonorrhoeae was compared with immunofluorescence and sugar degradation tests on 1710 gonococcal isolates, 72 of which produce beta-lactamase. The COA test gave a positive result for 98.6% of the strains. Treatment of suspensions with Streptomyces enzyme reduced the incidence of inconclusive results due to autoagglutination to 1.2%. Cross-reactivity of the gonococcal antiserum was minimised by absorption with meningococci and Moraxella species. The COA provide a simple, quick, and reliable method for identifying N gonorrhoeae in culture. PMID:6794854

  15. 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. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  16. 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.

  17. Pelvic inflammatory disease isolates of Neisseria gonorrhoeae are distinguished by C1q-dependent virulence for newborn rats and by the sac-4 region.

    PubMed Central

    Nowicki, S; Ram, P; Pham, T; Goluszko, P; Morse, S; Anderson, G D; Nowicki, B

    1997-01-01

    The virulence mechanism of Neisseria gonorrhoeae in pelvic inflammatory disease (PID) is not well understood, and an objective diagnostic method to identify patients with PID is lacking. We investigated the hypothesis that development of PID was associated with a C1q-dependent virulence property of gonococcal strains. Recent development of a C1q-dependent experimental model of gonococcal infection (S. Nowicki, M. Martens, and B. Nowicki, Infect. Immun. 63:4790-4794, 1995) created an opportunity to evaluate this hypothesis in vivo. Therefore, the virulence of 32 clinical isolates (18 PID isolates and 14 local infection [LI] isolates) was evaluated in experimental rat pups. A serum bactericidal assay was used to characterize a gonococcal serum-resistant (ser(r)) phenotype. PCR primers designed to amplify a suitable-size gonococcal sac-4 DNA fragment (unique for serum-resistant donor JC1) were used to evaluate the association of serum-resistant genotype sac-4 with two phenotypes: C1q-dependent virulence expressed in vivo and resistance to bactericidal activity of human serum expressed in vitro. Strains were also characterized by auxotyping and serotyping. Of 32 gonococcal strains, 15 (46.7%) caused C1q-dependent bacteremia in rat pups and were sac-4 positive and ser(r). However, of the 15 isolates, 13 (87%) represented strains associated with human PID and 2 (13%) were associated with LI. None of the strains that were completely serum-sensitive (ser(s)) and sac-4 negative produced C1q-dependent bacteremia in rat pups, suggesting that both ser(r) and sac-4 were required for infection. The serum-resistant recombinant recipient of sac-4 produced C1q-dependent bacteremia in the rat model similarly to the serum-resistant donor of sac-4; the serum-sensitive parent strain did not produce bacteremia. These data suggest that sac-4-mediated serum resistance conferred C1q-dependent virulence and is a unique characteristic associated with PID. These newly identified features may

  18. Real-Time PCR Targeting penA Mosaic XXXIV Type for Prediction of Extended-Spectrum Cephalosporins Susceptibility in Clinical Neisseria gonorrhoeae Isolates.

    PubMed

    Wong, L K; Hemarajata, P; Soge, O O; Humphries, R M; Klausner, J D

    2017-08-28

    N. gonorrhoeae with decreased susceptibility to extended-spectrum cephalosporins (ESCs) are increasing. We developed an assay to predict N. gonorrhoeae susceptibility to ESCs by targeting penA mosaic XXXIV, an allele prevalent among U.S. isolates with elevated ESC MICs. The assay was 97% sensitive and 100% specific in predicting at least one ESC minimum inhibitory concentration (MIC) above CDC alert value among clinical isolates, and could be multiplexed with previously validated gyrA PCR to predict ciprofloxacin susceptibility. Copyright © 2017 American Society for Microbiology.

  19. Comparative in vitro activity of Sch 29,482, a new oral penem, against Neisseria gonorrhoeae.

    PubMed Central

    Khan, M Y; Gruninger, R P; Nelson, S M; Simpson, M L

    1983-01-01

    The in vitro activity of Sch 29,482, a new oral beta-lactam antimicrobial agent, was compared with those of norfloxacin, rosoxacin, ampicillin, erythromycin, and tetracycline against 142 Neisseria gonorrhoeae strains. Sch 29,482 was as active as norfloxacin and rosoxacin. Its activity was greater than the other three antimicrobial agents. It inhibited 90% of the isolates, regardless of beta-lactamase activity, at a concentration of less than or equal to 0.06 micrograms/ml. PMID:6405687

  20. Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae.

    PubMed

    Cano, R J; Palomares, J C; Torres, M J; Klem, R E

    1992-07-01

    This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded gene cppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 of Neisseria gonorrhoeae. A total of 119 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All Neisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains of Neisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 x 10(4) cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100% while the specificity was 97.5%. Positive and negative predictive values were 91.2% and 100%, respectively. The fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.

  1. Neisseria gonorrhoeae : Detection and Typing by Probe Hybridization, LCR, and PCR.

    PubMed

    Gaydos, C A; Quinn, T C

    1999-01-01

    Neisseria gonorrhoeae, first described by Neisser in 1879, is a Gram-negative, nonmotile, nonspore-forming diplococcus, belonging to the family Neisseriaceae. It is the etiologic agent of gonorrhea. The other pathogenic species is Neisseria meningitidis, to which N. gonorrhoeae is genetically closely related. Although N. meningitidis is not usually considered to be a sexually transmitted disease, it may infect the mucous membranes of the anogenital area of homosexual men (1). The other members of the genus, which include Neisseria lactamic a, Neisseriapolysaccharea, Neisseria cinerea, and Neisseria flavescens, which are related to Neisseria gonorrhoeae, and saccharolytic strains, such as Neisseria subflava, Neisseria sicca, and Neisseria mucosa, which are less genetically related to the aforementioned, are considered to be nonpathogenic, being normal flora of the nasopharyngeal mucous membranes (2).

  2. 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.

  3. Inhibition of Neisseria gonorrhoeae by a Bacteriocin from Pseudomonas aeruginosa

    PubMed Central

    Morse, Stephen A.; Vaughan, Patrick; Johnson, Deanne; Iglewski, Barbara H.

    1976-01-01

    Supernatants from broth-grown cultures of Pseudomonas aeruginosa PA 103 exhibited bactericidal activity against Neisseria gonorrhoeae. The concentration of the bactericidal substance increased significantly after induction by mitomycin C. Purification was effected by salt fractionation, chromatography on diethylaminoethyl-cellulose, and sedimentation by centrifugation at 100,000 × g for 90 min. Electron microscopy of this purified preparation revealed structures resembling R-type pyocins in both the contracted and uncontracted state. Pyocins in the contracted state were observed in association with the gonococcal cell surface. No loss of bactericidal activity was observed after treatment with proteolytic enzymes. Standard pyocin typing procedures identified the pyocin pattern as 611 131. The bactericidal activity of this pyocin was examined on various species of Neisseria. Out of 56 strains of N. gonorrhoeae from disseminated and nondisseminated infections, all were susceptible to pyocin 611 131. However, only 3 of 20 strains of N. meningitidis and 5 of 16 strains of N. lactamica were susceptible. The bactericidal activity that pyocin 611 131 has for N. gonorrhoeae and other species of Neisseria is significant because it departs from the expected specificity that heretofore has distinguished bacteriocins from most “classical” antibiotics. Images PMID:825024

  4. 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.

  5. 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

  6. Effect of environment on sensitivity of Neisseria gonorrhoeae to Pseudomonas aeruginosa bacteriocins.

    PubMed Central

    Stein, D C; Hebeler, B H; Young, F E

    1980-01-01

    The effect of environmental variation on the susceptibility of Neisseria gonorrhoeae to pyocin produced by Pseudomonas aeruginosa was examined. Susceptibility to at least one pyocin was demonstrated in strains of N. gonorrhoeae (99%), N. meningitidis (35%), and N. lactamica (47%). The degree of sensitivity to pyocin displayed by N. gonorrhoeae was affected by varying the pH of the growth environment. Gonococcal strains were more sensitive to growth inhibition by pyocins at an alkaline pH and less sensitive to growth inhibition at an acid pH. Inhibitory titers fluctuated during nonselective subculture of fresh clinical isolates. There was no apparent correlation between auxotype and sensitivity to pyocin. Also, no relationship between colony morphology and pyocin sensitivity was seen. PMID:6783533

  7. NGMASTER: in silico multi-antigen sequence typing for Neisseria gonorrhoeae

    PubMed Central

    Gonçalves da Silva, Anders; Dyet, Kristin; Williamson, Deborah A.; Stinear, Timothy P.; Howden, Benjamin P.; Seemann, Torsten

    2016-01-01

    Whole-genome sequencing (WGS) provides the highest resolution analysis for comparison of bacterial isolates in public health microbiology. However, although increasingly being used routinely for some pathogens such as Listeria monocytogenes and Salmonella enterica, the use of WGS is still limited for other organisms, such as Neisseria gonorrhoeae. Multi-antigen sequence typing (NG-MAST) is the most widely performed typing method for epidemiological surveillance of gonorrhoea. Here, we present NGMASTER, a command-line software tool for performing in silico NG-MAST on assembled genome data. NGMASTER rapidly and accurately determined the NG-MAST of 630 assembled genomes, facilitating comparisons between WGS and previously published gonorrhoea epidemiological studies. The source code and user documentation are available at https://github.com/MDU-PHL/ngmaster. PMID:28348871

  8. [Evaluation of different methods of utilization of sugars in Neisseria gonorrhoeae].

    PubMed

    Rafael, Llanes; Sánchez, Idalmis; Díaz, Joel; Gutiérrez, Oderay; Guzmán, Daymi; Sosa, Jorge; Valdés, Eduardo A

    2003-01-01

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

  9. New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis.

    PubMed

    Ramsey, Meghan E; Hackett, Kathleen T; Kotha, Chaitra; Dillard, Joseph P

    2012-05-01

    We have created new complementation constructs for use in Neisseria gonorrhoeae and Neisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genes iga and trpB. In order to increase the available options for gene expression in Neisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-β-d-thiogalactopyranoside-inducible lac promoter, which has been widely used in Neisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcal opaB gene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter in Neisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenic Neisseria at very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy in N. gonorrhoeae as well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.

  10. Difficulties in differentiating Neisseria cinerea from Neisseria gonorrhoeae in rapid systems used for identifying pathogenic Neisseria species.

    PubMed

    Boyce, J M; Mitchell, E B

    1985-11-01

    Neisseria cinerea and Neisseria gonorrhoeae may occur at the same body sites and may have similar colony morphologies. Ideally, systems used for rapid identification of N. gonorrhoeae should be able to differentiate N. cinerea from gonococci. We tested seven N. cinerea strains using the Gonochek II (Du Pont Diagnostics), Minitek (BBL Microbiology Systems), RapID-NH (Innovative Diagnostics, Inc.), RIM-N (American Microscan), and Phadebact (Pharmacia Diagnostics) systems. We found that the reactions produced by N. cinerea in Gonochek II, Minitek, and RapID-NH kits could be confused with the results produced by some strains of N. gonorrhoeae. The susceptibility of N. cinerea to colistin, its ability to grow on tryptic soy or Mueller-Hinton agar, and its inability to grow on modified Thayer-Martin medium help differentiate it from gonococci.

  11. Distinct Neisseria gonorrhoeae transmission networks among men who have sex with men in Amsterdam, The Netherlands.

    PubMed

    Heymans, Raymond; A Matser, Amy; Bruisten, Sylvia M; Heijman, Titia; Geskus, Ronald B; Speksnijder, Adrianus G C L; Davidovich, Udi; de Vries, Henry J C; Coutinho, Roel A; Schim van der Loeff, Maarten F

    2012-08-15

    Molecular typing was used to elucidate Neisseria gonorrhoeae transmission networks among men who have sex with men (MSM) in Amsterdam, the Netherlands. We determined whether clusters of patients infected with specific N. gonorrhoeae genotypes were related to various epidemiological characteristics. MSM (age ≥18 years) visiting the sexually transmitted infections (STI) clinic between July 2008 and August 2009 were eligible. After STI screening, participants completed a behavioral questionnaire concerning the previous 6 months. N. gonorrhoeae cultures were genotyped using multiple-locus variable-number tandem repeat analysis typing. We obtained 278 N. gonorrhoeae-positive isolates from 240 MSM. Five large clusters (≥10 isolates), a unique sixth cluster (n = 9), and 8 smaller clusters (5-9 isolates) were identified. Prevalence of human immunodeficiency virus differed between clusters I and VI (P = .003), ranging from 27.8% to 100%. Receptive unprotected anal intercourse was frequently reported by MSM (51.8%) but did not differ significantly among clusters. Significant differences were identified concerning the participant's history of syphilis (P = .030), having met partners at a popular sex venue in Amsterdam (P = .048), and meeting partners outside Amsterdam (P = .036). Distinct N. gonorrhoeae transmission networks were present in a mixed high-risk MSM population; concordance between clusters and epidemiological characteristics was present but not marked.

  12. Multitarget PCR Assay for Direct Detection of Penicillinase-Producing Neisseria gonorrhoeae for Enhanced Surveillance of Gonococcal Antimicrobial Resistance

    PubMed Central

    Buckley, Cameron; Trembizki, Ella; Baird, Robert W.; Chen, Marcus; Donovan, Basil; Freeman, Kevin; Goire, Namraj; Guy, Rebecca; Lahra, Monica M.; Regan, David

    2015-01-01

    A multitarget PCR was developed for the direct detection of penicillinase-producing Neisseria gonorrhoeae (PPNG). The assay was validated by testing 342 PPNG isolates and 415 clinical samples. The method is suitable for routine detection of PPNG strains. Its multitarget approach reduces the potential for false-negative results caused by sequence variations. PMID:25994166

  13. 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

  14. Comparison of isolates of Neisseria gonorrhoeae causing meningitis and report of gonococcal meningitis in a patient with C8 deficiency.

    PubMed Central

    Del Rio, C; Stephens, D S; Knapp, J S; Rice, R J; Schalla, W O

    1989-01-01

    We studied a previously healthy 20-year-old woman who presented with gonococcal meningitis. The gonococcal isolate, HT-1, was prototrophic by auxotyping, was protein I serovar IB-1, and agglutinated with wheat germ lectin. This isolate differed from the proline-requiring, serovar IA-1 and IB-4, wheat germ-agglutination-negative gonococcal isolates recovered from three patients during a recent outbreak of gonococcal meningitis in Philadelphia. HT-1 was killed by normal pooled human sera (greater than or equal to 98% at 30 min) but not effectively killed by the convalescent-phase sera of the patient (greater than 30% survival at 30 min). Similar results were obtained when mucosal and cerebrospinal fluid isolates from a Philadelphia patient were exposed to these sera, but mucosal and blood isolates from another Philadelphia case showed increased resistance to killing by normal pooled human sera. Further characterization revealed multiple differences in outer membrane and cellular proteins and lipopolysaccharide between case isolates. Absence of the L8 lipopolysaccharide epitope was noted for all isolates. Sera of our patient were found to have low total hemolytic complement (CH100 = 21 U/ml; normal = 55 to 100 U/ml) due to deficiency of C8 (C8 less than 1,000 CH50 U/ml; normal = greater than or equal to 16,000 CH50 U/ml). This is the first reported case of gonococcal meningitis occurring in a patient with a terminal-complement deficiency. Gonococcal meningitis is a rare complication of gonococcal bacteremia. Both defects in host defenses (e.g., terminal-complement deficiency) and organisms with unusual virulence appear to contribute to the pathogenesis of this complication of gonococcal bacteremia. Images PMID:2473091

  15. Antimicrobial susceptibility/resistance and molecular epidemiological characteristics of Neisseria gonorrhoeae in 2009 in Belarus.

    PubMed

    Glazkova, Slavyana; Golparian, Daniel; Titov, Leonid; Pankratova, Nataliya; Suhabokava, Nataliya; Shimanskaya, Irina; Domeika, Marius; Unemo, Magnus

    2011-08-01

    Increased antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern, and ultimately gonorrhoea may become untreatable. Nonetheless, AMR data from East-Europe are scarce beyond Russia, and no AMR data or other characteristics of gonococci have been reported from Belarus for more than 20 years. The aim was to describe the prevalence of AMR, and report molecular epidemiological characteristics of gonococci circulating in 2009 in Belarus. In a sample of 80 isolates, resistance prevalences to antimicrobials used for gonorrhoea treatment in Belarus were: Ceftriaxone 0%, spectinomycin 0%, azithromycin 17.3%, tetracycline 25.9%, ciprofloxacin 34.6% and erythromycin 59.2%. The isolates displayed no penA mosaic alleles, 38 porB gene sequences and 35 N. gonorrhoeae multiantigen sequence types, of which 20 have not been described before worldwide. Due to the high levels of antimicrobial resistance, only ceftriaxone and spectinomycin can be recommended for empirical treatment of gonorrhoea in Belarus according to WHO recommendations. Continuous gonococcal AMR surveillance in Eastern Europe is crucial. This is now initiated in Belarus using WHO protocols.

  16. Fluoroquinolone-resistant Neisseria gonorrhoeae: the inevitable epidemic.

    PubMed

    Ghanem, Khalil G; Giles, Julie A; Zenilman, Jonathan M

    2005-06-01

    The worldwide incidence of fluoroquinolone-resistant Neisseria gonorrhoeae has increased dramatically in the last few years. Single doses of fluoroquinolones can no longer be used to treat N gonorrhoeae infections acquired in the Far East, parts of the Middle East, the Pacific Islands, and parts of Western Europe and the United States. Although California and Hawaii account for most of the current United States cases, the increased incidence of FQR in some high-risk groups independent of geography heralds an imminent spread of drug-resistant strains throughout the rest of the population. The use of molecular tests has revolutionized the diagnostic field in STIs. The main limitation of their application in N gonorrhoeae testing has been the loss of culture specimens that allow antimicrobial sensitivity testing. New molecular methods have made it possible to detect antimicrobial resistance without the use of live organisms. These tests hold the promise of improving epidemiologic tracking of N gonorrhoeae drug resistance, leading to better patient management at the local level. The loss of fluoroquinolones limits available oral regimens to a single CDC-recommended antibiotic, cefixime. Oral, inexpensive, single-dose alternatives are needed to ensure continued therapeutic success.

  17. TonB-Dependent Transporters Expressed by Neisseria gonorrhoeae.

    PubMed

    Cornelissen, Cynthia Nau; Hollander, Aimee

    2011-01-01

    Neisseria gonorrhoeae causes the common sexually transmitted infection, gonorrhea. This microorganism is an obligate human pathogen, existing nowhere in nature except in association with humans. For growth and proliferation, N. gonorrhoeae requires iron and must acquire this nutrient from within its host. The gonococcus is well-adapted for growth in diverse niches within the human body because it expresses efficient transport systems enabling use of a diverse array of iron sources. Iron transport systems facilitating the use of transferrin, lactoferrin, and hemoglobin have two components: one TonB-dependent transporter and one lipoprotein. A single component TonB-dependent transporter also allows N. gonorrhoeae to avail itself of iron bound to heterologous siderophores produced by bacteria within the same ecological niche. Other TonB-dependent transporters are encoded by the gonococcus but have not been ascribed specific functions. The best characterized iron transport system expressed by N. gonorrhoeae enables the use of human transferrin as a sole iron source. This review summarizes the molecular mechanisms involved in gonococcal iron acquisition from human transferrin and also reviews what is currently known about the other TonB-dependent transport systems. No vaccine is available to prevent gonococcal infections and our options for treating this disease are compromised by the emergence of antibiotic resistance. Because iron transport systems are critical for the survival of the gonococcus in vivo, the surface-exposed components of these systems are attractive candidates for vaccine development or therapeutic intervention.

  18. TonB-Dependent Transporters Expressed by Neisseria gonorrhoeae

    PubMed Central

    Cornelissen, Cynthia Nau; Hollander, Aimee

    2011-01-01

    Neisseria gonorrhoeae causes the common sexually transmitted infection, gonorrhea. This microorganism is an obligate human pathogen, existing nowhere in nature except in association with humans. For growth and proliferation, N. gonorrhoeae requires iron and must acquire this nutrient from within its host. The gonococcus is well-adapted for growth in diverse niches within the human body because it expresses efficient transport systems enabling use of a diverse array of iron sources. Iron transport systems facilitating the use of transferrin, lactoferrin, and hemoglobin have two components: one TonB-dependent transporter and one lipoprotein. A single component TonB-dependent transporter also allows N. gonorrhoeae to avail itself of iron bound to heterologous siderophores produced by bacteria within the same ecological niche. Other TonB-dependent transporters are encoded by the gonococcus but have not been ascribed specific functions. The best characterized iron transport system expressed by N. gonorrhoeae enables the use of human transferrin as a sole iron source. This review summarizes the molecular mechanisms involved in gonococcal iron acquisition from human transferrin and also reviews what is currently known about the other TonB-dependent transport systems. No vaccine is available to prevent gonococcal infections and our options for treating this disease are compromised by the emergence of antibiotic resistance. Because iron transport systems are critical for the survival of the gonococcus in vivo, the surface-exposed components of these systems are attractive candidates for vaccine development or therapeutic intervention. PMID:21747812

  19. Genetic basis of Neisseria gonorrhoeae lipooligosaccharide antigenic variation.

    PubMed Central

    Danaher, R J; Levin, J C; Arking, D; Burch, C L; Sandlin, R; Stein, D C

    1995-01-01

    Neisseria gonorrhoeae lipooligosaccharide (LOS) undergoes antigenic variation at a high rate, and this variation can be monitored by changes in a strain's ability to bind LOS-specific monoclonal antibodies. We report here the cloning and identification of a gene, lsi-2, that can mediate this variation. The DNA sequence of lsi-2 has been determined for N. gonorrhoeae 1291, a strain that expresses a high-molecular-mass LOS, and a derivative of this strain, RS132L, that produces a truncated LOS. In the parental strain, lsi-2 contains a string of 12 guanines in the middle of its coding sequence. In cells that had antigenically varied to produce a truncated LOS, the number of guanines in lsi-2 was altered. Site-specific deletions were constructed to verify that expression of a 3.6-kDa LOS is due to alterations in lsi-2. PMID:8522539

  20. Preservation of Neisseria gonorrhoeae by the gelatin-disc method.

    PubMed Central

    Yamai, S; Obara, Y; Nikkawa, T; Shimoda, Y; Miyamoto, Y

    1979-01-01

    Studies of Neisseria gonorrhoeae are difficult to perform because of the organism's poor survival in vitro. To solve this problem we tried to preserve the organism by a gelatin-disc method. The rate of survival and changes of variations in some biochemical properties of eight strains of N. gonorrhoeae were followed for three years. These studies proved that preservation was satisfactory with only a 1/10 reduction of the living cells. Another trial showed that the organism survived for over six months after being frozen at -20 degrees C. The colonial types, agglutination against red cells from rabbit and guinea pig, and antibiotic susceptibility to penicillin, chloramphenicol, tetracycline, kanamycin, and streptomycin did not change after three years' preservation. PMID:109165

  1. Molecular epidemiology, in 1994, of Neisseria gonorrhoeae in Manila and Cebu City, Republic of the Philippines.

    PubMed

    Knapp, J S; Mesola, V P; Neal, S W; Wi, T E; Tuazon, C; Manalastas, R; Perine, P L; Whittington, W L

    1997-01-01

    Failure of gonococcal infections to respond to 500 mg of ciprofloxacin or 400 mg of ofloxacin has been reported from Australia, the United Kingdom, and the United States. Recently, high rates of decreased susceptibility to the fluoroquinolones have been detected in penicillinase-producing Neisseria gonorrhoeae in the Republic of the Philippines. To assess the diversity of antimicrobial-resistant gonococcal strains isolated from female sex workers in Manila and Cebu City in the Republic of the Philippines in 1994. Isolates of N. gonorrhoeae isolated from 92 female sex workers in Manila (n = 28) and Cebu City (n = 64), respectively, were characterized by plasmid profile, auxotype/serovar class, and antimicrobial susceptibility profile. Plasmid-mediated resistance to penicillin or tetracycline was identified in 79.3% (73/92) of the isolates: penicillinase-producing N. gonorrhoeae (65/92; 70.7%), tetracycline-resistant N. gonorrhoeae (6/92; 6.5%), and penicillinase-producing/tetracycline-resistant N. gonorrhoeae (1/92; 1.1%). A beta-lactamase plasmid of 3.9 megadaltons was discovered. Of 54.3% (50/92) of strains resistant to nalidixic acid, 84% (42/50) of strains had minimum inhibitory concentrations of > or = 0.125 microgram/ml ciprofloxacin; penicillinase-producing N. gonorrhoeae (possessing the 3.05-, 3.2-, 3.9-, and 4.4-megadalton beta-lactamase plasmids, respectively) accounted for 68% (34/50) of these strains. In the Republic of the Philippines, gonococcal isolates resistant to penicillin or tetracycline accounted for 85.9% (79/92) of the isolates examined and included strains exhibiting resistance to fluoroquinolones. All gonococcal infections should be treated with antimicrobial therapies known to be active against all gonococcal strains to reduce the spread of strains exhibiting decreased susceptibilities to fluoroquinolones.

  2. [Antimicrobal resistance of Neisseria gonorrhoeae strains in Hungary].

    PubMed

    Nemes-Nikodém, Éva; Brunner, Alexandra; Tóth, Béla; Tóth, Veronika; Bánvölgyi, András; Ostorházi, Eszter

    2015-02-08

    Bevezetés: A Neisseria gonorrhoeae-infekciók kezelésére kiadott európai ajánlás elsősorban a nyugat-európai adatok alapján készült, és nem egyértelműen használható a magyarországi helyzet ismeretében. Célkitűzés: A szerzők 2011. január és 2014. június közötti időszakban a Semmelweis Egyetem, Bőr-, Nemikórtani és Bőronkológiai Klinika Országos Szexuális Úton Terjedő Betegségek Centrumában izolált Neisseria gonorrhoeae törzsek rezisztenciaadatait összevetették az izolált törzsek molekuláris tipizálási eredményeivel, azzal a céllal, hogy pontos adatokat kapjanak a hazánkban előforduló Neisseria gonorrhoeae törzsek antimikrobiális rezisztenciájáról. Módszer: Az antibiotikumrezisztencia-meghatározás minimális inhibitorkoncentráció-méréssel, a szekvenciameghatározás a Neisseria gonorrhoeae Multi Antigen Sequence Typing módszerrel történt. Eredmények: A jelenleg terápiának ajánlott széles spektrumú cefalosporinok elleni rezisztencia ritka, az utóbbi években az azithromycinrezisztencia előfordulása viszont rohamosan növekedett. Következtetések: Az új terápiás irányelvek készítésekor figyelembe kell venni, hogy a gyakran fertőzést okozó molekuláris típusba sorolható törzsek között kiemelkedően magas az azithromycinrezisztensek aránya. Orv. Hetil., 2015, 156(6), 226–229.

  3. Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network.

    PubMed

    Yu, Chunxiao; McClure, Ryan; Nudel, Kathleen; Daou, Nadine; Genco, Caroline Attardo

    2016-08-15

    The Neisseria gonorrhoeae ferric uptake regulator (Fur) protein controls expression of iron homeostasis genes in response to intracellular iron levels. In this study, using transcriptome sequencing (RNA-seq) analysis of an N. gonorrhoeae fur strain, we defined the gonococcal Fur and iron regulons and characterized Fur-controlled expression of an ArsR-like DNA binding protein. We observed that 158 genes (8% of the genome) showed differential expression in response to iron in an N. gonorrhoeae wild-type or fur strain, while 54 genes exhibited differential expression in response to Fur. The Fur regulon was extended to additional regulators, including NrrF and 13 other small RNAs (sRNAs), and two transcriptional factors. One transcriptional factor, coding for an ArsR-like regulator (ArsR), exhibited increased expression under iron-replete conditions in the wild-type strain but showed decreased expression across iron conditions in the fur strain, an effect that was reversed in a fur-complemented strain. Fur was shown to bind to the promoter region of the arsR gene downstream of a predicted σ(70) promoter region. Electrophoretic mobility shift assay (EMSA) analysis confirmed binding of the ArsR protein to the norB promoter region, and sequence analysis identified two additional putative targets, NGO1411 and NGO1646. A gonococcal arsR strain demonstrated decreased survival in human endocervical epithelial cells compared to that of the wild-type and arsR-complemented strains, suggesting that the ArsR regulon includes genes required for survival in host cells. Collectively, these results demonstrate that the N. gonorrhoeae Fur functions as a global regulatory protein to repress or activate expression of a large repertoire of genes, including additional transcriptional regulatory proteins. Gene regulation in bacteria in response to environmental stimuli, including iron, is of paramount importance to both bacterial replication and, in the case of pathogenic bacteria

  4. Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network

    PubMed Central

    Yu, Chunxiao; McClure, Ryan; Daou, Nadine

    2016-01-01

    ABSTRACT The Neisseria gonorrhoeae ferric uptake regulator (Fur) protein controls expression of iron homeostasis genes in response to intracellular iron levels. In this study, using transcriptome sequencing (RNA-seq) analysis of an N. gonorrhoeae fur strain, we defined the gonococcal Fur and iron regulons and characterized Fur-controlled expression of an ArsR-like DNA binding protein. We observed that 158 genes (8% of the genome) showed differential expression in response to iron in an N. gonorrhoeae wild-type or fur strain, while 54 genes exhibited differential expression in response to Fur. The Fur regulon was extended to additional regulators, including NrrF and 13 other small RNAs (sRNAs), and two transcriptional factors. One transcriptional factor, coding for an ArsR-like regulator (ArsR), exhibited increased expression under iron-replete conditions in the wild-type strain but showed decreased expression across iron conditions in the fur strain, an effect that was reversed in a fur-complemented strain. Fur was shown to bind to the promoter region of the arsR gene downstream of a predicted σ70 promoter region. Electrophoretic mobility shift assay (EMSA) analysis confirmed binding of the ArsR protein to the norB promoter region, and sequence analysis identified two additional putative targets, NGO1411 and NGO1646. A gonococcal arsR strain demonstrated decreased survival in human endocervical epithelial cells compared to that of the wild-type and arsR-complemented strains, suggesting that the ArsR regulon includes genes required for survival in host cells. Collectively, these results demonstrate that the N. gonorrhoeae Fur functions as a global regulatory protein to repress or activate expression of a large repertoire of genes, including additional transcriptional regulatory proteins. IMPORTANCE Gene regulation in bacteria in response to environmental stimuli, including iron, is of paramount importance to both bacterial replication and, in the case of pathogenic

  5. Antigenic variation in Neisseria gonorrhoeae: production of multiple lipooligosaccharides.

    PubMed Central

    Burch, C L; Danaher, R J; Stein, D C

    1997-01-01

    Individual cells of Neisseria gonorrhoeae may express a single lipooligosaccharide (LOS) component on their cell surfaces, or they may simultaneously express multiple LOS structures. Strain FA19 expresses LOS components that react with monoclonal antibodies (MAbs) 2-1-L8 and 1B2. The genetic locus responsible for this phenotype in FA19 was identified by isolating a clone that is able to impart the ability to simultaneously express both LOS molecules to strain 1291, a strain expressing only the MAb 1B2-reactive LOS. This clone, pCLB1, was characterized, and the gene responsible for the expression of both LOS components was determined to be lsi2. DNA sequence analysis of lsi2(Fa19) indicates that there are several differences between the DNA sequences of lsi2(FA19) and lsi2(1291). The region responsible for the LOS-specific phenotype change in lsi2(FA19) was identified by deletion and transformation analysis, mapping to a polyguanine tract within lsi2 where lsi2(FA19) possesses a +2 frameshift relative to lsi2(1291). The polyguanine tract in lsi2(FA19) was modified by site-directed mutagenesis to change the sequence to GGGAGGTGGCGGA to prevent frameshifting during DNA replication, transcription, and/or translation. Transformants of strain 1291 containing this DNA sequence express a single MAb 2-1-L8-reactive LOS component, the same phenotype exhibited by lsi2-defective strains. These data indicate that FA19 is able to generate a small amount of functional Lsi2 protein via transcriptional and/or translational frameshifting, and this limited amount of protein allows for the expression of MAb 1B2-reactive LOS molecules. PMID:9006061

  6. A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

    PubMed Central

    Hjelmevoll, Stig Ove; Olsen, Merethe Elise; Sollid, Johanna U. Ericson; Haaheim, Håkon; Unemo, Magnus; Skogen, Vegard

    2006-01-01

    Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection. PMID:17065426

  7. Emergence of decreased susceptibility and resistance to extended-spectrum cephalosporins in Neisseria gonorrhoeae in Korea

    PubMed Central

    Lee, Hyukmin; Unemo, Magnus; Kim, Hyo Jin; Seo, Younghee; Lee, Kyungwon; Chong, Yunsop

    2015-01-01

    Objectives Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern globally; however, no comprehensive AMR data for gonococcal isolates cultured after 2006 in Korea have been published internationally. We determined the susceptibility of N. gonorrhoeae isolates cultured in 2011–13, the mechanism of extended-spectrum cephalosporin (ESC) resistance and the molecular epidemiology of gonococcal strains in Korea. Methods In 2011–13, 210 gonococcal isolates were collected in Korea and their AMR profiles were examined by the agar dilution method. The penA, mtrR, penB, ponA and pilQ genes were sequenced in 25 isolates that were resistant to ESCs and 70 randomly selected isolates stratified by year. For molecular epidemiology, N. gonorrhoeae multiantigen sequence typing and MLST were performed. Results None of the N. gonorrhoeae isolates was susceptible to penicillin G and most were resistant to tetracycline (50%) and ciprofloxacin (97%). The rates of resistance to ceftriaxone, azithromycin, cefpodoxime and cefixime were 3%, 5%, 8% and 9%, respectively. However, all isolates were susceptible to spectinomycin. Twenty-one (84%) of the 25 ESC-resistant isolates contained the non-mosaic PBP2 XIII allele; however, the remaining 4 (16%) possessed the mosaic PBP2 X allele, which has been previously associated with ESC resistance including treatment failures. Conclusions In Korea, susceptibility to spectinomycin remains high. However, the recent emergence of ESC-resistant N. gonorrhoeae strains, including strains possessing the PBP2 mosaic X and non-mosaic XIII alleles, is a major concern and enhanced AMR surveillance is necessary to prevent transmission of these strains. PMID:26084303

  8. Detection of Neisseria gonorrhoeae in the pharynx and saliva: implications for gonorrhoea transmission.

    PubMed

    Chow, Eric P F; Lee, David; Tabrizi, Sepehr N; Phillips, Samuel; Snow, Anthony; Cook, Stuart; Howden, Benjamin P; Petalotis, Irene; Bradshaw, Catriona S; Chen, Marcus Y; Fairley, Christopher K

    2016-08-01

    This study aimed to determine the proportion of untreated pharyngeal swabs or saliva samples positive by culture or nucleic acid amplification tests (NAATs) for Neisseria gonorrhoeae up to 14 days after an initial culture-positive pharyngeal swab. Men who have sex with men who tested positive for pharyngeal gonorrhoea at Melbourne Sexual Health Centre (MSHC) and returned to MSHC for treatment within 14 days between 13 October 2014 and 25 March 2015 were included in this study. Pharyngeal swabs and saliva samples were collected for culture and NAAT. Of 33 initially culture-positive pharyngeal swabs, 32 saliva samples and 31 pharyngeal swabs were positive by NAAT and 14 pharyngeal and 6 saliva samples were positive by culture within 14 days. There was a significant decline in the proportion of repeated pharyngeal culture samples positive by culture over time (p<0.001). The rapid decline suggests pharyngeal gonorrhoea is short-lived, and the finding of gonorrhoea commonly in the saliva implicates this body fluid in its transmission without direct throat inoculation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  9. First Neisseria gonorrhoeae Genotyping Analysis in France: Identification of a Strain Cluster with Reduced Susceptibility to Ceftriaxone ▿

    PubMed Central

    Monfort, Laura; Caro, Valérie; Devaux, Zaelle; Delannoy, Anne-Sophie; Brisse, Sylvain; Sednaoui, Patrice

    2009-01-01

    Sexually transmitted infections are a major public health problem in France and other European countries. Particularly, surveillance data about Neisseria gonorrhoeae infections have clearly indicated an increase in the incidence of gonorrhoea in France in 2006. The French laboratories participated on voluntary basis in the RENAGO (Réseau National du Gonocoque) network and sent all of their collected strains to the National Reference Center for Neisseria gonorrhoeae. In this first French molecular epidemiological study, 93 isolates collected in 2006 and representative of the French gonorrhoea epidemiology were selected. Antibiotic susceptibility to six antibiotics was determined, and serotyping and N. gonorrhoeae multiantigen sequence typing (NG-MAST) were performed. NG-MAST identified 53 sequence types (STs), of which 13 STs contained 2 to 16 isolates. The major STs identified in France were previously described elsewhere. However, two newly described STs, ST1479 and ST1987, had only been found in France until now. ST1479 was characterized by a multiple-resistance phenotype, whereas ST1987 presented a susceptibility phenotype. Moreover, among the predominant French STs, ST225, which had already been described in many countries, comprised isolates (14/16) resistant to ciprofloxacin and with reduced susceptibility to ceftriaxone. Thus, the surveillance of resistance to antibiotics is a priority in order to adapt treatment and decrease the transmission of resistant strains. Of note, no predominant ST was identified among rectal isolates from men who have sex with men. PMID:19794054

  10. One-step staining of Neisseria gonorrhoeae in urethral discharge by methyl green-pyronin.

    PubMed Central

    Hanna, N F; Taylor-Robinson, D; Csonka, G W; Harris, J R; Al-Sowaygh, I A

    1980-01-01

    Methyl green-pyronin (MGP) was used in a one-step procedure to stain smears of urethral discharge from 169 men. Duplicate smears were stained by Gram's method and discharge was cultured for Neisseria gonorrhoeae. The organisms were isolated from 67 specimens and intracellular diplococci were seen in 74 smears after Gram staining and in 77 after staining by MGP. Furthermore, more extracellular and intracellular diplococci were seen in smears stained by MGP than by Gram's method and the proportion of polymorphonuclear leucocytes found to contain the organisms was greater after staining with MGP. Staining with MGP is simple, rapid, inexpensive, and easily automated. PMID:6159054

  11. In-vitro comparison of macrolides, lincosamides and synergistins on Neisseria gonorrhoeae.

    PubMed

    Thabaut, A; Meyran, M; Huerre, M

    1985-07-01

    The MIC of erythromycin, oleandomycin, spiramycin, josamycin, lincomycin and pristinamycin was determined for 100 strains of Neisseria gonorrhoeae isolated from cases of acute urethritis in men. The method of dilution in agar was used: blood agar with the addition of 'Polyvitex' and an innoculum of 10(3)-10(4) bacteria per spot. With respect to the break points defined by the C.F.A. all the strains of N. gonorrhoeae studied are sensitive to erythromycin, spiramycin, josamycin and pristinamycin, 12% strains are resistant to oleandomycin and 75% to lincomycin. The active antibiotics are classified as follows according to the active weight expressed by the MIC50: erythromycin, pristinamycin, 0.125 mg/l; josamycin, 0.5 mg/l; spiramycin, oleandomycin, 2 mg/l.

  12. Molecular epidemiology of drug-resistant Neisseria gonorrhoeae in Russia (Current Status, 2015).

    PubMed

    Kubanov, Alexey; Vorobyev, Denis; Chestkov, Aleksandr; Leinsoo, Arvo; Shaskolskiy, Boris; Dementieva, Ekaterina; Solomka, Viktoria; Plakhova, Xenia; Gryadunov, Dmitry; Deryabin, Dmitriy

    2016-08-09

    The widespread distribution of Neisseria gonorrhoeae strains that are resistant to previously used and clinically implemented antibiotics is a significant global public health problem. In line with WHO standards, the national Gonococcal Antimicrobial Surveillance Programme (RU-GASP) has been in existence in Russia since 2004; herein, the current status (2015) is described, including associations between N. gonorrhoeae antimicrobial susceptibility, primary genetic resistance determinants and specific strain sequence types. A total of 124 N. gonorrhoeae strains obtained from 9 regions in Russia in 2015 were examined using N. gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST), an antimicrobial susceptibility test according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria and an oligonucleotide microarray for the identification of mutations in the penA, ponA, rpsJ, gyrA and parC genes responsible for penicillin G, tetracycline, and fluoroquinolone resistance. Genogroup (G) isolates were evaluated based on their porB and tbpB sequence types (STs). NG-MAST analysis showed a diversified population of N. gonorrhoeae in Russia with 58 sequence types, 35 of which were described for the first time. The STs 807, 1544, 1993, 5714, 9476 and 12531, which were typical for some Russian Federation regions and several countries of the former Soviet Union, were represented by five or more isolates. The internationally widespread ST 1407 was represented by a single strain in the present study. Division into genogroups facilitated an exploration of the associations between N. gonorrhoeae sequence type, antimicrobial resistance spectra and genetic resistance determinant contents. Preliminarily susceptible (G-807, G-12531) and resistant (G-5714, G-9476) genogroups were revealed. The variability in the most frequently observed STs and genogroups in each participating region indicated geographically restricted antimicrobial susceptibility in N. gonorrhoeae

  13. Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication.

    PubMed

    Pagotto, F J; Salimnia, H; Totten, P A; Dillon, J R

    2000-02-22

    An origin of replication (ori) was obtained from a naturally occurring beta-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.

  14. Investigation of the Basis for Persistent Porin Serotypes of Neisseria Gonorrhoeae in Community Infections

    DTIC Science & Technology

    2006-01-01

    Davis, W. Fischer, J. C. Thomas, I. Martin , C. Ison, P.F. Sparling, and M. S. Cohen. 1998. Molecular Typing of Neisseria gonorrhoeae Causing Repeated...J. C. Thomas, I. Martin , C. Ison, P. F. Sparling, and M. S. Cohen. 1999. Molecular typing of Neisseria gonorrhoeae causing repeated infections...Bygraves, E. Feil , G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt. 1998

  15. In Vitro Activity of Ertapenem versus Ceftriaxone against Neisseria gonorrhoeae Isolates with Highly Diverse Ceftriaxone MIC Values and Effects of Ceftriaxone Resistance Determinants: Ertapenem for Treatment of Gonorrhea?

    PubMed Central

    Golparian, Daniel; Limnios, Athena; Whiley, David; Ohnishi, Makoto; Lahra, Monica M.; Tapsall, John W.

    2012-01-01

    Clinical resistance to the currently recommended extended-spectrum cephalosporins (ESCs), the last remaining treatment options for gonorrhea, is being reported. Gonorrhea may become untreatable, and new treatment options are crucial. We investigated the in vitro activity of ertapenem, relative to ceftriaxone, against N. gonorrhoeae isolates and the effects of ESC resistance determinants on ertapenem. MICs were determined using agar dilution technique or Etest for international reference strains (n = 17) and clinical N. gonorrhoeae isolates (n = 257), which included the two extensively drug-resistant (XDR) strains H041 and F89 and additional isolates with high ESC MICs, clinical ESC resistance, and other types of clinical high-level and multidrug resistance (MDR). Genetic resistance determinants for ESCs (penA, mtrR, and penB) were sequenced. In general, the MICs of ertapenem (MIC50 = 0.032 μg/ml; MIC90 = 0.064 μg/ml) paralleled those of ceftriaxone (MIC50 = 0.032 μg/ml; MIC90 = 0.125 μg/ml). The ESC resistance determinants mainly increased the ertapenem MIC and ceftriaxone MIC at similar levels. However, the MIC ranges for ertapenem (0.002 to 0.125 μg/ml) and ceftriaxone (<0.002 to 4 μg/ml) differed, and the four (1.5%) ceftriaxone-resistant isolates (MIC = 0.5 to 4 μg/ml) had ertapenem MICs of 0.016 to 0.064 μg/ml. Accordingly, ertapenem had in vitro advantages over ceftriaxone for isolates with ceftriaxone resistance. These in vitro results suggest that ertapenem might be an effective treatment option for gonorrhea, particularly for the currently identified ESC-resistant cases and possibly in a dual antimicrobial therapy regimen. However, further knowledge regarding the genetic determinants (and their evolution) conferring resistance to both antimicrobials, and clear correlates between genetic and phenotypic laboratory parameters and clinical treatment outcomes, is essential. PMID:22547617

  16. In Vitro Susceptibilities of Neisseria gonorrhoeae to Fatty Acids and Monoglycerides

    PubMed Central

    Bergsson, Gudmundur; Steingrímsson, Ólafur; Thormar, Halldor

    1999-01-01

    The susceptibility of Neisseria gonorrhoeae to several medium-chain fatty acids and their 1-monoglycerides was tested at a short inactivation time of 1 min. The results indicate that monocaprin, a monoglyceride of capric acid (10 carbon atoms, no double bonds), causes the fastest and most effective killing of all strains of N. gonorrhoeae tested. PMID:10543766

  17. Potential Therapy for Neisseria Gonorrhoeae Infections With Human Chorionic Gonadotropin.

    PubMed

    Rao, C V

    2015-12-01

    The scientific evidence suggests that Neisseria gonorrhoeae (NG) infects human fallopian tubes by molecular mimicry in which pathogens act like a ligand to bind to epithelial cell surface human chorionic gonadotrophin (hCG)/luteinizing hormone (LH) receptors. The hCG-like molecule has been identified as ribosomal protein L12 in NG coat surface. Human fallopian tube epithelial cells have been shown to contain functional hCG/LH receptors. As previously shown in human fallopian tube organ and cell culture studies, cellular invasion and infection can be prevented by exposing the cells to excess hCG, which would outnumber and outcompete NG for receptor binding. Based on these data, we suggest testing hCG in clinical trials on infected women.

  18. Antigenic and physical diversity of Neisseria gonorrhoeae lipooligosaccharides.

    PubMed Central

    Mandrell, R; Schneider, H; Apicella, M; Zollinger, W; Rice, P A; Griffiss, J M

    1986-01-01

    We used mouse monoclonal antibodies (MAbs) to characterize Neisseria gonorrhoeae lipooligosaccharide (LOS). LOSs that bound two or more MAbs in a solid-phase radioimmunoassay usually bound them to different LOS components, as separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); strains with multiple LOS components on SDS-PAGE usually bound more than one MAb. However, the LOS of some strains bound the same MAb to two LOS components with different relative molecular weights, and some individual LOS components bound more than one MAb. LOSs from different strains bound different amounts of the same MAb at saturation, reflecting differences in the quantitative expression of individual LOS components. Not all components recognized by MAbs were stained by silver after periodate oxidation. Treatment with NaOH variously affected epitopes defined by different MAbs. MAb 3F11 completely inhibited and MAb 2-1-L8 partially inhibited the binding of 125I-labeled 06B4 MAb to WR220 LOS and WR220 outer membranes in competitive binding studies. Other MAbs did not compete with the binding of 125I-labeled 06B4 to either antigen. We conclude that a strain of N. gonorrhoeae elaborates multiple LOSs that can be separated by SDS-PAGE and that are antigenically distinct. Epitope expression within these glycolipids is complex. Images PMID:2428752

  19. Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

    PubMed Central

    Rotman, Ella; Webber, David M.

    2016-01-01

    ABSTRACT Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance. Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). The N. gonorrhoeae chromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer pilS sequences into the pilE locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity. IMPORTANCE Measuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation. PMID:27381912

  20. Neisseria Species Identification Assay for the Confirmation of Neisseria gonorrhoeae-Positive Results of the COBAS Amplicor PCR▿

    PubMed Central

    Mangold, Kathy A.; Regner, MaryAnn; Tajuddin, Mohammed; Tajuddin, Aamair M.; Jennings, Lawrence; Du, Hongyan; Kaul, Karen L.

    2007-01-01

    Screening assays for Neisseria gonorrhoeae exhibit low positive predictive values, particularly in low-prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae, the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive for cppB. However, cppB was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or porA pseudogene (2%) assays. The porA pseudogene assay had the highest specificity for both genders but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppB, due to poor specificity. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non-N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay. PMID:17360838

  1. Transfer of plasmid-mediated ampicillin resistance from Haemophilus to Neisseria gonorrhoeae requires an intervening organism.

    PubMed

    McNicol, P J; Albritton, W L; Ronald, A R

    1986-01-01

    Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids.

  2. 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

  3. Isolation of Neisseria lactamica from the female genital tract. A case report.

    PubMed

    Telfer Brunton, W A; Young, H; Fraser, D R

    1980-10-01

    Neisseria lactamica was isolated from the genital tract of a young patient with a persistent vaginal discharge. Although infection with N lactamica occurs very rarely, the importance of complete biochemical identification of neisseriae is emphasised in view of the serious social and medicolegal consequences which could result from a misdiagnosis of gonorrhoea.

  4. Pharyngeal Neisseria gonorrhoeae detection in oral-throat wash specimens of male patients with urethritis.

    PubMed

    Takahashi, Satoshi; Kurimura, Yuichiro; Hashimoto, Jiro; Takeyama, Koh; Koroku, Mikio; Tanda, Hitoshi; Nishimura, Masahiro; Tsukamoto, Taiji

    2008-12-01

    Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in the pharynx has been highlighted in the prevention of the unexpected spread of sexually transmitted diseases. We tried to clarify the detection rate of Neisseria gonorrhoeae in the pharynx and the clinical relevance of oral-throat wash specimens to detect the organism in heterosexual men with gonococcal and nongonococcal urethritis. In our cohort of 79 male patients with urethritis, oral throat wash specimens were collected after they had gargled with normal saline for approximately 30 to 60 s. Positive pharyngeal N. gonorrhoeae was defined as a positive result on the strand displacement amplification test for the specimen from the oral-throat wash. N. gonorrhoeae was detected in the oral-throat wash specimens of 13 (31.7%) of the 41 male patients with gonococcal urethritis. Oral-throat wash with a nucleic acid amplification test can detect pharyngeal N. gonorrhoeae easily and efficiently.

  5. Neisseria gonorrhoeae activates the proteinase cathepsin B to mediate the signaling activities of the NLRP3 and ASC-containing inflammasome.

    PubMed

    Duncan, Joseph A; Gao, Xi; Huang, Max Tze-Han; O'Connor, Brian P; Thomas, Christopher E; Willingham, Stephen B; Bergstralh, Daniel T; Jarvis, Gary A; Sparling, P Frederick; Ting, Jenny P-Y

    2009-05-15

    Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1beta and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1beta-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1beta/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease cathepsin B as measured by the breakdown of a cathepsin B substrate. Inhibition of cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1beta production, pyronecrosis, and HMGB1 release. Nonpathogenic Neisseria strains (Neisseria cinerea and Neisseria flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned medium from N. gonorrhoeae contains factors capable of initiating the NLRP3-mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1beta secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae.

  6. Increasing Trend of Resistance to Penicillin, Tetracycline, and Fluoroquinolone Resistance in Neisseria gonorrhoeae from Pakistan (1992–2009)

    PubMed Central

    Jabeen, Kauser; Nizamuddin, Summiya; Irfan, Seema; Khan, Erum; Malik, Faisal; Zafar, Afia

    2011-01-01

    Emergence and spread of drug resistant Neisseria gonorrhoeae is global concern. We evaluated trends of antimicrobial resistance in Neisseria gonorrhoeae over years 1992–2009 in Pakistan. Resistance rates were compared between years (2007–2009) and (1992–2006). Antimicrobial susceptibility testing was performed and interpreted according to Clinical Laboratory Standards Institute (CLSI) criteria using the disk diffusion methodology against penicillin, ceftriaxone, tetracycline and ofloxacin. Additional antibiotics tested in 100 strains isolated during 2007–2009, included cefotaxime, cefoxitin, cefuroxime, cefipime, ceftazidime, ceftizoxime, cefixime, cefpodoxime, spectinomycin and azithromycin. Neisseria gonorrhoeae ATCC 49226 was used as control. Chi-square for trend analysis was conducted to assess resistance trend over the study period. During study period significant increase in combined resistance to penicillin, tetracycline and ofloxacin was observed (P value <0.01). Resistance rates during the two study period also increased significantly (P value <0.01). Ceftriaxone resistance was not observed. None of the isolates were found to be resistant or with intermediate sensitivity to additional antibiotics. Our findings suggest that penicillin, ciprofloxacin, tetracycline should not be used in the empirical treatment of gonorrhea in Pakistan. Ceftriaxone and cefixime should be the first line therapy; however periodic MICs should be determined to identify emergence of strains with reduced susceptibility. PMID:21941568

  7. 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.

  8. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry for the identification of Neisseria gonorrhoeae.

    PubMed

    Buchanan, R; Ball, D; Dolphin, H; Dave, J

    2016-09-01

    Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared with the API NH biochemical method for the identification of Neisseria gonorrhoeae in routine clinical samples. A retrospective review of laboratory records for 1090 isolates for which both biochemical and MALDI-TOF MS identifications were available was performed. Cases of discrepant results were examined in detail for evidence supportive of a particular organism identification. Of 1090 isolates, 1082 were identified as N. gonorrhoeae by API NH. MALDI-TOF MS successfully identified 984 (91%) of these after one analysis, rising to 1081 (99.9%) after two analyses, with a positive predictive value of 99.3%. For those isolates requiring a repeat analysis, failure to generate an identifiable proteomic signature was the reason in 76% of cases, with alternative initial identifications accounting for the remaining 24%. MALDI-TOF MS identified eight isolates as N. gonorrhoeae that were not identified as such by API NH-examination of these discrepant results suggested that the MALDI-TOF MS identification may be the more reliable. MALDI-TOF MS is at least as accurate and reliable a method of identifying N. gonorrhoeae as API NH. We propose that MALDI-TOF MS could potentially be used as a single method for N. gonorrhoeae identification in routine cases by laboratories with access to this technology.

  9. Whole-genome sequencing to determine Neisseria gonorrhoeae transmission: an observational study

    PubMed Central

    Cole, Kevin; Cole, Michelle J; Cresswell, Fiona; Dean, Gillian; Dave, Jayshree; Thomas, Daniel Rh; Foster, Kirsty; Waldram, Alison; Wilson, Daniel J; Didelot, Xavier; Grad, Yonatan H; Crook, Derrick W; Peto, Tim EA; Walker, A Sarah

    2016-01-01

    Background New approaches are urgently required to address increasing rates of gonorrhoea and the emergence and global spread of antibiotic-resistant Neisseria gonorrhoeae. Whole genome sequencing (WGS) can be applied to study transmission and track resistance. Methods We performed WGS on 1659 isolates from Brighton, UK, and 217 additional isolates from other UK locations. We included WGS data (n=196) from the USA. Estimated mutation rates, plus diversity observed within patients across anatomical sites and probable transmission pairs, were used to fit a coalescent model to determine the number of single nucleotide polymorphisms (SNPs) expected between sequences related by direct/indirect transmission, depending on the time between samples. Findings We detected extensive local transmission. 281/1061(26%) Brighton cases were indistinguishable (0 SNPs) to ≥1 previous case(s), and 786(74%) had evidence of a sampled direct or indirect Brighton source. There was evidence of sustained transmission of some lineages. We observed multiple related samples across geographic locations. Of 1273 infections in Brighton, 225(18%) were linked to another case from elsewhere in the UK, and 115(9%) to a case from the USA. Four lineages initially identified in Brighton could be linked to 70 USA sequences, including 61 from a lineage carrying the mosaic penA XXXIV associated with reduced cefixime susceptibility. Interpretation We present a WGS-based tool for genomic contact tracing of N. gonorrhoeae and demonstrate local, national and international transmission. WGS can be applied across geographical boundaries to investigate gonorrhoea transmission and to track antimicrobial resistance. Funding Oxford NIHR Health Protection Research Unit and Biomedical Research Centre. PMID:27427203

  10. Identification of TEM-135 β-Lactamase in Neisseria gonorrhoeae Strains Carrying African and Toronto Plasmids in Argentina

    PubMed Central

    Gianecini, R.; Oviedo, C.; Littvik, A.; Mendez, E.; Piccoli, L.; Montibello, S.

    2014-01-01

    One hundred forty-three penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates obtained in Argentina from 2008 and 2012 were examined to detect blaTEM-135 genes and to investigate plasmid profiles and multiantigen sequence types. Forty-two PPNG isolates were found to carry TEM-135, and two contained a new TEM derivative characterized as TEM-220. The blaTEM-135 allele was carried by the Toronto/Rio and African plasmids. Molecular epidemiology revealed that two blaTEM-135 isolates were related to previously described isolates from Thailand and China, indicating a common evolutionary origin. PMID:25367903

  11. Identification of TEM-135 β-lactamase in Neisseria gonorrhoeae strains carrying African and Toronto plasmids in Argentina.

    PubMed

    Gianecini, R; Oviedo, C; Littvik, A; Mendez, E; Piccoli, L; Montibello, S; Galarza, P

    2015-01-01

    One hundred forty-three penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates obtained in Argentina from 2008 and 2012 were examined to detect blaTEM-135 genes and to investigate plasmid profiles and multiantigen sequence types. Forty-two PPNG isolates were found to carry TEM-135, and two contained a new TEM derivative characterized as TEM-220. The blaTEM-135 allele was carried by the Toronto/Rio and African plasmids. Molecular epidemiology revealed that two blaTEM-135 isolates were related to previously described isolates from Thailand and China, indicating a common evolutionary origin.

  12. In vitro antibiotic susceptibility of Neisseria gonorrhoeae in Jakarta, Indonesia.

    PubMed

    Lesmana, M; Lebron, C I; Taslim, D; Tjaniadi, P; Subekti, D; Wasfy, M O; Campbell, J R; Oyofo, B 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 microg/ml), thiamphenicol (MIC >/= 1.0 microg/ml), kanamycin (MIC >/= 16.0 microg/ml), penicillin (MIC >/= 2.0 microg/ml), gentamicin (MIC >/= 16.0 microg/ml), and norfloxacin (MIC = 0.5 microg/ml). These data showed that certain antibiotics previously used in the treatment of gonorrhea are no longer effective.

  13. The U.S. military's Neisseria gonorrhoeae resistance surveillance initiatives in selected populations of five countries.

    PubMed

    Tsai, Alice Y; Dueger, Erica; Macalino, Grace E; Montano, Silvia M; Tilley, Drake H; Mbuchi, Margaret; Wurapa, Eyako K; Saylors, Karen; Duplessis, Christopher C; Puplampu, Naiki; Garges, Eric C; McClelland, R Scott; Sanchez, Jose L

    2013-02-01

    Multi-drug resistant Neisseria gonorrhoeae (GC) threatens the successful treatment of gonorrhea. This report presents preliminary findings with regard to the prevalence of laboratory-confirmed GC and the extent of drug-resistance among sample populations in five countries. Between October 2010 and January 2013, 1,694 subjects (54% male; 45% female; 1% unknown) were enrolled and screened for the presence of laboratory-confirmed GC in the United States, Djibouti, Ghana, Kenya, and Peru. Overall, 108 (6%) of enrolled subjects tested positive for GC. Antimicrobial susceptibility testing results were available for 66 GC isolates. Resistance to at least three antibiotics was observed at each overseas site. All isolates tested in Ghana (n=6) were resistant to ciprofloxacin, penicillin, and tetracycline. In Djibouti, preliminary results suggested resistance to penicillin, tetracycline, ciprofloxacin, cefepime, and ceftriaxone. The small sample size and missing data prevent comparative analysis and limit the generalizability of these preliminary findings.

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

    PubMed Central

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

    2016-01-01

    The emergence of ceftriaxone-resistant Neisseria gonorrhoeae is 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 in Neisseria 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 in penA (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 mutated penA gene into nonresistant strains increased the MIC between 2.0- and 5.3-fold, and transformation of mutated ftsX increased the MIC between 3.3- and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, while pilM, pilN, and pilQ were 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 of Neisseria gonorrhoeae to ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry. PMID:26787702

  15. Influence of conserved and hypervariable genetic markers on genotyping circulating strains of Neisseria gonorrhoeae.

    PubMed

    Vidovic, Sinisa; Horsman, Greg B; Liao, Mingmin; Dillon, Jo-Anne R

    2011-01-01

    Presently there is no vaccine against Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community (Saskatchewan, Canada) over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced.

  16. Cell envelope of Neisseria gonorrhoeae: penicillin enhancement of peptidoglycan hydrolysis.

    PubMed Central

    Wegener, W S; Hebeler, B H; Morse, S A

    1977-01-01

    The addition of 10 microgram of penicillin G per ml to log-phase cultures of Neisseria gonorrhoeae JW-31 (minimum inhibitory concentration for penicillin G, less than 0.007 microgram/ml) resulted in cellular lysis after a lag of 30 min. Penicillin markedly decreased the rate of peptidoglycan synthesis and enhanced the rate of hydrolysis of existing peptidoglycan. Hydrolysis was initiated immediately after addition of penicillin; cellular lysis did not occur until a considerable percentage of the peptidoglycan had been degraded. Cellular lysis was not due to penicillin per se but resulted from inhibition of cell wall synthesis. When cells were grown in media buffered with N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid at pH 6, penicillin did not cause lysis; however, at this pH, peptidoglycan hydrolysis occurred and cells lost viability at the same rate as in the control (pH 7.2). We suggest that the stability of gonococci grown at pH 6 is related to increased stability of the outer membrane. The penicillin-enhanced rate of peptidoglycan hydrolysis decreased approximately 50% at pH 6.0. Penicillin-enhanced lysis, peptidoglycan hydrolysis, and loss of viability were also markedly reduced in cells grown at 28 degrees C. PMID:22492

  17. Opa Expression Correlates with Elevated Transformation Rates in Neisseria gonorrhoeae

    PubMed Central

    Hill, Stuart A.

    2000-01-01

    Neisseria gonorrhoeae is naturally competent for DNA transformation. Under most conditions encountered in vivo, gonococci express one or more opacity (Opa) proteins on their surfaces. Recently, it was shown that DNA preferentially binds to the surfaces of Opa-expressing organisms compared to those of isogenic Opa-negative strains, presumably due to the numerous cationic residues in the predicted surface-exposed loops of the Opa protein. This study examined whether Opa-DNA interactions actually influence DNA transformation of the gonococcus. The data show that Opa-expressing gonococci are more efficient recipients of DNA for transformation and are more susceptible to exogenous DNase I treatment at early stages during the DNA transformation process than non-Opa expressors. Furthermore, inhibition of the transformation process was demonstrable for Opa+ populations when either nonspecific DNA or the polyanion heparin was used. Overall, the data suggest that Opa expression, with its presumptive positive surface charge contribution, promotes DNA transformation by causing a more prolonged sequestration of donor DNA at the cell surface, which translates into more efficient transformation over time. PMID:10613877

  18. Secreted single-stranded DNA is involved in the initial phase of biofilm formation by Neisseria gonorrhoeae.

    PubMed

    Zweig, Maria; Schork, Sabine; Koerdt, Andrea; Siewering, Katja; Sternberg, Claus; Thormann, Kai; Albers, Sonja-Verena; Molin, Søren; van der Does, Chris

    2014-04-01

    Neisseria gonorrhoeae is an obligate human pathogen that colonizes the genital tract and causes gonorrhoea. Neisseria gonorrhoeae can form biofilms during natural cervical infections, on glass and in continuous flow-chamber systems. These biofilms contain large amounts of extracellular DNA, which plays an important role in biofilm formation. Many clinical isolates contain a gonococcal genetic island that encodes a type IV secretion system (T4SS). The T4SS of N. gonorrhoeae strain MS11 secretes ssDNA directly into the medium. Biofilm formation, studied in continuous flow-chamber systems by confocal laser scanning microscopy (CLSM), was strongly reduced, especially in the initial phases of biofilm formation, in the presence of Exonuclease I, which specifically degrades ssDNA or in a ΔtraB strain that does not secrete ssDNA. To specifically detect ssDNA in biofilms using CLSM, a novel method was developed in which thermostable fluorescently labelled ssDNA- and ss/dsDNA-binding proteins were used to visualize ssDNA and total DNA in biofilms and planktonic cultures. Remarkably, mainly dsDNA was detected in biofilms of the ssDNA secreting strain. We conclude that the secreted ssDNA facilitates initial biofilm formation, but that the secreted ssDNA is not retained in mature biofilms.

  19. Incidence and treatment outcomes of pharyngeal Neisseria gonorrhoeae and Chlamydia trachomatis infections in men who have sex with men: a 13-year retrospective cohort study.

    PubMed

    Ota, Kaede V; Fisman, David N; Tamari, Itamar E; Smieja, Marek; Ng, Lai-King; Jones, Karen E; Diprima, Alessandro; Richardson, Susan E

    2009-05-01

    This study investigated the incidence and treatment outcomes of pharyngeal Neisseria gonorrhoeae and Chlamydia trachomatis cases at a Canadian clinic that mainly serves men who have sex with men. All patients with pharyngeal N. gonorrhoeae and C. trachomatis infections detected from 1 January 1995 through 31 December 2007 were identified. Original and test-of-cure N. gonorrhoeae culture isolates were compared using antibiotic susceptibility testing and N. gonorrhoeae multiantigen sequence typing. One hundred seventy-eight cases of pharyngeal N. gonorrhoeae infection and 97 cases of pharyngeal C. trachomatis infection were identified, primarily by culture methods. The mean incidence was 1.62 and 0.81 cases per 1000 visits per year for N. gonorrhoeae and C. trachomatis infection, respectively. Poisson regression modeling demonstrated a statistically significant surge of pharyngeal N. gonorrhoeae cases in 2007 after controlling for seasonal and long-term oscillation and long-term linear trends. Among patients with pharyngeal N. gonorrhoeae and C. trachomatis infection, 60.2% and 84.3%, respectively, would have been missed by relying on urine and urethral testing. Nine percent of patients with pharyngeal N. gonorrhoeae and 4.3% of patients with pharyngeal C. trachomatis infection who underwent test-of-cure procedures had at least 1 positive result. Antibiograms were not different in 8 of 10 pretreatment and posttreatment N. gonorrhoeae isolate pairs. N. gonorrhoeae multiantigen sequence typing results were identical in 2 of these cases. Public health records documented abstinence in both individuals. Nine percent of cases with pharyngeal N. gonorrhoeae and 4.3% of cases with pharyngeal C. trachomatis infection that underwent tests of cure had positive results. Available typing results suggest antibiotic treatment failure rather than reinfection. Specific antibiotic treatment regimens for pharyngeal N. gonorrhoeae and C. trachomatis infections need to be developed and

  20. 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.

  1. Neisseria gonorrhoeae survives within and modulates apoptosis and inflammatory cytokine production of human macrophages.

    PubMed

    Château, Alice; Seifert, H Steven

    2016-04-01

    The human-adapted organism Neisseria gonorrhoeae is the causative agent of gonorrhoea, a sexually transmitted infection. It readily colonizes the genital, rectal and nasalpharyngeal mucosa during infection. While it is well established that N. gonorrhoeae recruits and modulates the functions of polymorphonuclear leukocytes during infection, how N. gonorrhoeae interacts with macrophages present in infected tissue is not fully defined. We studied the interactions of N. gonorrhoeae with two human monocytic cell lines, THP-1 and U937, and primary monocytes, all differentiated into macrophages. Most engulfed bacteria were killed in the phagolysosome, but a subset of bacteria was able to survive and replicate inside the macrophages suggesting that those cells may be an unexplored cellular reservoir for N. gonorrhoeae during infection. N. gonorrhoeae was able to modulate macrophage apoptosis: N. gonorrhoeae induced apoptosis in THP-1 cells whereas it inhibited induced apoptosis in U937 cells and primary human macrophages. Furthermore, N. gonorrhoeae induced expression of inflammatory cytokines in macrophages, suggesting a role for macrophages in recruiting polymorphonuclear leukocytes to the site of infection. These results indicate macrophages may serve as a significant replicative niche for N. gonorrhoeae and play an important role in gonorrheal pathogenesis.

  2. Comparison of Mismatch Amplification Mutation Assay with DNA Sequencing for Characterization of Fluoroquinolone Resistance in Neisseria gonorrhoeae

    PubMed Central

    Sultan, Zafar; Nahar, Shamsun; Wretlind, Bengt; Lindback, Emma; Rahman, Motiur

    2004-01-01

    A mismatch amplification mutation assay (MAMA) was developed for identification of point mutations in quinolone resistance-determining region (QRDR) of gyrA at codons 91 and 95. MAMA PCR was used to detect mutations at codons 91 and 95 of gyrA in 117 Neisseria gonorrhoeae isolates (with ciprofloxacin MICs of 0.004 to >32 μg/ml) from Bangladesh during 1997 to 2001. The QRDR regions of the gyrA genes from 31 randomly selected isolates were sequenced, and the results were compared with those of MAMA PCR. Using mismatch PCR, a mutation at Ser91 could be detected in all 27 (resistant and intermediate) isolates, and an Asp95-to-Gly95 mutation could be detected in all 15 isolates, as detected by sequencing. MAMA PCR offers a simple, inexpensive, rapid, and easier alternative for detection of point mutations in fluoroquinolone resistance in N. gonorrhoeae. PMID:14766821

  3. Examination of Neisseria Gonorrhoeae Opacity Protein Expression During Experimental Murine Genital Tract Infection

    DTIC Science & Technology

    2005-01-01

    of wild-type Chinese hamster ovary ( CHO ) cells and isogenic mutants with deficiencies in HSPG biosynthesis was used to identify the HSPG-binding...34Vitronectin mediates internalization of Neisseria gonorrhoeae by Chinese hamster ovary cells ." Infect Immun 65(3): 964-70. 57. Duensing, T. D. and J. P...Seifert, Northwestern University) was implemented to insert the opaB::phoA fusion into a non- essential locus of the genome of N. gonorrhoeae strain

  4. 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.

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

    PubMed Central

    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.

    2016-01-01

    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

  6. Neisseria gonorrhoeae with reduced susceptibility to azithromycin--San Diego County, California, 2009.

    PubMed

    2011-05-13

    A single 2 g dose of azithromycin effectively treats genitourinary infections caused by susceptible Neisseria gonorrhoeae and has been used to treat uncomplicated gonorrhea in persons with cephalosporin allergy. However, azithromycin is not recommended as monotherapy because of concern over the emergence of resistance. Instead, a 1 g dose of azithromycin is recommended as a component of dual therapy for gonorrhea, in conjunction with a cephalosporin (i.e., 250 mg of ceftriaxone or 400 mg of cefixime, if ceftriaxone is not an option). During January 1992--July 2009, of 87,566 N. gonorrhoeae isolates tested for azithromycin susceptibility by CDC's national Gonoccoccal Isolate Surveillance Project (GISP), only 39 (0.04%) had minimum inhibitory concentrations (MICs) ≥8 µg/mL (including 25 with 8 µg/mL and 14 with 16 µg/mL), indicating reduced susceptibility; none of the isolates were collected in San Diego County, California (CDC, unpublished data, 2011). During August--October 2009, five of 55 (9.1%) N. gonorrhoeae isolates obtained from men with symptomatic urethritis tested at San Diego County's main municipal sexually transmitted disease (STD) clinic had high azithromycin MICs: three with 8µg/mL and two with 16 µg/mL. This report summarizes the laboratory and epidemiologic findings associated with this reduced susceptibility to azithromycin. In San Diego County, clinicians treating cephalosporin-allergic patients with a 2 g dose of azithromycin for uncomplicated gonorrhea are advised to obtain tests of cure 3 weeks after treatment and to recommend sexual abstinence until a negative test result for gonorrhea is achieved. Continued surveillance for antibiotic resistance and effective control efforts are critical for gonorrhea prevention.

  7. First nationwide study regarding ceftriaxone resistance and molecular epidemiology of Neisseria gonorrhoeae in China.

    PubMed

    Chen, Shao-Chun; Yin, Yue-Ping; Dai, Xiu-Qin; Unemo, Magnus; Chen, Xiang-Sheng

    2016-01-01

    Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health concern worldwide. This is the first nationwide study, performed within the China Gonococcal Antimicrobial Susceptibility Programme (China-GASP), regarding AMR, including ceftriaxone genetic resistance determinants, and molecular epidemiology of gonococci in China. Gonococcal isolates (n = 1257) from consecutive patients were collected at 11 sentinel sites distributed across China during 2012-13. Susceptibility to ceftriaxone, spectinomycin, ciprofloxacin and tetracycline was determined using the agar dilution method. Ceftriaxone resistance determinants penA and penB were examined using sequencing. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed for molecular epidemiology. Among isolates, 0.2% were resistant to spectinomycin, 4.4% to ceftriaxone, 42.9% to tetracyclines (high-level resistance) and 99.8% to ciprofloxacin. Among 890 sequenced isolates, 16 (1.8%) possessed a penA mosaic allele; 4 of these isolates belonged to the MDR internationally spread NG-MAST genogroup G1407 (first description in China). Non-mosaic penA alleles with an A501T mutation and an A102D alteration in porB1b were statistically associated with decreased susceptibility/resistance to ceftriaxone. NG-MAST G10339, G1424 and G1053 were associated with decreased susceptibility/resistance to ceftriaxone. In China, ceftriaxone and spectinomycin can continue to be recommended for gonorrhoea treatment, with the possible exception of Hainan and Sichuan provinces where ceftriaxone resistance exceeded 5% and AMR surveillance needs to be strengthened. Molecular approaches including genotyping and AMR determinant analysis can be valuable to supplement and enhance conventional surveillance of gonococcal AMR in China. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Neisseria gonorrhoeae downregulates expression of the human antimicrobial peptide LL-37.

    PubMed

    Bergman, Peter; Johansson, Linda; Asp, Vendela; Plant, Laura; Gudmundsson, Gudmundur H; Jonsson, Ann-Beth; Agerberth, Birgitta

    2005-07-01

    Neisseria gonorrhoeae is a human pathogen causing the sexually transmitted disease gonorrhoeae. The bacteria preferentially attach to and invade epithelial cells of the genital tract. As these cells previously have been shown to express the human cathelicidin LL-37, we wanted to investigate the role of LL-37 during N. gonorrhoeae infection. The cervical epithelial cell line ME180 was utilized and the expression of LL-37 was confirmed on both peptide and transcriptional levels. Moreover, LL-37 exhibited potent in vitro activity against N. gonorrhoeae. Interestingly, the transcript and peptide levels of LL-37 were downregulated during infection, according to quantitative real-time polymerase chain reaction (PCR) and immunocyto-chemistry. The downregulation was most prominent with pathogenic strains of Neisseria, while non-pathogenic strains such as Neisseria lactamica and Escherichia coli only exhibited moderate effects. Heat-killed N. gonorrhoeae had no impact on the downregulation, emphasizing the importance of live bacteria. The results in this study suggest that pathogenic Neisseria may gain a survival advantage in the female genital tract by downregulating LL-37 expression.

  9. Effects of tampon components on growth and dissemination of Neisseria gonorrhoeae.

    PubMed Central

    Arko, R J; Wong, K H; Finley-Price, K G; Rasheed, J K

    1982-01-01

    Six components used in vaginal tampons were tested for their effects on a strain of Neisseria gonorrhoeae isolated from a patient with disseminated infection. Tampon components containing carboxymethyl cellulose or its derivative prolonged the in-vitro survival of gonococci and, when injected with mucin into mice, significantly (P less than 0.0001) increased the dissemination of gonococci from the peritoneal cavity. In contrast, a component extracted from rayon tampons reduced in-vitro survival and appeared to suppress gonococcal dissemination in mice. Since tampons are used by a large number of women at a time when the risk of developing complications from venereal infections are increased, their effects on potential urogenital pathogens warrant further study. PMID:6802439

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

    PubMed Central

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

    2016-01-01

    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 103 to 104 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

  11. 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.

  12. 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.

  13. 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

  14. Lectin characterization of gonococci from an outbreak caused by penicillin-resistant Neisseria gonorrhoeae.

    PubMed Central

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

    1985-01-01

    A total of 40 Neisseria gonorrhoeae isolates, representing 19 penicillin-resistant isolates (from 8 heterosexual patients and 11 homosexual patients) and 21 penicillin-susceptible isolates (from 15 heterosexual patients and 6 homosexual patients) and obtained from the same geographic area, were examined. Lectin agglutination patterns were based on the reactivity of the isolates with the following 14 lectins: concanavalin A, Lens culinaris, Trichosanthes kinlowii, Griffonia simplicifolia I, Arachis hypogeae (peanut agglutinin), Glycine max (soybean agglutinin), Dolichos bifloris, Griffonia simplicifolia II, Solanum tuberosum (potato starch agglutinin), Triticum vulgaris (wheat germ agglutinin), Limax flavus, Phaseolus vulgaris, Ulex europaeus I, and Lotus tetragonolobus. All isolates were serotyped with monoclonal antibodies specific for gonococcal outer membrane protein I and auxotyped, and the plasmid content was determined. Resistant patient isolates were selected for their decreased penicillin susceptibility, and control isolates were selected for their penicillin susceptibility. Even though the patient isolates demonstrated resistance to penicillin, no phenotypic differences in lectin-grouping patterns were demonstrated between the two study groups; i.e., two predominant lectin groups were observed. No resistance-associated plasmids were detected. All patient isolates were serogroup IB (serovars IB-1, IB-2, and IB-4), whereas 12 of 21 control isolates were serogroup IA (P less than 0.05). Isolates obtained from different anatomical sites in the same patient (cervical and rectal) agreed with regard to lectin patterns and serovars but not auxotypes. PMID:3935658

  15. 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

  16. 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.

  17. Neisseria gonorrhoeae survives within and modulates apoptosis and inflammatory cytokine production of human macrophages

    PubMed Central

    Château, Alice; Seifert, H. Steven

    2017-01-01

    The human-adapted organism Neisseria gonorrhoeae is the causative agent of gonorrhea a sexually transmitted infection. It readily colonizes the genital, rectal, and nasalpharyngal mucosa during infection. While it is well-established that N. gonorrhoeae recruits and modulates the functions of polymorphonuclear leukocytes (PMNs) during infection, how N. gonorrhoeae interacts with macrophages present in infected tissue is not fully defined. We studied the interactions of N. gonorrhoeae with two human monocytic cell lines, THP-1 and U937, and primary monocytes, all differentiated into macrophages. Most engulfed bacteria were killed in the phagolysosome, but a subset of bacteria were able to survive and replicate inside the macrophages suggesting that those cells may be an unexplored cellular reservoir for N. gonorrhoeae during infection. N. gonorrhoeae was able to modulate macrophage apoptosis, N. gonorrhoeae induced apoptosis in THP-1 cells whereas it inhibited induced apoptosis in U937 cells and primary human macrophages. Furthermore, N. gonorrhoeae induced expression of inflammatory cytokines in macrophages, suggesting a role for macrophages in recruiting PMNs to the site of infection. These results indicate macrophages may serve as a significant replicative niche for N. gonorrhoeae and play an important role in gonorrheal pathogenesis. PMID:26426083

  18. pilS loci in Neisseria gonorrhoeae are transcriptionally active

    PubMed Central

    Wachter, Jenny; Masters, Thao L.; Wachter, Shaun; Mason, Joanna

    2015-01-01

    Piliation is an important virulence determinant for Neisseria gonorrhoeae. PilE polypeptide is the major protein subunit in the pilus organelle and engages in extensive antigenic variation due to recombination between pilE and a pilS locus. pilS were so-named as they are believed to be transcriptionally silent, in contrast to the pilE locus. In this study, we demonstrate the presence of a small, pil-specific RNA species. Through using a series of pilE deletion mutants, we show by Northern blotting and quantitative reverse transcriptase PCR analysis (qRT-PCR), that these smaller RNA species are not derived from the primary pilE transcript following some processing events, but rather, arose through transcription of the pilS loci. Small transcriptome analysis, in conjunction with analysis of pilS recombinants, identified both sense and anti-sense RNAs originating from most, but not all, of the pilS gene copies. Focusing on the MS11 pilS6 locus, we identified by site-directed mutagenesis a sense promoter located immediately upstream of pilS6 copy 2, as well as an anti-sense promoter immediately downstream of pilS6 copy 1. Whole transcriptome analysis also revealed the presence of pil-specific sRNA in both gonococci and meningococci. Overall, this study reveals an added layer of complexity to the pilE/pilS recombination scheme by demonstrating pil-specific transcription within genes that were previously thought to be transcriptionally silent. PMID:25701734

  19. Neisseria gonorrhoeae and Chlamydia trachomatis among Women Reporting Extragenital Exposures

    PubMed Central

    Trebach, Joshua D.; Chaulk, C. Patrick; Page, Kathleen R.; Tuddenham, Susan; Ghanem, Khalil G.

    2015-01-01

    Introduction The CDC recommends pharyngeal screening of Neisseria gonorrhoeae (GC) and rectal screening of GC and Chlamydia trachomatis (CT) in HIV-infected and at-risk men who have sex with men (MSM). There are currently no recommendations to routinely screen women at extragenital sites. We define the prevalence of extragenital GC and CT in women attending two urban STD clinics in Baltimore City and compare it to the prevalence of extragenital infections in MSM and men who have sex with women (MSW). Methods All patients who reported extragenital exposures in the preceding 3 months, who presented for care between 6/1/2011 and 5/31/2013, and were tested for GC and CT using nucleic acid amplification tests at all sites of exposure were included in the analyses. We used logistic regression models to identify risk factors for extragenital infections. Results 10,389 patients were included in this analysis (88% African American, mean age 29 years, 42% women, 7% MSM, 2.5% HIV infected). The prevalence estimates of any extragenital GC and CT were: 2.4% GC and 3.7% CT in women; 2.6% GC and 1.6% CT in MSW; 18.9% GC and 11.8% CT in MSM. Among women, 30.3% of GC infections and 13.8% of CT infections would have been missed with urogenital-only testing. Unlike MSM, age ≤ 18 years was the strongest predictor of extragenital infections in women. Conclusions Although the prevalence of extragenital gonorrhea and chlamydia is highest in MSM, a significant number of GC and CT infections in young women would be missed with genital-only testing. Cost-effectiveness analyses are needed to help inform national guidelines on extragenital screening in young women. PMID:25868133

  20. Neisseria gonorrhoeae and extended-spectrum cephalosporins in California: surveillance and molecular detection of mosaic penA

    PubMed Central

    2013-01-01

    Background The spread of Neisseria gonorrhoeae strains with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins is a major public health problem. While much work has been performed internationally, little is known about the genetics or molecular epidemiology of N. gonorrhoeae isolates with reduced susceptibility to extended-spectrum cephalosporins in the United States. The majority of N. gonorrhoeae infections are diagnosed without a live culture. Molecular tools capable of detecting markers of extended-spectrum cephalosporin resistance are needed. Methods Urethral N. gonorrhoeae isolates were collected from 684 men at public health clinics in California in 2011. Minimum inhibitory concentrations (MICs) to ceftriaxone, cefixime, cefpodoxime and azithromycin were determined by Etest and categorized according to the U.S. Centers for Disease Control 2010 alert value breakpoints. 684 isolates were screened for mosaic penA alleles using real-time PCR (RTPCR) and 59 reactive isolates were subjected to DNA sequencing of their penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). To increase the specificity of the screening RTPCR in detecting isolates with alert value extended-spectrum cephalosporin MICs, the primers were modified to selectively amplify the mosaic XXXIV penA allele. Results Three mosaic penA alleles were detected including two previously described alleles (XXXIV, XXXVIII) and one novel allele (LA-A). Of the 29 isolates with an alert value extended-spectrum cephalosporin MIC, all possessed the mosaic XXXIV penA allele and 18 were sequence type 1407, an internationally successful strain associated with multi-drug resistance. The modified RTPCR detected the mosaic XXXIV penA allele in urethral isolates and urine specimens and displayed no amplification of the other penA alleles detected in this study. Conclusion N. gonorrhoeae isolates with mosaic penA alleles and reduced susceptibility to extended

  1. Neisseria gonorrhoeae and extended-spectrum cephalosporins in California: surveillance and molecular detection of mosaic penA.

    PubMed

    Gose, Severin; Nguyen, Duylinh; Lowenberg, Daniella; Samuel, Michael; Bauer, Heidi; Pandori, Mark

    2013-12-04

    The spread of Neisseria gonorrhoeae strains with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins is a major public health problem. While much work has been performed internationally, little is known about the genetics or molecular epidemiology of N. gonorrhoeae isolates with reduced susceptibility to extended-spectrum cephalosporins in the United States. The majority of N. gonorrhoeae infections are diagnosed without a live culture. Molecular tools capable of detecting markers of extended-spectrum cephalosporin resistance are needed. Urethral N. gonorrhoeae isolates were collected from 684 men at public health clinics in California in 2011. Minimum inhibitory concentrations (MICs) to ceftriaxone, cefixime, cefpodoxime and azithromycin were determined by Etest and categorized according to the U.S. Centers for Disease Control 2010 alert value breakpoints. 684 isolates were screened for mosaic penA alleles using real-time PCR (RTPCR) and 59 reactive isolates were subjected to DNA sequencing of their penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). To increase the specificity of the screening RTPCR in detecting isolates with alert value extended-spectrum cephalosporin MICs, the primers were modified to selectively amplify the mosaic XXXIV penA allele. Three mosaic penA alleles were detected including two previously described alleles (XXXIV, XXXVIII) and one novel allele (LA-A). Of the 29 isolates with an alert value extended-spectrum cephalosporin MIC, all possessed the mosaic XXXIV penA allele and 18 were sequence type 1407, an internationally successful strain associated with multi-drug resistance. The modified RTPCR detected the mosaic XXXIV penA allele in urethral isolates and urine specimens and displayed no amplification of the other penA alleles detected in this study. N. gonorrhoeae isolates with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins are currently

  2. 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

  3. Anaerobiosis increases resistance of Neisseria gonorrhoeae to O2-independent antimicrobial proteins from human polymorphonuclear granulocytes.

    PubMed Central

    Casey, S G; Shafer, W M; Spitznagel, J K

    1985-01-01

    We investigated the in vitro resistance of Neisseria gonorrhoeae FA19 to the O2-independent antimicrobial systems of human polymorphonuclear leukocytes. Acid extracts of polymorphonuclear leukocyte granules (crude granule extracts) and a purified granule protein (57 kilodaltons) were, at low concentrations, bactericidal for gonococci under aerobic conditions that permitted growth. However, they were less effective under anaerobic conditions that imposed bacteriostasis. We found that adding sodium nitrite to reduced growth media permitted the growth of strain FA19 in an anaerobic environment. Under these conditions with nitrite, anaerobic cultures of strain FA19 were no more resistant to the crude granule extract and the 57-kilodalton protein than aerobic cultures. In contrast, Salmonella typhimurium SL-1004, a facultative anaerobe, was readily killed by both the crude granule extract and the 57-kilodalton antimicrobial protein regardless of the presence or absence of free molecular oxygen. This is the first demonstration that an isolated antimicrobial protein from polymorphonuclear leukocyte granules is active against bacteria under anaerobic conditions. Our results also indicated that the efficacy of human polymorphonuclear leukocyte O2-independent killing of N. gonorrhoeae may, in part, be inhibited by bacteriostatic conditions imposed by hypoxia. Images PMID:3917976

  4. Conservation of peptide structure of outer membrane protein-macromolecular complex from Neisseria gonorrhoeae.

    PubMed Central

    Hansen, M V; Wilde, C E

    1984-01-01

    The structural conservation of an outer membrane protein of Neisseria gonorrhoeae called OMP-MC (outer membrane protein-macromolecular complex) was investigated by determining the isoelectric point and amino-terminal amino acid sequence of the protein and by using high-performance liquid chromatography for comparative tryptic peptide mapping. The 76,000-dalton subunits generated by reduction and alkylation of the native 800,000-dalton complex from six test strains focused in ultrathin gels as bands of restricted heterogeneity at an approximate pI of 7.6. Dansyl chloride labeling indicated that all strains shared glycine as the amino-terminal amino acid. Sequence analysis of OMP-MC from two strains revealed no amino acid differences within the first 11 residues. Dual-label peptide maps revealed an extremely high degree of conservation of peptide structure. The results indicate that (i) OMP-MCs isolated from various strains of N. gonorrhoeae share structural homology and (ii) the 800,000-dalton complex is a homopolymer composed of 10 to 12 apparently identical 76,000-dalton subunits. Images PMID:6421738

  5. Biological properties of two distinct pilus types produced by isogenic variants of Neisseria gonorrhoeae P9.

    PubMed Central

    Lambden, P R; Robertson, J N; Watt, P J

    1980-01-01

    Isogenic variants from a single strain of Neisseria gonorrhoeae were shown to produce two distinct types of pili. These pili, designated alpha and beta, differed in both subunit molecular weight and in ability to attach to buccal epithelial cells. Images PMID:6101593

  6. Mutation of a single lytic transglycosylase causes aberrant septation and inhibits cell separation of Neisseria gonorrhoeae.

    PubMed

    Cloud, Karen A; Dillard, Joseph P

    2004-11-01

    The function of lytic peptidoglycan transglycosylases is poorly understood. Single lytic transglycosylase mutants of Escherichia coli have no growth phenotype. By contrast, mutation of Neisseria gonorrhoeae ltgC inhibited cell separation without affecting peptidoglycan monomer production. Thus, LtgC has a dedicated function in gonococcal cell division.

  7. Comparison of Serologic and Genetic porB-Based Typing of Neisseria gonorrhoeae: Consequences for Future Characterization

    PubMed Central

    Unemo, Magnus; Olcén, Per; Albert, Jan; Fredlund, Hans

    2003-01-01

    Due to temporal changes in the epidemiology of gonorrhea, a precise characterization of Neisseria gonorrhoeae is essential. In the present study genetic heterogeneity in the porB genes of N. gonorrhoeae was examined, and serovar determination was compared to porB gene sequencing. Among 108 N. gonorrhoeae isolates, phylogenetic analysis of the entire porB alleles (924 to 993 bp) identified 87 unique sequences. By analyzing only the four to six most heterogeneous porB gene regions (174 to 363 bp), 86 out of these 87 genetic variants were identified. Consequently, analysis of shorter highly variable regions of the porB gene generates high-level discriminatory ability as well as fast, objective, reproducible, and portable data for epidemiological characterization of N. gonorrhoeae. Regarding putative antigenic epitopes of PorB for Genetic Systems monoclonal antibodies (MAbs), some of the previous findings were confirmed, but new findings were also observed. For several of the MAbs, however, the precise amino acid residues of PorB critical for single-MAb reactivity were difficult to identify. In addition, repeated serovar determination of 108 N. gonorrhoeae isolates revealed discrepancies for 34 isolates, mostly due to nonreproducible reactivity with single MAbs. Thus, the prospects of a genetic typing system with congruent translation of the serovar determination seem to be limited. In conclusion, analysis of short highly variable regions of the porB gene could form the basis for a fast molecular epidemiological tool for the examination of emergence and transmission of N. gonorrhoeae strains within the community. PMID:12958238

  8. Neisseria gonorrhoeae Crippled Its Peptidoglycan Fragment Permease To Facilitate Toxic Peptidoglycan Monomer Release

    PubMed Central

    Chan, Jia Mun

    2016-01-01

    ABSTRACT Neisseria gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci) are human pathogens that cause gonorrhea and meningococcal meningitis, respectively. Both N. gonorrhoeae and N. meningitidis release a number of small peptidoglycan (PG) fragments, including proinflammatory PG monomers, although N. meningitidis releases fewer PG monomers. The PG fragments released by N. gonorrhoeae and N. meningitidis are generated in the periplasm during cell wall remodeling, and a majority of these fragments are transported into the cytoplasm by an inner membrane permease, AmpG; however, a portion of the PG fragments are released into the extracellular environment through unknown mechanisms. We previously reported that the expression of meningococcal ampG in N. gonorrhoeae reduced PG monomer release by gonococci. This finding suggested that the efficiency of AmpG-mediated PG fragment recycling regulates the amount of PG fragments released into the extracellular milieu. We determined that three AmpG residues near the C-terminal end of the protein modulate AmpG's efficiency. We also investigated the association between PG fragment recycling and release in two species of human-associated nonpathogenic Neisseria: N. sicca and N. mucosa. Both N. sicca and N. mucosa release lower levels of PG fragments and are more efficient at recycling PG fragments than N. gonorrhoeae. Our results suggest that N. gonorrhoeae has evolved to increase the amounts of toxic PG fragments released by reducing its PG recycling efficiency. IMPORTANCE Neisseria gonorrhoeae and Neisseria meningitidis are human pathogens that cause highly inflammatory diseases, although N. meningitidis is also frequently found as a normal member of the nasopharyngeal microbiota. Nonpathogenic Neisseria, such as N. sicca and N. mucosa, also colonize the nasopharynx without causing disease. Although all four species release peptidoglycan fragments, N. gonorrhoeae is the least efficient at recycling and releases

  9. Whole genome sequencing of Neisseria gonorrhoeae reveals transmission clusters involving patients of mixed HIV serostatus.

    PubMed

    Peters, Joanna; Cresswell, Fiona; Amor, Lauren; Cole, Kevin; Dean, Gillian; Didelot, Xavier; De Silva, Dilrini; Eyre, David W; Paul, John

    2017-10-11

    Prevention and control of gonorrhoea depends on understanding the nature of sexual networks and risk factors for infection. We aimed to use high-resolution typing (whole genome sequencing (WGS)) of Neisseria gonorrhoeae isolates plus patient questionnaire data to gain insights into transmission patterns in a high prevalence setting. During a 9-month period (July 2014-March 2015), patients diagnosed with gonorrhoea attending sexual health service in Brighton, UK, were invited to provide anonymised detailed information by questionnaire about risk factors for infection. Questionnaire data plus WGS data from cultured isolates were analysed to yield information about sexual networks and risk factors for infection. 104/149 individuals who consented to participate in the study were culture positive. 97/104 (93%) were male. 80 self-reported to be men who have sex with men (MSM). 35/104 (34%) of patients were HIV positive. 51/104 (49%) individuals reported using geosocial networking applications to facilitate contact. Sex under the influence of drugs was reported by 16/34 (46%) of HIV-positive MSM, 17/41 (41%) of HIV-negative MSM and 5/15 (31%) of heterosexuals. WGS data were available for 100 isolates from 83 patients. 55 isolates (66%) belonged to genetically related subtypes involving one or more patients, who could be plausibly linked through recent direct or indirect transmission. Four transmission clusters containing 3-12 individuals were composed of MSM of mixed HIV serostatus. We show that data obtained from WGS of N. gonorrhoeae and enhanced epidemiological data obtained from patient questionnaires are mutually supportive and reveal insights into sexual networks. Our findings suggest that serosorting may have declined as a practice and indicate the importance of designing public health interventions that target infection risks associated with recreational drug use and contact made using geosocial networking applications. © Article author(s) (or their employer

  10. Neisseria gonorrhoeae-Mediated Inhibition of Apoptotic Signalling in Polymorphonuclear Leukocytes▿

    PubMed Central

    Chen, Adrienne; Seifert, H. Steven

    2011-01-01

    The human pathogen Neisseria gonorrhoeae recruits and interacts extensively with polymorphonuclear leukocytes (PMNs) during infection. N. gonorrhoeae is able to survive the bactericidal activity of these innate immune cells and can actively modulate PMN functions in vitro. PMNs are short-lived cells which readily undergo apoptosis, and thus the effect of N. gonorrhoeae infection on PMN survival has implications for whether PMNs might serve as an important site of bacterial replication during infection. We developed and validated an HL-60 myeloid leukemia cell culture model for PMN infection and used both these cells and primary PMNs to show that N. gonorrhoeae infection alone does not induce apoptosis and furthermore that N. gonorrhoeae can inhibit both spontaneous apoptosis and apoptosis induced by the intrinsic and extrinsic apoptosis inducers staurosporine (STS) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), respectively. N. gonorrhoeae infection also results in the activation of NF-κB signaling in neutrophils and induces secretion of an identical profile of proinflammatory cytokines and chemokines in both HL-60 cells and primary PMNs. Our data show that the HL-60 cell line can be used to effectively model N. gonorrhoeae-PMN interactions and that N. gonorrhoeae actively inhibits apoptosis induced by multiple stimuli to prolong PMN survival and potentially facilitate bacterial survival, replication, and transmission. PMID:21844239

  11. Neisseria gonorrhoeae Induces a Tolerogenic Phenotype in Macrophages to Modulate Host Immunity

    PubMed Central

    Candia, Enzo; Reyes-Cerpa, Sebastian; Villegas-Valdes, Bélgica; Neira, Tanya; Lopez, Mercedes; Maisey, Kevin; Tempio, Fabián; Ríos, Miguel; Acuña-Castillo, Claudio; Imarai, Mónica

    2013-01-01

    Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF-β1) but not the production of proinflammatory cytokine TNF-α, indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response. PMID:24204097

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

    PubMed

    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

    2016-04-01

    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. 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. 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. 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 surveillance. © The Author 2016. Published by Oxford

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

    PubMed Central

    Hemarajata, P.; Yang, S.; Soge, O. O.; Klausner, J. D.

    2016-01-01

    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

  14. Neisseria gonorrhoeae activates the proteinase Cathepsin B to mediate the signaling activities of the NLRP3 and ASC - containing inflammasome1

    PubMed Central

    Duncan, Joseph A.; Gao, Xi; Huang, Max; O’Connor, Brian P.; Thomas, Christopher E.; Willingham, Stephen B.; Bergstralh, Daniel T.; Jarvis, Gary A.; Sparling, P. Frederick; Ting, Jenny P-Y.

    2009-01-01

    Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1β and other inflammatory cytokines. Several NLR (Nucleotide binding domain, Leucine Rich Repeat) proteins are implicated in the formation of pro-IL-1β-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin,NALP3) is the primary NLR required for IL-1β/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease Cathepsin-B as measured by the breakdown of a Cathepsin B substrate. Inhibition of Cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1β production, pyronecrosis, and HMGB1 release. Non-pathogenic Neisseria strains (N. cinerea and N. flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned media from N. gonorrhoeae contains factors capable of initiating the NLRP3 mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1β secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae. PMID:19414800

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

    PubMed Central

    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

    2016-01-01

    The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZMr) 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 AZMr strains. The genomes of 213 AZMr 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 AZMr (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 AZMr 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 AZMr (MICs = 2 to 4 and 8 to 32 μg/ml, respectively). Low-level AZMr 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 AZMr strains arise independently and can then rapidly expand clonally in a region through local sexual networks. PMID:26935729

  16. Protocol for the molecular detection of antibiotic resistance mechanisms in Neisseria gonorrhoeae.

    PubMed

    Goire, Namraj; Sloots, Theo P; Nissen, Michael D; Whiley, David M

    2012-01-01

    Gonorrhoea is no longer an easily treatable ailment but rather is now a challenging disease in terms of antimicrobial resistance (AMR) with treatment options rapidly diminishing. The causative agent of gonorrhoea, Neisseria gonorrhoeae, has managed to develop resistance to almost every single drug used against it with the sole exception of extended spectrum cephalosporins. The situation is further exacerbated by the fact that not only are the rates of gonococcal infections on a steady rise globally, but tracking AMR is being undermined by the growing popularity of molecular methods at the expense of traditional bacterial culture in diagnostic laboratories. Recently, concerns have been raised over the emergence of a multi-resistant gonococci and the potential for untreatable gonorrhoea. Maintaining optimal epidemiological surveillance of gonococcal AMR remains an important aspect of gonorrhoea control. The development of molecular tools for tracking AMR in N. gonorrhoeae has the potential to further enhance such surveillance. In this chapter, we discuss nucleic acid amplification-based detection of AMR in gonorrhoea with a particular emphasis on chromosomal-mediated resistance to beta-lactam antibiotics.

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

    PubMed Central

    Fedler, Kelley A.; Scangarella-Oman, Nicole E.; Ross, James E.; Flamm, Robert K.

    2016-01-01

    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

  18. Proteolysis of bacterial membrane proteins by Neisseria gonorrhoeae type 2 immunoglobulin A1 protease.

    PubMed Central

    Shoberg, R J; Mulks, M H

    1991-01-01

    The immunoglobulin A1 (IgA1) proteases of Neisseria gonorrhoeae have been defined as having human IgA1 as their single permissive substrate. However, in recent years there have been reports of other proteins which are susceptible to the proteolytic activity of these enzymes. To examine the possibility that gonococcal membrane proteins are potential substrates for these enzymes, isolated outer and cytoplasmic membranes of N. gonorrhoeae were treated in vitro with exogenous pure IgA1 protease. Analysis of silver-stained sodium dodecyl sulfate-polyacrylamide gels of outer membranes indicated that there were two outer membrane proteins of 78 and 68 kDa which were cleaved by IgA1 protease in vitro in GCM 740 (a wild-type strain) and in two isogenic IgA1 protease-negative variants. Similar results were observed with a second gonococcal strain, F62, and its isogenic IgA1 protease-negative derivative. When GCM 740 cytoplasmic membranes were treated with protease, three minor proteins of 24.5, 23.5, and 21.5 kDa were cleaved. In addition, when outer membranes of Escherichia coli DH1 were treated with IgA1 protease, several proteins were hydrolyzed. While the identities of all of these proteolyzed proteins are unknown, the data presented indicate that there are several proteins found in the isolated membranes of gram-negative bacteria which are permissive in vitro substrates for gonococcal IgA1 protease. Images PMID:1713195

  19. Recall of LCx Neisseria gonorrhoeae assay and implications for laboratory testing for N. gonorrhoeae and Chlamydia trachomatis.

    PubMed

    2002-08-16

    On July 18, 2002, Abbott Laboratories (Abbott Park, IL) initiated a voluntary recall of its LCx Neisseria gonorrhoeae Assay (List Numbers 8A48-81 and 8A48-82) because, during routine quality assurance testing, several reagent lots failed to meet the analytical sensitivity described in the product insert. The cause of the failure is under investigation by the company. Abbott Laboratories has sent a letter to its customers informing them of this recall and the specific reagent lot numbers not meeting the analytical sensitivity.

  20. A comparison of agar dilution with the Calibrated Dichotomous Sensitivity (CDS) and Etest methods for determining the minimum inhibitory concentration of ceftriaxone against Neisseria gonorrhoeae.

    PubMed

    Enriquez, Rodney P; Goire, Namraj; Kundu, Ratan; Gatus, Barrie J; Lahra, Monica M

    2016-09-01

    The objective of this study was to compare the Calibrated Dichotomous Sensitivity (CDS) based agar dilution (CDS AD) method with the Etest (bioMérieux SA) methods using 2 method protocols for determining the minimum inhibitory concentration (MIC) of ceftriaxone against Neisseria gonorrhoeae. The two method protocols were the manufacturer's protocol for which the Clinical and Laboratory Standards Institute (CLSI) interpretative criteria for Neisseria gonorrhoeae could be applied, and the CDS-adapted protocol. Comparability of MIC data is critical for situation analysis and monitoring trends in global antimicrobial analysis. Two hundred and forty eight clinical isolates of N. gonorrhoeae and the World Health Organisation (WHO) N. gonorrhoeae reference strains were tested using the three methods. When compared, CDS AD and CDS Etest gave a regression R(2) value of 94%, the Pearson's correlation coefficient was 97% and a paired comparison within one log2 dilution was 98%. The CDS AD and the Etest (CLSI) comparison gave a regression R(2) value of 90%, a Pearson's correlation coefficient of 95% and a paired comparison within one log2 dilution was 98%. The comparison of the CDS Etest and CLSI Etest gave a regression R(2) value of 91%, a Pearson's correlation coefficient of 95% and a paired comparison within one log2 dilution of 99%. Importantly, there was robust agreement between all three methods for the categorization of susceptibility of Neisseria gonorrhoeae isolates using the WHO nominated breakpoint for decreased susceptibility to ceftriaxone (≥0.125 μg/mL). The CDS Etest method is comparable to agar dilution and the Etest methods for determining the MIC of ceftriaxone against N. gonorrhoeae. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Molecular and phenotypic characterization of penicillinase-producing Neisseria gonorrhoeae from Canadian sources.

    PubMed Central

    Dillon, J R; Duck, P; Thomas, D Y

    1981-01-01

    The incidence of penicillinase-producing Neisseria gonorrhoeae (PPNG) infections has increased in Canada during the past 2 years. Most of these cases were imported from abroad. The PPNG strains from these cases were characterized with respect to susceptibility to 11 antibiotics, auxotype, and plasmid content. Rosaramicin and cefuroxime proved to be the most potent of the antibiotics tested. The molecular characterization of the isolates indicated that all carried a 2.6-megadalton cryptic plasmid. Most of the PPNG isolates (87%) harbored a 4.5-megadalton penicillinase-producing plasmid, whereas only 13% harbored the 3.2-megadalton penicillinase-producing plasmid. In those cases where contact tracing was possible, the correlation linking strains of Far Eastern etiology with carriage of the 4.5-megadalton plasmid was upheld. The penicillinase-producing strains were typed auxanographically in either the proline-requiring (57%) or prototrophic groups (42%). Substrate hydrolysis profiles and analytical isoelectric focusing of crude beta-lactamase extracts of several isolates has reconfirmed that these strains elaborate a type TEM-1 enzyme. Several of the penicillinase-producing plasmids were also examined for plasmid stability. PMID:6791587

  2. In-vitro activity of 21 antimicrobial agents against Neisseria gonorrhoeae in Brussels.

    PubMed

    Gordts, B; Vanhoof, R; Hubrechts, J M; Dierickx, R; Coignau, H; Butzler, J P

    1982-02-01

    The minimum inhibitory concentrations (MIC) of 21 antimicrobial agents was measured for 80 strains of Neisseria gonorrhoeae isolated in Brussels in 1978. Bimodal distributions were found for penicillin G, ampicillin, amoxycillin, carbenicillin, and cephalexin. Of the strains, 17.5% were relatively resistant to penicillin G (MIC greater than 0.08 microgram/ml) 27.5% to ampicillin (MIC greater than 0.16 microgram/ml), 23.8% to amoxycillin, and 43.3% to carbenicillin. Cefotaxime was the most active antibiotic, with MICs in the nanogram range; 3.8% and 5% of the strains were relatively resistant to cephaloridine and cephalexin respectively, but no strains were resistant to cefazolin, cefuroxime, or cefotaxime. Resistance to tetracycline, doxycycline, minocycline, erythromycin, and spiramycin (MIC greater than 1 microgram/ml) was found in 6.3%, 2.5%, 5%, and 51.3% of the strains respectively. A very good correlation was present between chloramphenicol and thiamphenicol, with 16.3% and 10% of relatively resistant strains respectively. Only two isolates showed an MIC greater than 1.25 microgram/ml for rifampicin, and 10% of the strains needed greater than or equal to 12 microgram/ml of spectinomycin for complete inhibition of growth. A very high energy was found for the 20 : 1 combination of sulphamethoxazole and trimethoprim, with only one isolate resistant to this combination. None of the strains tested produced beta-lactamase.

  3. Molecular and biological analysis of eight genetic islands that distinguish Neisseria meningitidis from the closely related pathogen Neisseria gonorrhoeae.

    PubMed

    Klee, S R; Nassif, X; Kusecek, B; Merker, P; Beretti, J L; Achtman, M; Tinsley, C R

    2000-04-01

    The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis.

  4. Characterization of Neisseria cinerea, a nonpathogenic species isolated on Martin-Lewis medium selective for pathogenic Neisseria spp.

    PubMed

    Knapp, J S; Totten, P A; Mulks, M H; Minshew, B H

    1984-01-01

    An asaccharolytic, gram-negative, oxidase-positive diplococcus was isolated on Martin-Lewis medium from the cervix of a patient attending an arthritis clinic at Seattle Public Health Hospital, Seattle, Wash. This strain, NRL 32165, did not produce detectable acid from glucose, maltose, sucrose, fructose, mannitol, or lactose in either cystine Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) or modified oxidation-fermentation medium and was identified presumptively as a glucose-negative Neisseria gonorrhoeae strain, but was identified later as Neisseria cinerea on the basis of its biochemical reactions. Nitrate was not reduced, nitrite (0.001%, wt/vol) was reduced, and polysaccharide was not produced from sucrose. Proline, arginine, and cystine-cysteine were required for growth on defined medium. Strain NRL 32165 did not react with antigonococcal protein I monoclonal antibodies and did not produce immunoglobulin A protease. In DNA:DNA homology studies with N. gonorrhoeae NRL 8038 (F62) and N. cinerea type strain NRL 30003, strain NRL 32165 showed 95% homology relative to N. cinerea and 44% homology relative to N. gonorrhoeae. Thus, the identity of strain NRL 32165 was confirmed as N. cinerea (von Lingelsheim 1906) Murray 1939. Of all Neisseria spp., N. cinerea is most likely to be misidentified as a glucose-negative N. gonorrhoeae strain.

  5. 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

  6. An unusual case of penicillinase-producing Neisseria gonorrhoeae resistant to spectinomycin in California.

    PubMed

    Pon, E; Batchelor, R A; Howell, H B; Blakley, K G; Lake, J; Rice, R J; Biddle, J W; Conwill, D E

    1986-01-01

    We report a case of gonorrhea due to a penicillinase-producing strain of Neisseria gonorrhoeae resistant to spectinomycin in a 26-year-old man who had not been out of the United States for a year-and-a-half. His sexual contact also had no recent travel out of the United States. The genital and oropharyngeal infections were successfully treated with cefoxitin (1 g im) plus probenecid (1 g orally) and trimethoprim-sulfamethoxazole (80 mg of trimethoprim and 400 mg of sulfamethoxazole). The patient took nine of the latter tablets daily for five days. The organism was a serovar IB-3, proline-requiring auxotype. The patient's isolate contained both 2.6-megadalton and 4.4-megadalton plasmids. Measurement of minimal inhibitory concentrations (MICs) of antibiotics for the isolate confirmed the penicillin resistance and showed an MIC of spectinomycin of greater than 256 micrograms/ml. The epidemiologic investigation suggested that the source of the infection was a male contact with unusual clinical features, including bloody urethral discharge and a possible incubation period of 28 days.

  7. [Cloning and prokaryotic expression of the outer membrane protein gene PorB of Neisseria gonorrhoeae].

    PubMed

    Wang, Yan; Zhang, Lei; Zhang, Li; Wang, Han

    2011-07-01

    To construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB. A pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting. Restriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli. The prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.

  8. A Putatively Phase Variable Gene (dca) Required for Natural Competence in Neisseria gonorrhoeae but Not Neisseria meningitidis Is Located within the Division Cell Wall (dcw) Gene Cluster

    PubMed Central

    Snyder, Lori A. S.; Saunders, Nigel J.; Shafer, William M.

    2001-01-01

    A cluster of 18 open reading frames (ORFs), 15 of which are homologous to genes involved in division and cell wall synthesis, has been identified in Neisseria gonorrhoeae and Neisseria meningitidis. The three additional ORFs, internal to the dcw cluster, are not homologous to dcw-related genes present in other bacterial species. Analysis of the N. meningitidis strain MC58 genome for foreign DNA suggests that these additional ORFs have not been acquired by recent horizontal exchange, indicating that they are a long-standing, integral part of the neisserial dcw gene cluster. Reverse transcription-PCR analysis of RNA extracted from N. gonorrhoeae strain FA19 confirmed that all three ORFs are transcribed in gonococci. One of these ORFs (dca, for division cluster competence associated), located between murE and murF, was studied in detail and found to be essential for competence in the gonococcal but not in the meningococcal strains tested. Computer analysis predicts that dca encodes an inner membrane protein similar to hypothetical proteins produced by other gram-negative bacteria. In some meningococcal strains dca is prematurely terminated following a homopolymeric tract of G's, the length of which differs between isolates of N. meningitidis, suggesting that dca is phase variable in this species. A deletion and insertional mutation was made in the dca gene of N. gonorrhoeae strain FA19 and N. meningitidis strain NMB. This mutation abrogated the ability of the gonococci to be transformed with chromosomal DNA. Thus, we conclude that the dca-encoded gene product is an essential competence factor for gonococci. PMID:11157935

  9. A putatively phase variable gene (dca) required for natural competence in Neisseria gonorrhoeae but not Neisseria meningitidis is located within the division cell wall (dcw) gene cluster.

    PubMed

    Snyder, L A; Saunders, N J; Shafer, W M

    2001-02-01

    A cluster of 18 open reading frames (ORFs), 15 of which are homologous to genes involved in division and cell wall synthesis, has been identified in Neisseria gonorrhoeae and Neisseria meningitidis. The three additional ORFs, internal to the dcw cluster, are not homologous to dcw-related genes present in other bacterial species. Analysis of the N. meningitidis strain MC58 genome for foreign DNA suggests that these additional ORFs have not been acquired by recent horizontal exchange, indicating that they are a long-standing, integral part of the neisserial dcw gene cluster. Reverse transcription-PCR analysis of RNA extracted from N. gonorrhoeae strain FA19 confirmed that all three ORFs are transcribed in gonococci. One of these ORFs (dca, for division cluster competence associated), located between murE and murF, was studied in detail and found to be essential for competence in the gonococcal but not in the meningococcal strains tested. Computer analysis predicts that dca encodes an inner membrane protein similar to hypothetical proteins produced by other gram-negative bacteria. In some meningococcal strains dca is prematurely terminated following a homopolymeric tract of G's, the length of which differs between isolates of N. meningitidis, suggesting that dca is phase variable in this species. A deletion and insertional mutation was made in the dca gene of N. gonorrhoeae strain FA19 and N. meningitidis strain NMB. This mutation abrogated the ability of the gonococci to be transformed with chromosomal DNA. Thus, we conclude that the dca-encoded gene product is an essential competence factor for gonococci.

  10. Frequency of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma species in cervical samples.

    PubMed

    Rodrigues, M M; Fernandes, P Á; Haddad, J P; Paiva, M C; Souza, M Do Carmo M; Andrade, T C A; Fernandes, A P

    2011-01-01

    We investigated the relative frequencies of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma sp. in cervical samples. PCR analyses were performed in ectocervical and endocervical samples from 224 patients attending public health services in Belo Horizonte and Contagem, Minas Gerais Brazil. A high prevalence of colonisation of the cervix (6.3% for C. trachomatis, 4.0% for N. gonorrhoeae, 0.9% for M. genitalium, 21.9% for M. hominis, 38.4% for Ureaplasma sp.) was demonstrated not only for pathogens classically associated to cervicitis (C. trachomatis and N. gonorrhoeae), but also for M. hominis and Ureaplasma sp. These findings may be useful to guide more adequate diagnosis to interrupt transmission and to avoid negative impacts on the female reproductive tract.

  11. 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.

  12. Proposed interpretive criteria and quality control parameters for ofloxacin susceptibility testing of Neisseria gonorrhoeae.

    PubMed Central

    Fuchs, P C; Barry, A L; Baker, C; Murray, P R; Washington, J A

    1992-01-01

    A multilaboratory study designed to determine the in vitro susceptibility criteria and quality control parameters for ofloxacin against Neisseria gonorrhoeae was conducted according to the guidelines of the National Committee for Clinical Laboratory Standards. Proposed susceptibility breakpoints are MICs of less than or equal to 0.25 microgram/ml for the agar dilution test and greater than or equal to 31 mm for the disk diffusion test. A category for resistance could not be defined. Proposed acceptable quality control MICs for N. gonorrhoeae ATCC 49226 and Staphylococcus aureus ATCC 29213 range from 0.004 to 0.03 microgram/ml and 0.25 to 1.0 microgram/ml, respectively. With 5-micrograms ofloxacin disks, acceptable inhibitory zone diameters for S. aureus ATCC 25923 and the N. gonorrhoeae control strains range from 22 to 27 mm and 43 to 51 mm, respectively. PMID:1572960

  13. Meeting the public health challenge of multidrug- and extensively drug-resistant Neisseria gonorrhoeae.

    PubMed

    Tapsall, John W; Ndowa, Francis; Lewis, David A; Unemo, Magnus

    2009-09-01

    Globally, antimicrobial resistance (AMR) in Neisseria gonorrhoeae is increasing in prevalence, both within and across antibiotic classes, including extended-spectrum cephalosporins, raising concerns that gonorrhea may become untreatable in certain circumstances. The AMR surveillance that is essential to optimize standard treatments is often lacking or of poor quality in countries with high disease rates. Recent initiatives by the WHO to enhance global AMR surveillance that focus on multidrug- and extensively drug-resistant N. gonorrhoeae through revision of surveillance standards and use of a new panel of N. gonorrhoeae control strains are described. Keys to meeting these new challenges posed by gonococcal AMR remain the reduction in global burden of gonorrhea combined with implementation of wider strategies for general AMR control, and better understanding of mechanisms of emergence and spread of AMR.

  14. 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

  15. Potent and rapid antigonococcal activity of the venom peptide BmKn2 and its derivatives against different Maldi biotype of multidrug-resistant Neisseria gonorrhoeae.

    PubMed

    Arpornsuwan, Teerakul; Buasakul, Brisana; Jaresitthikunchai, Janthima; Roytrakul, Sittiruk

    2014-03-01

    The emergence of multidrug-resistant strains of Neisseria gonorrhoeae constitutes a serious threat to public health and necessitates the discovery of new types of antimicrobial agents. Among the 18 clinical isolates of N. gonorrhoeae with susceptible to spectinomycin, ceftriaxone and cefixime, 14 isolates were resistance to penicillin, tetracycline and ciprofloxacin, while 2 isolates were susceptible to tetracycline and another was penicillin intermediate isolate. Significant differences between laboratory strain and multidrug resistant strains were revealed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling and bioinformatics examination using the MALDI BioTyper software. However, Maldi Biotyper was not successfully separated ciprofloxacin-penicillin resistance and ciprofloxacin-tetracycline resistance from ciprofloxacin-penicillin-tetracycline resistant N. gonorrhoeae isolates. BmKn2 is a basic, alpha-helical peptide with no disulfide-bridge venom peptides that was first isolated from Buthus martensii Kasch. A panel of BmKn2 scorpion venom peptide and its derivatives of varying length and characteristics were synthesized chemically and evaluated for their ability to inhibit the growth of clinical N. gonorrhoeae isolates. Synthetic BmKn2 displayed potent activity against 18 clinical isolates of N. gonorrhoeae with MIC50 values of 6.9-27.6 μM. BmKn2 exerted its antibacterial activity via a bactericidal mechanism. Cyclic BmKn1 did not show antigonococcal activity. Decreasing the cationicity and helix percentage at the C-terminus of BmKn2 reduced the potency against N. gonorrhoeae. Taken together, the BmKn1 peptide can be developed as a topical therapeutic agent for treating multidrug-resistant strains of N. gonorrhoeae infections. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. 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

  17. Microwave-Accelerated Method for Ultra-Rapid Extraction of Neisseria gonorrhoeae DNA for Downstream Detection

    PubMed Central

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

    2016-01-01

    Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by the detection of the genomic target often involving 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 (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 present 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 minutes 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 towards the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

  18. 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.

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

    PubMed Central

    Unemo, Magnus; Shafer, William M.

    2015-01-01

    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

  20. 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. © 2011 New York Academy of Sciences.

  1. 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

  2. Molecular characterization of Neisseria gonorrhoeae on non-cultured specimens from multiple anatomic sites.

    PubMed

    Carannante, Anna; Ghisetti, Valeria; Dal Conte, Ivano; Gregori, Gabriella; Stella, Maria Laura; Vacca, Paola; Del Re, Simonetta; Stefanelli, Paola

    2017-01-01

    The aim of this study was to molecularly characterize Neisseria gonorrhoeae on non-cultured specimens collected from multiple anatomic sites. N. gonorrhoeae multiantigen sequence typing (NG-MAST) together with the gene sequence analysis of antimicrobial resistance (AMR) target genes were used. Seventeen genital and extra-genital samples from eight patients (7 were men who have sex with men, MSM, and 1 women who have sex with men, WSM) with gonorrhoea symptoms were analyzed. For 7, of the 8 patients, conventional culture method has been used to identify gonorrhoea. All the samples were tested with the rapid molecular method CEPHEID. Amplification and sequencing of porB and tbpB, to identify the Sequence Type (ST) by NG-MAST, and penA, mtrR, porB1b, ponA genes were also performed. Antimicrobial susceptibility by Etest, for the available culture positive samples, was carried out. For 7 patients the ST was obtained and for 6 the complete sequence analysis of the AMR target genes was also defined. For the majority of them, samples collected from multiple sites (oropharynx, rectum, vaginal and urethra) confirm the presence of the same gonorrhoea strain. In particular, for 5 patients the same STs and changes in the AMR target genes were identified. Molecular characterization on non-cultured or culture negative specimens for gonorrhoea can successfully be applied directly to genital and extra-genital samples. Thus permit to identify the presence of the same strain in patients with gonorrhoea infection in multiple anatomic sites and to predict the antimicrobial susceptibility pattern.

  3. A misleading false-negative result using Neisseria gonorrhoeae opa MGB multiplex PCR assay in patient's rectal sample due to partial mutations of the opa gene.

    PubMed

    Vahidnia, Ali; van Empel, Pieter Jan; Costa, Sandra; Oud, Rob T N; van der Straaten, Tahar; Bliekendaal, Harry; Spaargaren, Joke

    2015-07-01

    A 53-year-old homosexual man presented at his general practitioner (GP) practice with a suspicion of sexually transmitted infection. Initial NAAT screening was performed for Chlamydia trachomatis and Neisseria gonorrhoeae. The patient was positive for Neisseria gonorrhoeae both for his urine and rectal sample. The subsequent confirmation test for Neisseria gonorrhoeae by a second laboratory was only confirmed for the urine sample and the rectal sample was negative. We report a case of a potential false-negative diagnosis of Neisseria gonorrhoeae due to mutations of DNA sequence in the probe region of opa-MGB assay of the rectal sample. The patient did not suffer any discomfort as diagnosis of Neisseria gonorrhoeae in his urine sample had already led to treatment by prescribing the patient with Ceftriaxone 500 mg IV dissolved in 1 ml lidocaine 2% and 4 mL saline. The patient also received a prescription for Azithromycin (2x500 mg).

  4. ComE, a competence protein from Neisseria gonorrhoeae with DNA-binding activity.

    PubMed

    Chen, I; Gotschlich, E C

    2001-05-01

    Neisseria gonorrhoeae is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in N. gonorrhoeae, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene comE was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent Bacillus subtilis. The gene comE is present in four identical copies in the genomes of both N. gonorrhoeae and Neisseria meningitidis, located downstream of each of the rRNA operons. Single-copy deletion of comE in N. gonorrhoeae did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 x 10(4)-fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of comE has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of N. gonorrhoeae and N. meningitidis. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function.

  5. 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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. TaqMan real-time quantitative PCR assay for detection of fluoroquinolone-resistant Neisseria gonorrhoeae.

    PubMed

    Zhao, LiHong; Zhao, ShuPing

    2012-12-01

    It is noted that more than 99 % of fluoroquinolone resistance in Neisseria gonorrhoeae (QRNG) specimens have been shown to have the mutation of Ser91/Phe in the gyrA gene. In order to detect QRNG isolates as quickly as possible, the real-time TaqMan quantitative PCR assay was established for detection of the point mutation of Ser91/Phe in gyrA gene. The standard curve was generated automatically on ABI Prism PE7500. The correlation coefficient (r) of the standard curve was -0.9984 (R(2) = 0.9968), indicating a quietly precise log-linear relationship between the concentration of target DNA and the Ct value. Presently, correlated, cultured antimicrobial susceptibility testing of N. gonorrhoeae isolates continues to be the gold standard method for the detection of antimicrobial resistance. Comparison to the correlated, cultured antimicrobial susceptibility testing, the sensitivity and specificity of the established TaqMan assay for the detection of the QRNG specimens were 100 and 99 %, respectively. The TaqMan assay also allows for rapid detection of QRNG isolates without complex laboratory techniques. Therefore, real-time TaqMan quantitative PCR assay is a rapid, simple, highly sensitive, highly specific, and easy-to-perform method for the detection of the QRNG specimens. It can be applied as a quick screening method for QRNG isolates to help clinical determination of optimal treatment prescription.

  7. Identification of penicillinase producing Neisseria gonorrhoeae in Chile during clinical and microbiological study of gonococcal susceptibility to antimicrobial agents.

    PubMed Central

    Garcia Moreno, J; Dillon, J R; Arroyave, R; Maldonado, A; Fich, F; Salvo, A; Villalobos, D; Vincent, P; Pauze, M

    1987-01-01

    The first penicillinase producing isolates of Neisseria gonorrhoeae (PPNG) identified in Chile were discovered during a clinical and microbiological study to compare the efficacy of penicillin (4.8 MIU aqueous procaine penicillin G plus 1 g oral probenecid) and tetracycline (1.5 g followed by 500 mg four times daily for four days) treatment regimens for acute uncomplicated gonorrhoea. Penicillin treatment was effective in 93.1% (282) of 303 patients, whereas tetracycline was effective in 98.3% (233) of 237 patients. Six of the penicillin treatment failures were attributable to PPNG strains. In all, 21 PPNG strains were identified during the study. They were genetically identical, having a wild type auxotype, a WII/III serotype (serovar Bajk), and carrying cryptic and transfer plasmids and an Asian type penicillinase producing plasmid. In addition, 674 non-PPNG isolates were tested for their susceptibility to eight antimicrobials. Over 95% were sensitivie to 1 mg/l of penicillin, ampicillin, cefotaxime, cefuroxime, and erythromycin, over 90% were sensitive to 1 mg/l of tetracycline and 2 mg/l of thiamphenicol, and all were sensitive to spectinomycin. Of 226 non-PPNG isolates characterised for plasmid content and auxotype, 90% (205) were either wild type or proline requiring, 67% (153) carried only the cryptic plasmid, and a further 31% (71) carried both cryptic and transfer plasmids. Unusually, three of four isolates lacking the cryptic plasmid carried only the transfer plasmid. Images PMID:3102348

  8. Phase variable DNA repeats in Neisseria gonorrhoeae influence transcription, translation, and protein sequence variation

    PubMed Central

    Zelewska, Marta A.; Pulijala, Madhuri; Spencer-Smith, Russell; Mahmood, Hiba-Tun-Noor A.; Norman, Billie; Churchward, Colin P.; Calder, Alan

    2016-01-01

    There are many types of repeated DNA sequences in the genomes of the species of the genus Neisseria, from homopolymeric tracts to tandem repeats of hundreds of bases. Some of these have roles in the phase-variable expression of genes. When a repeat mediates phase variation, reversible switching between tract lengths occurs, which in the species of the genus Neisseria most often causes the gene to switch between on and off states through frame shifting of the open reading frame. Changes in repeat tract lengths may also influence the strength of transcription from a promoter. For phenotypes that can be readily observed, such as expression of the surface-expressed Opa proteins or pili, verification that repeats are mediating phase variation is relatively straightforward. For other genes, particularly those where the function has not been identified, gathering evidence of repeat tract changes can be more difficult. Here we present analysis of the repetitive sequences that could mediate phase variation in the Neisseria gonorrhoeae strain NCCP11945 genome sequence and compare these results with other gonococcal genome sequences. Evidence is presented for an updated phase-variable gene repertoire in this species, including a class of phase variation that causes amino acid changes at the C-terminus of the protein, not previously described in N. gonorrhoeae. PMID:28348872

  9. [Molecular epidemiology of Neisseria gonorrhoeae: effect of sex and sexual orientation on the distribution of gonococcal types].

    PubMed

    Kohl, P K; Henze, I; Kamionek, I; Krahl, D; Petzoldt, D

    1994-05-01

    Auxotype/serovar (A/S) classification enables precise characterisation of Neisseria gonorrhoeae. In the present study we evaluated whether sex and sexual preference of the patient influence the auxotype/serovar class of the infecting gonococcal strain. In male patients prototrophic/IB-3 was the most frequently isolated A/S class. By contrast, in female patients the A/S class (P)AH(U)/IA-1/2 was significantly (p < 0.005) more frequently isolated than in male patients. Analysis of our data according to sexual preference of the patients showed that in heterosexual patients the two mentioned A/S classes were leading, whereas in homo- and bisexual patients A/S classes prototrophic/IB-2 (p < 0.0001) and Pro/IB-2/16 (p < 0.0001) were isolated significantly more often. Our data are a strong indication that the host environment is also responsible for the selection of N. gonorrhoeae strains with certain typing characteristics.

  10. Benefits of Rapid Molecular Diagnosis of Chlamydia Trachomatis and Neisseria Gonorrhoeae Infections in Women Attending Family Planning Clinics.

    PubMed

    Bourgeois-Nicolaos, Nadège; Jaureguy, Françoise; Pozzi-Gaudin, Stéphanie; Masson, Claire; Guillet-Caruba, Christelle; Lavisse, Frédérique; Larmignat, Philippe; Benachi, Alexandra; Picard, Bertrand; Doucet-Populaire, Florence

    2015-11-01

    We evaluated the benefits of on-demand systematic screening for Chlamydia trachomatis and Neisseria gonorrhoeae using the Xpert CT/NG assay in 589 women attending family planning clinics. The sexually transmitted infection prevalence was 16.5% with 15.1% C. trachomatis and 3.1% N. gonorrhoeae infections. The on-demand test allowed for a quicker management of patients at high risk for sexually transmitted infections.

  11. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    PubMed

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  12. A novel factor H-Fc chimeric immunotherapeutic molecule against Neisseria gonorrhoeae

    PubMed Central

    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

    2015-01-01

    Neisseria gonorrhoeae (Ng), 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 Ng, 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 utility 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 Ng, 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 Ng isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10/15 (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 Ng. PMID:26773149

  13. In-vitro activity of antimicrobial agents against Neisseria gonorrhoeae in Brussels.

    PubMed

    Vanhoof, R; Vanderlinden, M P; Hubrechts, J M; Butzler, J P; Yourassowsky, E

    1978-10-01

    The minimum inhibitory concentrations (MICs) of 18 antimicrobial agents against 104 strains of Neisseria gonorrhoeae isolated in the Brussels area between January and October 1976 have been measured. The MICs for penicillin G, ampicillin, amoxycillin, carbenicillin, and cephalexin showed a bimodal distribution. The second modus strains of cephalexin (MIC = 6.25 microgram/ml) were relatively resistant to penicillin G (MIC greater than or equal to 0.08 microgram/ml). About 51% of all strains were relatively resistant to penicillin G, 40.5% to ampicillin (MIC greater than or equal to 0.16 microgram/ml), 46% to amoxycillin, and 47.5% to carbenicillin. For cephalexin and cephaloridine, 25% and 8.5% respectively of all strains were relatively resistant (MIC greater than 3.12 microgram/ml). For cefazolin all MICs fell into a range of 0.097--3.12 microgram/ml. Resistance to tetracycline, doxycycline, minocycline, erythromycin, and spiramycin (MIC greater than or equal to 1 microgram/ml) was found in 9.5%, 7%, 6%, 36.5%, and 71% respectively of all isolates. No strains were resistant to rifampicin. For chloramphenicol and thiamphenicol the MICs ranged from 0.39 to 12.5 microgram/ml and from 0.195 to 3.12 microgram/ml respectively. The results for sulphamethoxazole, trimethoprim, and the combination of sulphamethoxazole and trimethoprim in a 20:1 ratio are given and discussed. The fractional inhibitory concentration (FIC) indices have also been calculated. No beta-lactamase-producing strains were found, and a contingency coefficient C has been determined for all the pairs of antibiotics investigated.

  14. The Neisseria gonorrhoeae biofilm matrix contains DNA, and an endogenous nuclease controls its incorporation.

    PubMed

    Steichen, Christopher T; Cho, Christine; Shao, Jian Q; Apicella, Michael A

    2011-04-01

    Neisseria gonorrhoeae has been shown to produce biofilms both in experimental flow chambers and in the human host. Our laboratory has shown that extracellular DNA is an essential component of the gonococcal matrix. We have also identified a gene in N. gonorrhoeae, which we designated nuc. This gene has homology with the staphylococcus-secreted thermonuclease. Our laboratory has characterized nuc through phenotypic analysis of a nuc deletion mutant. Biofilms grown with this strain are significantly thicker and of greater biomass than the N. gonorrhoeae 1291 parent strain. Confocal microscopy indicates that the increased size of the mutant biofilms appears to be due to elevated amounts of extracellular DNA in the biofilm matrix. Chromosomal complementation of the nuc mutation restored the wild-type biofilm phenotype. In addition, we have cloned and expressed the Nuc protein in Escherichia coli, and our data indicate that it has the ability to digest multiple forms of DNA and is a thermonuclease. The ability of Nuc to digest DNA also extends to its ability to disrupt established gonococcal biofilms through degradation of the DNA in the biofilm matrix. Our studies indicate that the N. gonorrhoeae biofilm contains DNA and that the Nuc protein appears to play a role in biofilm formation and remodeling.

  15. Chlamydia trachomatis and Neisseria gonorrhoeae Infections Among Men and Women Entering California Prisons

    PubMed Central

    Bernstein, Kyle T.; Chow, Joan M.; Ruiz, Juan; Schachter, Julius; Horowitz, Evalyn; Bunnell, Rebecca; Bolan, Gail

    2006-01-01

    Objective. We estimated the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae infection among newly arriving inmates at 6 California prisons. Methods. In this cross-sectional study in 1999, urine specimens collected from 698 men aged 18 to 25 years and 572 women aged 18 years or older were tested at intake for C trachomatis and N gonorrhoeae using ligase chain reaction. An analysis of demographic and arrest-related correlates of C trachomatis and N gonorrhoeae infection was performed. Results. The overall C trachomatis prevalence was 9.9% (95% CI=7.8%, 12.3%) among men aged 18 to 25 years, 8.9% (95% CI = 2.9%, 22.1%) among women aged 18 to 25 years, and 3.3% (95% CI=2.0%, 5.1%) among women overall. Three N gonorrhoeae cases were detected with an overall prevalence of 0.24% (95% CI=0.05%, 0.69%). Conclusions. The prevalence of C trachomatis infection at entry to California prisons, especially among young female and male inmates, was high, which supports routine screening at entry into prison. In addition, screening in a jail setting where most detainees are incarcerated before entry into the prison setting may provide an excellent earlier opportunity to identify these infections and treat disease to prevent complications and burden of infection in this high-risk population. PMID:17008584

  16. Metronidazole and spiramycin therapy of mixed Bacteroides spp. and Neisseria gonorrhoeae infection in mice.

    PubMed

    Brook, I

    1989-01-01

    The in vitro and in vivo activity of metronidazole and spiramycin, used singly or in combination, was tested in the eradication of infection caused by Bacteroides spp. and Neisseria gonorrhoeae alone or in combination. The in vitro tests consisted of determinations of the minimal inhibitory concentrations (MIC), carried out with or without the addition of a constant amount of the other antimicrobials. The MIC of both Bacteroides bivius and Bacteroides fragilis for metronidazole were significantly reduced by the addition of spiramycin (from 0.5 to 0.125 micrograms/ml). The in vivo tests were carried out in mice and consisted of measurements of the effects of the antimicrobial agents on the bacterial contents of abscesses induced by subcutaneous injection of bacterial suspension. Synergism between metronidazole and spiramycin was noted against Bacteroides spp. in abscesses caused by either Bacteroides spp. alone, or in combination with N. gonorrhoeae. Furthermore, an additional reduction in the number of N gonorrhoeae was noted in mixed infection with Bacteroides that was treated with metronidazole alone. This study demonstrates the in vitro and in vivo efficacy of the combination of metronidazole and spiramycin in the treatment of infections caused by either Bacteroides spp. alone or in combination with N. gonorrhoeae.

  17. Attenuation of the Type IV Pilus Retraction Motor Influences Neisseria gonorrhoeae Social and Infection Behavior.

    PubMed

    Hockenberry, Alyson M; Hutchens, Danielle M; Agellon, Al; So, Magdalene

    2016-12-06

    Retraction of the type IV pilus (Tfp) mediates DNA uptake, motility, and social and infection behavior in a wide variety of prokaryotes. To date, investigations into Tfp retraction-dependent activities have used a mutant deleted of PilT, the ATPase motor protein that causes the pilus fiber to retract. ΔpilT cells are nontransformable, nonmotile, and cannot aggregate into microcolonies. We tested the hypothesis that these retraction-dependent activities are sensitive to the strength of PilT enzymatic activity by using the pathogen Neisseria gonorrhoeae as a model. We constructed an N. gonorrhoeae mutant with an amino acid substitution in the PilT Walker B box (a substitution of cysteine for leucine at position 201, encoded by pilTL201C). Purified PilTL201C forms a native hexamer, but mutant hexamers hydrolyze ATP at half the maximal rate. N. gonorrhoeae pilTL201C cells produce Tfp fibers, crawl at the same speed as the wild-type (wt) parent, and are equally transformable. However, the social behavior of pilTL201C cells is intermediate between the behaviors of wt and ΔpilT cells. The infection behavior of pilTL201C is also defective, due to its failure to activate the epidermal growth factor receptor (EGFR)-heparin-binding EGF-like growth factor (HB-EGF) pathway. Our study indicates that pilus retraction, per se, is not sufficient for N. gonorrhoeae microcolony formation or infectivity; rather, these activities are sensitive to the strength of PilT enzymatic activity. We discuss the implications of these findings for Neisseria pathogenesis in the context of mechanobiology.

  18. Attenuation of the Type IV Pilus Retraction Motor Influences Neisseria gonorrhoeae Social and Infection Behavior

    PubMed Central

    Hutchens, Danielle M.; Agellon, Al

    2016-01-01

    ABSTRACT Retraction of the type IV pilus (Tfp) mediates DNA uptake, motility, and social and infection behavior in a wide variety of prokaryotes. To date, investigations into Tfp retraction-dependent activities have used a mutant deleted of PilT, the ATPase motor protein that causes the pilus fiber to retract. ΔpilT cells are nontransformable, nonmotile, and cannot aggregate into microcolonies. We tested the hypothesis that these retraction-dependent activities are sensitive to the strength of PilT enzymatic activity by using the pathogen Neisseria gonorrhoeae as a model. We constructed an N. gonorrhoeae mutant with an amino acid substitution in the PilT Walker B box (a substitution of cysteine for leucine at position 201, encoded by pilTL201C). Purified PilTL201C forms a native hexamer, but mutant hexamers hydrolyze ATP at half the maximal rate. N. gonorrhoeae pilTL201C cells produce Tfp fibers, crawl at the same speed as the wild-type (wt) parent, and are equally transformable. However, the social behavior of pilTL201C cells is intermediate between the behaviors of wt and ΔpilT cells. The infection behavior of pilTL201C is also defective, due to its failure to activate the epidermal growth factor receptor (EGFR)-heparin-binding EGF-like growth factor (HB-EGF) pathway. Our study indicates that pilus retraction, per se, is not sufficient for N. gonorrhoeae microcolony formation or infectivity; rather, these activities are sensitive to the strength of PilT enzymatic activity. We discuss the implications of these findings for Neisseria pathogenesis in the context of mechanobiology. PMID:27923924

  19. Differential effects of DNA gyrase inhibitors on the genetic transformation of Neisseria gonorrhoeae.

    PubMed Central

    Parham, C; Cunningham, E; McGinnis, E

    1988-01-01

    Inhibitors of DNA gyrase in Escherichia coli exerted differential effects on the genetic transformation of Neisseria gonorrhoeae. When competent cells of the gonococcus were exposed to novobiocin before the uptake of transforming antibiotic resistance DNA, there was a 50 to 60% reduction in the number of transformants compared with the number of control untreated cells. Norfloxacin, a more potent inhibitor of DNA gyrase and an analog of nalidixic acid, nearly abolished the production of transformants by recipient cells. On the contrary, exposure of competent cells to nalidixic acid had no effect on transformant yield. The target of these inhibitors appears to be at the level of recombination. Possible mechanisms are discussed. PMID:2854452

  20. Characterization of the novel DNA gyrase inhibitor AZD0914: low resistance potential and lack of cross-resistance in Neisseria gonorrhoeae.

    PubMed

    Alm, Richard A; Lahiri, Sushmita D; Kutschke, Amy; Otterson, Linda G; McLaughlin, Robert E; Whiteaker, James D; Lewis, Lisa A; Su, Xiaohong; Huband, Michael D; Gardner, Humphrey; Mueller, John P

    2015-03-01

    The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections.

  1. Characterization of the Novel DNA Gyrase Inhibitor AZD0914: Low Resistance Potential and Lack of Cross-Resistance in Neisseria gonorrhoeae

    PubMed Central

    Kutschke, Amy; Otterson, Linda G.; McLaughlin, Robert E.; Whiteaker, James D.; Lewis, Lisa A.; Su, Xiaohong; Huband, Michael D.; Gardner, Humphrey; Mueller, John P.

    2014-01-01

    The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections. PMID:25534723

  2. 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

  3. 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

  4. Most but not all laboratories can detect the recently emerged Neisseria gonorrhoeae porA mutants - results from the QCMD 2013 N. gonorrhoeae external quality assessment programme.

    PubMed

    Luijt, D; Di Lorenzo, C; van Loon, A M; Unemo, M

    2014-02-27

    We describe the results of the Quality Control for Molecular Diagnostics 2013 Neisseria gonorrhoeae external quality assessment programme that included an N. gonorrhoeae strain harbouring an N. meningitidis porA gene which causes false-negative results in molecular diagnostic assays targeting the gonococcal porA pseudogene. Enhanced awareness of the international transmission of such gonococcal strains is needed to avoid false-negative results in both in-house and commercial molecular diagnostic assays used in laboratories worldwide, but particularly in Europe.

  5. Antigenic diversity of the serotype antigen complex of Neisseria gonorrhoeae: analysis by an indirect enzyme-linked immunoassay.

    PubMed Central

    Johnston, K H

    1980-01-01

    An indirect enzyme-linked immunoassay (ELISA) has been developed to analyze the antigenic profile of the outer membrane serotype complex of Neisseria gonorrhoeae. Antisera raised in rabbits to serotype-specific vesicles (SSV) reacted primarily with homologous SSV; however, there was significant cross-reactivity (less than 50%) with heterologous SSV. N. meningitidis SSV cross-reacted with all antigonococcal SSV but at a lower degree (less than 20%). Preimmune sera did not cross-react significantly with all antigonoccoccal SSV. The sensitivity of the ELISA was enhanced when the integral SSV proteins 1a and 2 were used as adsorbed antigen. Heterologous anti-SSV cross-reacted slightly, having ELISA values less than 15% of the homologous reaction. Antisera prepared by immunoabsorbent affinity columns were highly specific. Homologous affinity anti-SSV reacted only with proteins 1a and 2. The reaction of immune sera was inhibited by homologous proteins 1a and 2; lipopolysaccharide and proteins 1a and 2 isolated from heterologous serotypes did not inhibit the reaction. The reaction of affinity-purified antisera could be inhibited only by homologous protein 1a. By the use of affinity-purified antisera, a specific and highly sensitive ELISA was developed to analyze the antigenic profile of strains of N. gonorrhoeae. PMID:6769815

  6. Differences in Neisseria gonorrhoeae population structure and antimicrobial resistance pattern between men who have sex with men and heterosexuals.

    PubMed

    Serra-Pladevall, J; Barberá, M J; Callarisa, A E; Bartolomé-Comas, R; Andreu, A

    2017-01-01

    This study compared the antimicrobial susceptibility and genotypes of strains of Neisseria gonorrhoeae isolated from men who have sex with men (MSM) and from heterosexuals. One hundred and eleven strains were characterized from 107 patients, comprising 57 strains from 54 heterosexuals and 54 strains from 53 MSM. Antimicrobial resistance rates were higher in strains from heterosexual patients, with resistance to cefixime (P = 0·0159) and ciprofloxacin (P = 0·002) being significantly higher. Typing by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) showed that the most prevalent sequence types (ST) and genogroups (G) respectively were ST2400, ST2992, and ST5793, and G1407, G2992, and G2400. A statistically significant association was observed for MSM and genogroups G2400 (P = 0·0005) and G2992 (P = 0·0488), and G1407 with heterosexuals (P = 0·0002). We conclude that in our region distinct populations of gonococci are circulating among subjects with different sexual practices, with their corresponding transmission patterns. Furthermore, the high prevalence of genotype G2400 in MSM, has not to our knowledge been previously described.

  7. Substrate specificity and kinetic characterization of peptidoglycan O-acetyltransferase B from Neisseria gonorrhoeae.

    PubMed

    Moynihan, Patrick J; Clarke, Anthony J

    2014-06-13

    The O-acetylation of the essential cell wall polymer peptidoglycan is a major virulence factor identified in many bacteria, both Gram-positive and Gram-negative, including Staphylococcus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neisseria meningitidis. With Gram-negative bacteria, the translocation of acetyl groups from the cytoplasm is performed by an integral membrane protein, PatA, for its transfer to peptidoglycan by O-acetyltransferase PatB, whereas a single bimodal membrane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro biochemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate, such as peptidoglycan. In this study, we present the first biochemical and kinetic analysis of a peptidoglycan O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tri- and tetrapeptide stems on muramyl residues. With chitooligosaccharides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. This information will be valuable for the identification and development of peptidoglycan O-acetyltransferase inhibitors that could represent potential leads to novel classes of antibiotics.

  8. Difference in DNA-binding abilities of Fur-homolog DNA binding protein from Neisseria gonorrhoeae.

    PubMed

    Bagchi, Angshuman

    2016-10-01

    Gonorrhea is a severe disease infecting both men and women worldwide. The causative agent of the disease is Neisseria gonorrhoeae. The organism mostly affects human beings in iron restricted environments. In such an environment the organism produces a set of proteins which are mostly absent in iron rich environments. The expressions of the genes for the proteins are regulated by the transcription factor (TF) belonging to the Fur family. Interestingly, the same TF acts as the activator and repressor of genes. In this present work, an attempt has been made to analyze the molecular details of the differential DNA-binding activities of the TF from Neisseria gonorrhoeae to come up with a plausible molecular reason behind the difference DNA binding activities of the same TF. Computational modelling technique was used to build the three dimensional structure of the TF. Molecular docking and molecular dynamics simulations were employed to determine the binding interactions between the TF and the promoter DNA. With the help of the computational techniques, the biochemical reason behind the different modes of DNA binding by the TF was analyzed. Results from this analysis may be useful to future drug development endeavours to curtail the spread of Gonorrhea.

  9. 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

  10. 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.

  11. 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.

  12. An anthropologically based model of the impact of asymptomatic cases on the spread of Neisseria gonorrhoeae

    PubMed Central

    Hazel, Ashley; Marino, Simeone; Simon, Carl

    2015-01-01

    Neisseria gonorrhoeae (GC) remains a serious burden in many high-sexual-activity, undertreated populations. Using empirical data from a 2009 study of GC burden among pastoralists in Kaokoveld, Namibia, we expand the standard gonorrhoea transmission model by using locally derived sexual contact data to explore transmission dynamics in a population with high rates of partner exchange and low treatment-seeking behaviour. We use the model to generate ball-park estimates for transmission probabilities and other parameter values for low-level (i.e. less than approx. 1200 copies/20 µl PCR reaction) asymptomatic infections, which account for 74% of all GC infections found in Kaokoveld in 2009, and to describe the impact of asymptomatic, low-level infections on overall prevalence patterns. Our results suggest that GC transmission probabilities are higher than previously estimated, that untreated infections take longer to clear than previously estimated and that a high prevalence of low-level infections is partially due to larger numbers of untreated, asymptomatic infections. These results provide new insights into the natural history of GC and the challenge of syndromic management programmes for the eradication of endemic gonorrhoea. PMID:25808340

  13. Identification of ZipA, a Signal Recognition Particle-Dependent Protein from Neisseria gonorrhoeae

    PubMed Central

    Du, Ying; Arvidson, Cindy Grove

    2003-01-01

    A genetic screen designed to identify proteins that utilize the signal recognition particle (SRP) for targeting in Escherichia coli was used to screen a Neisseria gonorrhoeae plasmid library. Six plasmids were identified in this screen, and each is predicted to encode one or more putative cytoplasmic membrane (CM) proteins. One of these, pSLO7, has three open reading frames (ORFs), two of which have no similarity to known proteins in GenBank other than sequences from the closely related N. meningitidis. Further analyses showed that one of these, SLO7ORF3, encodes a protein that is dependent on the SRP for localization. This gene also appears to be essential in N. gonorrhoeae since it was not possible to generate null mutations in the gene. Although appearing unique to Neisseria at the DNA sequence level, SLO7ORF3 was found to share some features with the cell division gene zipA of E. coli. These features included similar chromosomal locations (with respect to linked genes) as well as similarities in the predicted protein domain structures. Here, we show that SLO7ORF3 can complement an E. coli conditional zipA mutant and therefore encodes a functional ZipA homolog in N. gonorrhoeae. This observation is significant in that it is the first ZipA homolog identified in a non-rod-shaped organism. Also interesting is that this is the fourth cell division protein (the others are FtsE, FtsX, and FtsQ) shown to utilize the SRP for localization, which may in part explain why the genes encoding the three SRP components are essential in bacteria. PMID:12644481

  14. Hexa-acylated lipid A is required for host inflammatory response to Neisseria gonorrhoeae in experimental gonorrhea.

    PubMed

    Zhou, Xiyou; Gao, Xi; Broglie, Peter M; Kebaier, Chahnaz; Anderson, James E; Thom, Natalie; Apicella, Michael A; Sempowski, Gregory D; Duncan, Joseph A

    2014-01-01

    Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted infection characterized by inflammation of the cervix or urethra. However, a significant subset of patients with N. gonorrhoeae remain asymptomatic, without evidence of localized inflammation. Inflammatory responses to N. gonorrhoeae are generated by host innate immune recognition of N. gonorrhoeae by several innate immune signaling pathways, including lipooligosaccharide (LOS) and other pathogen-derived molecules through activation of innate immune signaling systems, including toll-like receptor 4 (TLR4) and the interleukin-1β (IL-1β) processing complex known as the inflammasome. The lipooligosaccharide of N. gonorrhoeae has a hexa-acylated lipid A. N. gonorrhoeae strains that carry an inactivated msbB (also known as lpxL1) gene produce a penta-acylated lipid A and exhibit reduced biofilm formation, survival in epithelial cells, and induction of epithelial cell inflammatory signaling. We now show that msbB-deficient N. gonorrhoeae induces less inflammatory signaling in human monocytic cell lines and murine macrophages than the parent organism. The penta-acylated LOS exhibits reduced toll-like receptor 4 signaling but does not affect N. gonorrhoeae-mediated activation of the inflammasome. We demonstrate that N. gonorrhoeae msbB is dispensable for initiating and maintaining infection in a murine model of gonorrhea. Interestingly, infection with msbB-deficient N. gonorrhoeae is associated with less localized inflammation. Combined, these data suggest that TLR4-mediated recognition of N. gonorrhoeae LOS plays an important role in the pathogenesis of symptomatic gonorrhea infection and that alterations in lipid A biosynthesis may play a role in determining symptomatic and asymptomatic infections.

  15. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR): a novel antimicrobial resistance multilocus typing scheme for tracking the global dissemination of N. gonorrhoeae strains.

    PubMed

    Demczuk, W; Sidhu, S; Unemo, M; Whiley, D M; Allen, V G; Dillon, J R; Cole, M; Seah, C; Trembizki, E; Trees, D L; Kersh, E N; Abrams, A J; de Vries, H J C; van Dam, A P; Medina, I; Bharat, A; Mulvey, M R; Van Domselaar, G; Martin, I

    2017-02-22

    A curated web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible at https://ngstar.canada.ca The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence combining the historical nomenclature for penA types I-XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA, porB, gyrA and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n=660) consisting of 29 CCs having a maximum of a single locus variation; and 76 NG-STAR STs (n=109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index of 96.5% (CI 95%=0.959-0.969). The most common STs were NG-STAR: ST-90 (n=100, 13.0%), ST-42 and ST-91 (n=45, 5.9%), ST-64 (n=44, 5.72%), and ST-139 (n=42, 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91 and ST-139(n=156, 92.3%); decreased susceptibility to cephalosporins with NG-STAR ST-90, ST-91 and ST-97 (n=162, 94.2%); and ciprofloxacin resistance with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150 and ST-158 (n=196, 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n=106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial resistant N. gonorrhoeae strains.

  16. Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA.

    PubMed

    Sikora, Aleksandra E; Mills, Robert H; Weber, Jacob V; Hamza, Adel; Passow, Bryan W; Romaine, Andrew; Williamson, Zachary A; Reed, Robert W; Zielke, Ryszard A; Korotkov, Konstantin V

    2017-08-01

    Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of N. gonorrhoeae necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of N. gonorrhoeae anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC50 value of C7-3 and C7-3m2 against anaerobically grown N. gonorrhoeae was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development. Copyright © 2017 American Society for Microbiology.

  17. Intracellular Survival of Neisseria gonorrhoeae in Male Urethral Epithelial Cells: Importance of a Hexaacyl Lipid A

    PubMed Central

    Post, Deborah M. B.; Phillips, Nancy J.; Shao, Jian Q.; Entz, David D.; Gibson, Bradford W.; Apicella, Michael A.

    2002-01-01

    Neisseria gonorrhoeae is a strict human pathogen that invades and colonizes the urogenital tracts of males and females. Lipooligosaccharide (LOS) has been shown to play a role in gonococcal pathogenesis. The acyl transferase MsbB is involved in the biosynthesis of the lipid A portion of the LOS. In order to determine the role of an intact lipid A structure on the pathogenesis of N. gonorrhoeae, the msbB gene was cloned and sequenced, a deletion and insertion mutation was introduced into N. gonorrhoeae, and the mutant strain was designated 1291A11K3. Mass spectrometric analyses of 1291A11K3 LOS determined that this mutation resulted in a pentaacyl rather than a hexaacyl lipid A structure. These analyses also demonstrated an increase in the phosphorylation of lipid A and an increase in length of the oligosaccharide of a minor species of the msbB LOS. The interactions of this mutant with male urethral epithelial cells (uec) were examined. Transmission and scanning electron microscopy studies indicated that the msbB mutants formed close associations with and were internalized by the uec at levels similar to those of the parent strain. Gentamicin survival assays performed with 1291A11K3 and 1291 bacteria demonstrated that there was no difference in the abilities of the two strains to adhere to uec; however, significantly fewer 1291A11K3 bacteria than parent strain bacteria were recovered from gentamicin-treated uec. These studies suggest that the lipid A modification in the N. gonorrhoeae msbB mutant may render it more susceptible to innate intracellular killing mechanisms when internalized by uec. PMID:11796626

  18. 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.

  19. 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.

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

    PubMed Central

    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.

    2015-01-01

    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

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

    PubMed Central

    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

  2. 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

  3. 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

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

    PubMed

    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

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

    PubMed

    Lahra, Monica M

    2012-03-31

    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

  6. Gonorrhoea

    PubMed Central

    2014-01-01

    Introduction In 2012, the diagnosis rates for gonorrhoea among adults aged 20 to 24 years in the UK were 249 per 100,000 for men and 140 per 100,000 for women. Resistance to one or more antimicrobial agent is reported in more than one quarter of isolates. Co-infection with Chlamydia trachomatis is reported in 10% to 40% of people with gonorrhoea in the US and UK. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of treatments for uncomplicated infections in men and non-pregnant women, and in pregnant women? What are the effects of treatments for disseminated gonococcal infection? What are the effects of dual treatment for gonorrhoea and chlamydia infection? We searched: Medline, Embase, The Cochrane Library, and other important databases up to September 2013 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 7 studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review we present information relating to the effectiveness and safety of the following interventions: antibiotic regimens (dual treatment, multiple dose, single dose). PMID:24559849

  7. Isolation of Bacteriophages Active Against Neisseria meningitidis

    PubMed Central

    Cary, Sylvia G.; Hunter, Donald H.

    1967-01-01

    Five distinct bacteriophages have been isolated from strains of Neisseria meningitidis. Filtrates with titers of 10−4 to 10−6 were produced with a modified Swanstrom and Adams semisolid agar procedure, employing Eugonbroth with added agar and an incubation temperature of 30 C. Of 49 strains of N. meningitidis (groups B and C), 25 were lysed by one or more of the phages, but there was no lysis of other Neisseria and Mima polymorpha strains. Images PMID:4990042

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

    PubMed Central

    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

  9. 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.

  10. Gentamicin in vitro activity and tentative gentamicin interpretation criteria for the CLSI and calibrated dichotomous sensitivity disc diffusion methods for Neisseria gonorrhoeae.

    PubMed

    Bala, Manju; Singh, Vikram; Philipova, Ivva; Bhargava, Aradhana; Chandra Joshi, Naveen; Unemo, Magnus

    2016-07-01

    XDR Neisseria gonorrhoeae imposes the threat of untreatable gonorrhoea. Gentamicin is considered for future treatment; however, no interpretation criteria for the CLSI and calibrated dichotomous sensitivity (CDS) disc diffusion (DD) techniques are available for N. gonorrhoeae. We investigated the in vitro gentamicin activity by MIC and DD methods, proposed DD breakpoints and determined DD ranges for 10 international quality control (QC) strains. Gentamicin susceptibility of 333 N. gonorrhoeae isolates, including 323 clinical isolates and 10 QC strains, was determined. MIC determination (Etest) and DD methods (CLSI and CDS) were performed. The relationship between MIC, inhibition zone diameter and annular radius was determined by linear regression analysis and the correlation coefficient (r) was calculated. Gentamicin MICs for the QC strains were within published ranges. Of the 323 clinical isolates, according to published breakpoints 75.9%, 23.5% and 0.6% were susceptible, intermediately susceptible and resistant, respectively. Based on error minimization with MICs of ≤4, 8-16 and ≥32 mg/L, breakpoints proposed are susceptible ≥16 mm, intermediately susceptible 13-15 mm and resistant ≤12 mm for the CLSI method and susceptible ≥6 mm, less susceptible 3-5 mm and resistant ≤2 mm for the CDS technique. Low resistance to gentamicin was identified and gentamicin might be a future treatment option for gonorrhoea. Tentative gentamicin zone breakpoints were defined for two DD methods and QC ranges for 10 international reference strains were established. Our findings suggest that in resource-poor settings where MIC testing is not a feasible option, the DD methods can be used to indicate gentamicin resistance. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. Gene conversion variations generate structurally distinct pilin polypeptides in Neisseria gonorrhoeae.

    PubMed

    Swanson, J; Robbins, K; Barrera, O; Koomey, J M

    1987-04-01

    Pilus+ to pilus- phenotype change occurs in Neisseria gonorrhoeae through gene conversion of the gonococcus' complete, expressed pilin gene by nucleotides homologous to the pilS1 copy 5 partial pilin gene; assembly missense pilin is synthesized but pili are not. Reversion to pilus+ occurs by a subsequent recombinational event that replaces the complete pilin gene's pilS1 copy 5-like sequence with nucleotides from a different partial gene to effect expression of an orthodox (i.e., pilus producing) pilin. Sibling pilus+ revertants of common parentage can carry different sequences in their expressed pilin genes because they have undergone nonidentical gene conversion events such as recombinations with sequences from different partial genes, or recombinations with different length nucleotide stretches of the same partial gene; either can yield structurally and antigenically variant pilin polypeptides.

  12. Stability and viability of Neisseria gonorrhoeae in various solutions and buffers.

    PubMed Central

    Norrod, P; Williams, R P

    1979-01-01

    The stability and viability of Neisseria gonorrhoeae WP (T4) was tested in a variety of buffers and solutions, many of which are commonly used in gonococcal research. Each solution was tested at room temperature for its ability to maintain stability and viability of gonococci in concentrated suspensions and to maintain viability of gonococci in dilute suspensions. The 14 buffers and solutions tested could be divided into four groups based upon these criteria. Only a few solutions satisfied all three criteria. Of those tested, Gey salt solution and bovine serum albumin (0.01%) and proteose peptone (1%) in saline were the only two in which the gonococci retained viability in dilute suspensions for 25 min. Most of the solutions were not able to maintain viability of gonococci in dilute suspensions, even when the same solution was capable of maintaining stability and viability in concentrated suspensions. PMID:107855

  13. Differential intracellular efficacies of ciprofloxacin and cefixime against Neisseria gonorrhoeae in human fallopian tube organ culture.

    PubMed

    Phanucharas, J P; Gorby, G L

    1997-07-01

    This study compared the abilities of ciprofloxacin and cefixime to kill intracellular Neisseria gonorrhoeae in a human fallopian tube organ culture assay. When invasion was inhibited by cytochalasin D, 0.996% of the tissue-associated gonococci survived ciprofloxacin exposure compared to 1.70% of gonococci exposed to cefixime (95% confidence interval for the ratio of the means, 0.267 to 1.30), indicating that the two antibiotics did not significantly differ in the ability to kill extracellular attached organisms. In the absence of cytochalasin D, 1.63% survived ciprofloxacin exposure while 9.76% survived cefixime treatment (95% confidence interval for the ratio of the means, 0.067 to 0.418). These results suggest that ciprofloxacin penetrated epithelial cells and killed intracellular gonococci better than did cefixime. Thus, at concentrations achievable in serum, ciprofloxacin was more effective in total gonococcal killing than cefixime in this human fallopian tube organ culture model.

  14. The major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, Pan 1, is a lipoprotein.

    PubMed Central

    Hoehn, G T; Clark, V L

    1992-01-01

    Pan 1 is an acidic outer membrane protein of Neisseria gonorrhoeae that is expressed only when gonococci are grown anaerobically. On silver-stained sodium dodecyl sulfate-polyacrylamide gels, Pan 1 migrates as an intense but diffuse 54-kDa protein. The deduced amino acid sequence of Pan 1 from the aniA (anaerobically induced protein) open reading frame reveals a lipoprotein consensus sequence, Ala-Leu-Ala-Ala-Cys, and a processed molecular mass of 39 kDa. Furthermore, there is strong homology at the N terminus and C terminus of Pan 1 to the termini of the gonococcal outer membrane lipoproteins Lip and Laz. [3H]palmitic acid labeling of gonococci grown under oxygen-limited conditions demonstrated specific incorporation of label into Pan 1, suggesting further that Pan 1 is a lipoprotein. Images PMID:1398981

  15. Risk factors for antimicrobial-resistant Neisseria gonorrhoeae and characteristics of patients infected with gonorrhea.

    PubMed

    Fuertes de Vega, Irene; Baliu-Piqué, Carola; Bosch Mestres, Jordi; Vergara Gómez, Andrea; Vallés, Xavier; Alsina Gibert, Mercè

    2017-01-13

    There are very few data available regarding risk factors associated with antibiotic resistant-Neisseria gonorrhoeae. A study was conducted on 110 samples from 101 patients with gonococcal infection, in order to describe their characteristics and compare them with the antimicrobial susceptibility profile of their samples. An association was observed between resistant infections and heterosexual men, older age, concurrent sexually transmitted infection, and unsafe sexual behaviors. There is a need for improved data on the risk factors associated with antibiotic resistant gonococcal infection in order to identify risk groups, and to propose public health strategies to control this infection. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  16. DNA uptake sequences in Neisseria gonorrhoeae as intrinsic transcriptional terminators and markers of horizontal gene transfer

    PubMed Central

    Gurung, Neesha

    2016-01-01

    DNA uptake sequences are widespread throughout the Neisseria gonorrhoeae genome. These short, conserved sequences facilitate the exchange of endogenous DNA between members of the genus Neisseria. Often the DNA uptake sequences are present as inverted repeats that are able to form hairpin structures. It has been suggested previously that DNA uptake sequence inverted repeats present 3′ of genes play a role in rho-independent termination and attenuation. However, there is conflicting experimental evidence to support this role. The aim of this study was to determine the role of DNA uptake sequences in transcriptional termination. Both bioinformatics predictions, conducted using TransTermHP, and experimental evidence, from RNA-seq data, were used to determine which inverted repeat DNA uptake sequences are transcriptional terminators and in which direction. Here we show that DNA uptake sequences in the inverted repeat configuration occur in N. gonorrhoeae both where the DNA uptake sequence precedes the inverted version of the sequence and also, albeit less frequently, in reverse order. Due to their symmetrical configuration, inverted repeat DNA uptake sequences can potentially act as bi-directional terminators, therefore affecting transcription on both DNA strands. This work also provides evidence that gaps in DNA uptake sequence density in the gonococcal genome coincide with areas of DNA that are foreign in origin, such as prophage. This study differentiates for the first time, to our knowledge, between DNA uptake sequences that form intrinsic transcriptional terminators and those that do not, providing characteristic features within the flanking inverted repeat that can be identified. PMID:28348864

  17. 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

  18. 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

  19. Neisseria gonorrhoeae Elicits Extracellular Traps in Primary Neutrophil Culture While Suppressing the Oxidative Burst

    PubMed Central

    Gunderson, Carl W.

    2015-01-01

    ABSTRACT  Neisseria gonorrhoeae (the gonococcus) causes gonorrhea and is uniquely adapted to survive within the human reproductive tract. Gonococci evade host immune surveillance in part by varying their pili and opacity-associated proteins. These variable surface antigens influence interactions with host epithelial and immune cells. A potent polymorphonuclear leukocyte (PMN) response is a hallmark of symptomatic gonococcal infection, with vast numbers of PMNs recruited to the site of infection. A large body of literature describes gonococcus-PMN interactions, but the factors driving the outcome of infection are not fully understood. Gonococci have been described to both induce and suppress the PMN oxidative burst, but we determined that gonococci differentially affect induction of the PMN oxidative burst depending on the multiplicity of infection (MOI). Infecting PMN at an MOI of <20 gonococci elicits an oxidative burst, while an MOI of >20 suppresses the burst. Oxidative burst in response to gonococci is enhanced by, but does not require, expression of pili or opacity proteins. Neutrophil extracellular traps (NETs) were observed in gonococcus-infected PMNs, a process which requires an oxidative burst, yet gonococci induced NETs under suppressing conditions. The NETs were unable to kill gonococci despite killing the common vaginal bacterium Lactobacillus crispatus. Thus, gonococci influence PMN biology to promote their own survival by suppressing the oxidative burst of PMNs and stimulating the formation of NETs, which do not effectively kill gonococci, illustrating how N. gonorrhoeae has evolved to modulate PMN responses to promote infection. PMID:25670773

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

    DOE PAGES

    Su, Chih -Chia; Bolla, Jani  Reddy; Kumar, Nitin; ...

    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 tomore » 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.« less

  1. Is Neisseria gonorrhoeae Initiating a Future Era of Untreatable Gonorrhea?: Detailed Characterization of the First Strain with High-Level Resistance to Ceftriaxone ▿ †

    PubMed Central

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

    2011-01-01

    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

  2. Comparative assessment of CDS, CLSI disc diffusion and Etest techniques for antimicrobial susceptibility testing of Neisseria gonorrhoeae: a 6-year study

    PubMed Central

    Singh, Vikram; Kakran, Monika; Ramesh, V

    2012-01-01

    Background A variety of techniques are available for antimicrobial susceptibility testing of Neisseria gonorrhoeae. Objective The aim of this study was to find a cost-effective, reliable and easily applicable microbiological method to detect antimicrobial susceptibilities of N. gonorrhoeae in resource-poor countries. Design Prospective study. Setting Male and female STD clinic of Regional STD Teaching, Training and Research Centre, New Delhi, India. Participants N. gonorrhoeae isolates from all male and female patients presenting with acute gonococcal urethritis and cervical discharge. Material and methods A total of 295 consecutive N. gonorrhoeae isolates during 2005–2010 was used to compare the Clinical and Laboratory Standards Institute (CLSI) and CDS disc diffusion technique with Etest by performing antimicrobial susceptibility testing in parallel for penicillin, tetracycline, ceftriaxone, ciprofloxacin and spectinomycin. WHO reference strains were used as controls. Results CDS disc diffusion zones of inhibition showed that complete percentage agreement for penicillin, ciprofloxacin and tetracycline was high with their analogous Etest minimal inhibitory concentrations in comparison to CLSI disc diffusion technique, that is, 91.5%, 92.9% and 99.3% versus 87.5%, 88.5% and 74.9%, respectively. CDS results had less number of major and minor category discrepancies in comparison to CLSI and CDS method showed excellent correlation coefficient (r=1) with Etest for all five antimicrobial agents tested in comparison to CLSI (r=0.92). It was very poor (r=0.61) by CLSI method for tetracycline. The correlation coefficients between the two methods and the Etest were identical if tetracycline was removed from the CLSI analysis. Conclusions The CDS technique is an attractive alternative for N. gonorrhoeae susceptibility testing and is recommended for monitoring the antimicrobial susceptibility in less developed and resource-poor settings to facilitate enhanced antimicrobial

  3. Comparative assessment of CDS, CLSI disc diffusion and Etest techniques for antimicrobial susceptibility testing of Neisseria gonorrhoeae: a 6-year study.

    PubMed

    Singh, Vikram; Bala, Manju; Kakran, Monika; Ramesh, V

    2012-01-01

    A variety of techniques are available for antimicrobial susceptibility testing of Neisseria gonorrhoeae. The aim of this study was to find a cost-effective, reliable and easily applicable microbiological method to detect antimicrobial susceptibilities of N. gonorrhoeae in resource-poor countries. Prospective study. Male and female STD clinic of Regional STD Teaching, Training and Research Centre, New Delhi, India. N. gonorrhoeae isolates from all male and female patients presenting with acute gonococcal urethritis and cervical discharge. A total of 295 consecutive N. gonorrhoeae isolates during 2005-2010 was used to compare the Clinical and Laboratory Standards Institute (CLSI) and CDS disc diffusion technique with Etest by performing antimicrobial susceptibility testing in parallel for penicillin, tetracycline, ceftriaxone, ciprofloxacin and spectinomycin. WHO reference strains were used as controls. CDS disc diffusion zones of inhibition showed that complete percentage agreement for penicillin, ciprofloxacin and tetracycline was high with their analogous Etest minimal inhibitory concentrations in comparison to CLSI disc diffusion technique, that is, 91.5%, 92.9% and 99.3% versus 87.5%, 88.5% and 74.9%, respectively. CDS results had less number of major and minor category discrepancies in comparison to CLSI and CDS method showed excellent correlation coefficient (r=1) with Etest for all five antimicrobial agents tested in comparison to CLSI (r=0.92). It was very poor (r=0.61) by CLSI method for tetracycline. The correlation coefficients between the two methods and the Etest were identical if tetracycline was removed from the CLSI analysis. The CDS technique is an attractive alternative for N. gonorrhoeae susceptibility testing and is recommended for monitoring the antimicrobial susceptibility in less developed and resource-poor settings to facilitate enhanced antimicrobial resistance surveillance when the WHO Gonococcal Antimicrobial Surveillance Programme is

  4. Phenotypic and genetic characterization of the first two cases of extended-spectrum-cephalosporin-resistant Neisseria gonorrhoeae infection in South Africa and association with cefixime treatment failure.

    PubMed

    Lewis, David A; Sriruttan, Charlotte; Müller, Etienne E; Golparian, Daniel; Gumede, Lindy; Fick, Donald; de Wet, Johan; Maseko, Venessa; Coetzee, Jennifer; Unemo, Magnus

    2013-06-01

    To describe the phenotypic and genetic characteristics of the first two cases of extended-spectrum cephalosporin (ESC)-resistant Neisseria gonorrhoeae in South Africa, one of which was associated with verified cefixime treatment failure. Two ESC-resistant N. gonorrhoeae isolates were cultured from the urethral discharge of two men who have sex with men (MSM). One man reported a persistent urethral discharge that had failed to respond to previous therapy with oral cefixime. Agar dilution MICs were determined for eight antibiotics. β-Lactam-associated resistance mutations were identified through PCR-based amplification and sequencing for several key genes: penA, mtrR and its promoter, porB1b (penB), ponA and pilQ. For molecular epidemiological characterization, full-length porB gene sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) were performed. Both isolates were resistant to cefixime, ciprofloxacin, penicillin and tetracycline and intermediate/resistant to azithromycin, but susceptible to ceftriaxone, gentamicin and spectinomycin. Both isolates had the type XXXIV penA mosaic allele in addition to previously described resistance mutations in the mtrR promoter (A deletion), porB1b (penB) (G101K and A102N) and ponA1 (L421P). Both isolates had an identical NG-MAST sequence type (ST4822) and MLST sequence type (ST1901). Both isolates were resistant to cefixime and possessed a number of identical mutations in key genes contributing to ESC resistance in N. gonorrhoeae. The two isolates contained the type XXXIV penA mosaic allele and belonged to a successful international MSM-linked multidrug-resistant gonococcal clone (MLST ST1901) associated with several cefixime treatment failures in Europe and North America.

  5. Rapid antigen tests for Neisseria gonorrhoeae and Chlamydia trachomatis are not accurate for screening women with disturbed vaginal lactobacillary flora.

    PubMed

    Donders, G G; van Gerven, V; de Wet, H G; van Straten, A M; de Boer, F

    1996-01-01

    We studied the accuracy of the rapid antigen detection tests Gonozyme and Chlamydiazyme in high-risk women in an outpatient prenatal clinic, Kalafong University Hospital, Pretoria, South Africa. Women (n = 433) presenting with uneventful pregnancy (n = 324), unavoidable miscarriage (n = 41) or infertility of 1 year's duration (n = 68) had a Pap smear for lactobacillary grading and detection of pathogens like Candida albicans or Trichomonas vaginalis, a swab for culture of Neisseria gonorrhoeae, and a swab for Gonozyme, Chlamydiazyme and Chlamydia immunofluorescence collected from the endocervix. Specificities of both antigen tests were high, but sensitivities and positive predictive values were disappointingly low. Chlamydial antigen was recovered in only 37% of samples with positive immunofluorescence, gonococcal antigen was detected in only 50% of samples with positive culture for N. gonorrhoeae. Although prevalence of N. gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis was higher in women with disturbed lactobacillary grades on the Pap smears, sensitivities of the antigen tests were lower in this group. We conclude that detection of endocervical antigens of C. trachomatis and N. gonorrhoeae lacked sensitivity in pregnant and infertile women living in an area with high prevalence of chlamydial cervicitis, gonorrhoea and Trichomonas vaginitis. Furthermore, the rapid antigen tests lack accuracy when the lactobacillary flora is disturbed and are, therefore, not suitable for detection of C. trachomatis or N. gonorrhoeae in pre-screened patients.

  6. recA and catalase in H sub 2 O sub 2 -mediated toxicity in Neisseria gonorrhoeae

    SciTech Connect

    Hassett, D.J.; Charniga, L.; Cohen, M.S. )

    1990-12-01

    Neisseria gonorrhoeae cells defective in the biosynthesis of the recA gene product are no more sensitive to hydrogen peroxide than wild-type cells. Although gonococci possess nearly 100-fold-greater catalase levels than Escherichia coli, they are more susceptible to hydrogen peroxide than this organism. The natural niche of gonococci undoubtedly results in exposure to oxidant stress; however, they do not demonstrate particularly efficient antioxidant defense systems.

  7. Comparison of Gram stain with DNA probe for detection of Neisseria gonorrhoeae in urethras of symptomatic males.

    PubMed Central

    Juchau, S V; Nackman, R; Ruppart, D

    1995-01-01

    The comparison of Gram-stained urethral smears with Gen-Probe for the detection of Neisseria Gonorrhoeae in the urethras of males with symptomatic urethritis revealed a 99.6% correlation between the two methods. A simple Gram stain would appear to be the method of choice for the detection of gonorrhea in symptomatic males, because it is much less expensive and much more rapid than the Gen-Probe method. PMID:8576380

  8. Antimicrobial resistance monitoring in Neisseria gonorrhoeae and strategic use of funds from the Global Fund to set up a systematic Moroccan gonococcal antimicrobial surveillance programme.

    PubMed

    Hançali, Amina; Ndowa, Francis; Bellaji, Bahija; Bennani, Aziza; Kettani, Amina; Charof, Reda; El Aouad, Rajae

    2013-12-01

    The aims of this study were to assess antimicrobial resistance in Neisseria gonorrhoeae infections and update the treatment in the national guidelines for the syndromic management of sexually transmitted infections in Morocco. 171 men complaining of urethral discharge were recruited from basic health services during 2009. Urethral swab samples were collected and N gonorrhoeae identification was performed by culture. Antimicrobial susceptibility testing was performed using the Etest method and the antimicrobial agents tested were ciprofloxacin, penicillin, spectinomycin, tetracycline, ceftriaxone and cefixime. A total of 72 isolates were examined. Significant resistance to tetracycline (92.8%) and ciprofloxacin (86.8%), which was used as first-line treatment in gonococcal infections, was noted. No resistance to spectinomycin, ceftriaxone or cefixime was detected in all the isolates. Following these results the Ministry of Health of Morocco replaced ciprofloxacin and introduced ceftriaxone 250 mg as a single dose in the treatment of gonococcal infections. Using funds from the Global Fund to Fight AIDS, Tuberculosis and Malaria (the Global Fund), a surveillance programme was set up for antimicrobial resistance testing in N gonorrhoeae.

  9. Neisseria gonorrhoeae Metalloprotease NGO1686 Is Required for Full Piliation, and Piliation Is Required for Resistance to H2O2- and Neutrophil-Mediated Killing

    PubMed Central

    Stohl, Elizabeth A.; Dale, Erin M.; Criss, Alison K.; Seifert, H. Steven

    2013-01-01

    ABSTRACT The sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogen Neisseria gonorrhoeae. Type IV pili are an essential virulence factor uniformly expressed on clinical gonococcal isolates and are required for several aspects of gonococcal pathogenesis, including adherence to host tissues, autoagglutination, twitching motility, and the uptake of DNA during transformation. Symptomatic gonococcal infection is characterized by the influx of neutrophils or polymorphonuclear leukocytes (PMNs) to the site of infection. PMNs are a key component of gonococcal pathogenesis, mediating the innate immune response through the use of oxidative and nonoxidative killing mechanisms. The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan. We report that the effect of NGO1686 on survival after exposure to H2O2 and PMNs is mediated through its role in elaborating pili and that nonpiliated mutants of N. gonorrhoeae are less resistant to killing by H2O2, LL-37, and PMNs than the corresponding piliated strains. These findings add to the various virulence-associated functions attributable to gonococcal pili and may explain the selection basis for piliation in clinical isolates of N. gonorrhoeae. PMID:23839218

  10. Identification and characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae

    PubMed Central

    Post, Deborah M. B.; Schilling, Birgit; Reinders, Lorri M.; D’Souza, Alexandria K.; Ketterer, Margaret R.; Kiel, Steven J.; Chande, Aroon T.; Apicella, Michael A.; Gibson, Bradford W.

    2017-01-01

    Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as “AckA-dependent”. Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies

  11. 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

  12. Organization and transcription of the division cell wall (dcw) cluster in Neisseria gonorrhoeae.

    PubMed

    Francis, F; Ramirez-Arcos, S; Salimnia, H; Victor, C; Dillon, J R

    2000-06-27

    A cluster of genes involved in cell division and cell wall (dcw) biosynthesis was identified in Neisseria gonorrhoeae using genomic analysis and through verification of gene order by polymerase chain reaction (PCR) analysis. The gonococcal dcw cluster consists of 17 genes, in the order 5'-mraZ-mraW-ftsI-murE-hyp1-murF- mraY-hyp2-murD-ftsW-murG-murC-ddl -ft sQ-ftsA-ftsZ-hyp3-3'. The gene organization of the dcw cluster of N. gonorrhoeae is more similar to that observed in Gram-negative rods such as Escherichia coli and Haemophilus influenzae than in Gram-positive bacteria. The cluster is characterized by several intergenic spaces. Compared with E. coli, two genes, ftsL and envA, are absent in the gonococcal dcw cluster and three hypothetical genes are novel to the cluster. The cluster is flanked by two transcriptional terminators consisting of paired neisserial uptake sequences and also includes four internal terminators, three of which are paired neisserial uptake sequences. We also found that a repeated sequence on the gonococcal genome, commonly called a Correia element, acts as the fourth transcriptional terminator. All termination sequences were shown to be fully functional by using reverse transcription PCR experiments. Transcriptional start sites upstream of ftsQ, ftsA and ftsZ were determined by primer extension and six promoters were identified; three promoters were located upstream of ftsZ in the intergenic space, two were upstream of ftsA within ftsQ and one was upstream of ftsQ within ddl. Some of these promoters were preferentially used under anaerobic conditions. The location of these promoters differed from those described in E. coli indicating dissimilar transcriptional regulation.

  13. Neisseria gonorrhoeae FA1090 Carries Genes Encoding Two Classes of Vsr Endonucleases ▿

    PubMed Central

    Kwiatek, Agnieszka; Łuczkiewicz, Maciej; Bandyra, Katarzyna; Stein, Daniel C.; Piekarowicz, Andrzej

    2010-01-01

    A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize T:G mismatches in every nucleotide context tested, whereas V.NgoAXIV recognized T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC, and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25, His64, and Asp97 of V.NgoAXIV and Glu24, Asp63, and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However, Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished. PMID:20511499

  14. Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

    PubMed

    Kwiatek, Agnieszka; Luczkiewicz, Maciej; Bandyra, Katarzyna; Stein, Daniel C; Piekarowicz, Andrzej

    2010-08-01

    A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize T:G mismatches in every nucleotide context tested, whereas V.NgoAXIV recognized T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC, and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25, His64, and Asp97 of V.NgoAXIV and Glu24, Asp63, and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However, Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished.

  15. Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

    PubMed Central

    Brettin, Thomas; Altherr, Michael R; Du, Ying; Mason, Roxie M; Friedrich, Alexandra; Potter, Laura; Langford, Chris; Keller, Thomas J; Jens, Jason; Howie, Heather; Weyand, Nathan J; Clary, Susan; Prichard, Kimberly; Wachocki, Susi; Sodergren, Erica; Dillard, Joseph P; Weinstock, George; So, Magdalene; Arvidson, Cindy Grove

    2005-01-01

    Background The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system. Results The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci. Conclusion This archived set of Gateway® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome. PMID:16137322

  16. A new rapid resazurin-based microdilution assay for antimicrobial susceptibility testing of Neisseria gonorrhoeae.

    PubMed

    Foerster, Sunniva; Desilvestro, Valentino; Hathaway, Lucy J; Althaus, Christian L; Unemo, Magnus

    2017-07-01

    Rapid, cost-effective and objective methods for antimicrobial susceptibility testing of Neisseria gonorrhoeae would greatly enhance surveillance of antimicrobial resistance. Etest, disc diffusion and agar dilution methods are subjective, mostly laborious for large-scale testing and take ∼24 h. We aimed to develop a rapid broth microdilution assay using resazurin (blue), which is converted into resorufin (pink fluorescence) in the presence of viable bacteria. The resazurin-based broth microdilution assay was established using 132 N. gonorrhoeae strains and the antimicrobials ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and penicillin. A regression model was used to estimate the MICs. Assay results were obtained in ∼7.5 h. The EC 50 of the dose-response curves correlated well with Etest MIC values (Pearson's r  = 0.93). Minor errors resulting from misclassifications of intermediate strains were found for 9% of the samples. Major errors (susceptible strains misclassified as resistant) occurred for ceftriaxone (4.6%), cefixime (3.3%), azithromycin (0.6%) and tetracycline (0.2%). Only one very major error was found (a ceftriaxone-resistant strain misclassified as susceptible). Overall the sensitivity of the assay was 97.1% (95% CI 95.2-98.4) and the specificity 78.5% (95% CI 74.5-82.9). A rapid, objective, high-throughput, quantitative and cost-effective broth microdilution assay was established for gonococci. For use in routine diagnostics without confirmatory testing, the specificity might remain suboptimal for ceftriaxone and cefixime. However, the assay is an effective low-cost method to evaluate novel antimicrobials and for high-throughput screening, and expands the currently available methodologies for surveillance of antimicrobial resistance in gonococci.

  17. [Prevalence of anti-Chlamydia trachomatis and anti-Neisseria gonorrhoeae antibodies in Mexican populations].

    PubMed

    Cravioto, María del Carmen; Matamoros, Oscar; Villalobos-Zapata, Yvonne; Peña, Oscar; García-Lara, Enrique; Martínez, Maribel; Castelo, Julio; Sifuentes-Osornio, José

    2003-01-01

    To estimate the prevalence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection in groups of individuals at different risks of sexually transmitted infections (STI). Between January 1992 and December 1993, a cross-sectional multicentric study was carried out at the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (National Institute of Medical Sciences and Nutrition "Salvador Zubirán") in Mexico City. The study population consisted of 945 reproductive age subjects (585 females and 360 males). Low and high risk groups were classified according to their risk for STI. High risk groups included infertile women with tubal damage, women with a history of ectopic pregnancy or abortion, infertile men, HIV/AIDS patients, homo- or bisexual men, and female commercial sex workers. Low risk groups included primigravidae, fertile men, and infertile women with no tubal damage. Serum anti-NG and anti-CT IgG and IgA were determined, in duplicate by immune-enzymatic assay, using as antigens NG pili and the LI fraction of CT. Descriptive analysis is presented as percentages. NG prevalence in females was 13.7% by IgG and 14.3% by IgA. CT prevalence was 11.4% by IgG and 4.4% by IgA. In males, NG prevalences were 3.3% and 13.3% by IgG and IgA, respectively; CT prevalences were 7.2% and 5.5%, respectively. In commercial sex workers, NG prevalences were 31.2% by IgG and 28.4% by IgA, and CT 25.0% and 5.7% by IgG and IgA, respectively. In women with infertility due to tubal damage the prevalences of NG were 5.6% and 9.8%, respectively, and those of CT were 8.4% and 1.4%, respectively. In 110 young primigravid NG prevalences were 4.5% and 10.0%, respectively, and CT 3.6% and 9.1%. These data confirm the high prevalence of Neisseria gonorrhoeae and Chlamydia trachomatis in female commercial sex workers and homo- or bisexual men, but not in other high-risk groups like infertile women or women with a history of ectopic pregnancy or abortion. The English

  18. Lost opportunity to save newborn lives: variable national antenatal screening policies for Neisseria gonorrhoeae and Chlamydia trachomatis.

    PubMed

    Medline, Alexandra; Joseph Davey, Dvora; Klausner, Jeffrey D

    2017-06-01

    Unfavorable pregnancy outcomes caused by Chlamydia trachomatis or Neisseria gonorrhoeae infection are well known. The first step in addressing antenatal C. trachomatis and N. gonorrhoeae infection is a national policy to screen all pregnant women for C. trachomatis and N. gonorrhoeae, regardless of symptoms. The aim of this study was to inform policy makers on the presence of antenatal screening recommendations for C. trachomatis and N. gonorrhoeae infection. We conducted a three-part study from June 2015 to February 2016. We analyzed English and French language information online on Ministry of Health websites regarding C. trachomatis and N. gonorrhoeae antenatal screening. We referenced both primary official country and regional policy documents. We contacted the Ministry of Health directly if the information on the national antenatal screening was outdated or unavailable. In parallel, we sent a survey to the regional representative from the World Health Organization to help collect country-level data. Fourteen countries have current policies for antenatal screening of C. trachomatis and/or N. gonorrhoeae infection: Australia, the Bahamas, Bulgaria, Canada, Estonia, Japan, Germany, Latvia, New Zealand, Democratic People's Republic of Korea, Romania, Sweden, the United Kingdom, and the United States. Australia, New Zealand, and Latvia and the United States restricted antenatal screening to women ≤25 years old and those of higher risk. Several countries responded that they had policies to treat pregnant women with symptoms. This is the currently recommended WHO guideline but is not the same as universal screening. North Korea had policies in place which were not implemented due to lack of personnel and/or supplies. National level policies to support routine screening for C. trachomatis and N. gonorrhoeae infection to prevent adverse pregnancy and newborn outcomes are uncommon.

  19. Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae--a review.

    PubMed

    Fussenegger, M; Rudel, T; Barten, R; Ryll, R; Meyer, T F

    1997-06-11

    In Neisseria gonorrhoea (Ngo), the processes of type-4 pilus biogenesis and DNA transformation are functionally linked and play a pivotal role in the life style of this strictly human pathogen. The assembly of pili from its main subunit pilin (PilE) is a prerequisite for gonococcal infection since it allows the first contact to epithelial cells in conjunction with the pilus tip-associated PilC protein. While the components of the pilus and its assembly machinery are either directly or indirectly involved in the transport of DNA across the outer membrane, other factors unrelated to pilus biogenesis appear to facilitate further DNA transfer across the murein layer (ComL, Tpc) and the inner membrane (ComA) before the transforming DNA is rescued in the recipient bacterial chromosome in a RecA-dependent manner. Interestingly, PilE is essential for the first step of transformation, i.e., DNA uptake, and is itself also subject to transformation-mediated phase and antigenic variation. This short-term adaptive mechanism allows Ngo to cope with changing micro-environments in the host as well as to escape the immune response during the course of infection. Given the fact that Ngo has no ecological niche other than man, horizontal genetic exchange is essential for a successful co-evolution with the host. Horizontal exchange gives rise to heterogeneous populations harboring clones which better withstand selective forces within the host. Such extended horizontal exchange is reflected by a high genome plasticity, the existence of mosaic genes and a low linkage disequilibrium of genetic loci within the neisserial population. This led to the concept that rather than regarding individual Neisseria species as independent traits, they comprise a collective of species interconnected via horizontal exchange and relying on a common gene pool.

  20. High-Throughput Screening for Novel Inhibitors of Neisseria gonorrhoeae Penicillin-Binding Protein 2

    PubMed Central

    Fedarovich, Alena; Djordjevic, Kevin A.; Swanson, Shauna M.; Peterson, Yuri K.; Nicholas, Robert A.; Davies, Christopher

    2012-01-01

    The increasing prevalence of N. gonorrhoeae strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for β-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of N. gonorrhoeae. It also provides a set of lead compounds for optimization of anti-gonococcal agents. PMID:23049763

  1. Changes in the six most common sequence types of Neisseria gonorrhoeae, including ST4378, identified by surveillance of antimicrobial resistance in northern Taiwan from 2006 to 2013.

    PubMed

    Cheng, Ching-Wai; Li, Lan-Hui; Su, Chen-Yi; Li, Shu-Ying; Yen, Muh-Yong

    2016-10-01

    There has been no longitudinal study of drug susceptibility in Neisseria gonorrhoeae in Taiwan since 2006. We collected 1090 gonococcal isolates from Taipei City Hospital, Taiwan from April 2006 to August 2013. We used a disk diffusion assay to determine the susceptibility to five antibiotics and an E-test to determine the minimum inhibitory concentrations for cefixime and ceftriaxone in isolates with resistance. Neisseria gonorrhoeae-multi Antigen Sequence Typing and DNA sequencing of the por and tbpB genes were used to identify sequence types. Among the 1090 isolates, the resistances to penicillin, ciprofloxacin, cefpodoxime, cefixime, and ceftriaxone were 61.01%, 83.39%, 9.63%, 6.70%, and 2.39%, respectively. The highest minimum inhibitory concentrations of cefixime and ceftriaxone were 0.19 mg/L and 0.50 mg/L, respectively. There were 327 sequence types. The four most common sequence types in homosexuals were ST4378, ST359, ST4654, and ST547; the two most common sequence types in heterosexuals were ST421 and ST419. Each of these sequence types had more than 25 isolates. There were significant differences in the sequence types in patients with different sexual orientations (p < 0.001). Oral cefixime or ceftriaxone injections were used as first-line drugs for the treatment of gonorrhea from 2006 to 2013 because gonorrhea isolates had low minimum inhibitory concentrations for these two drugs. The abrupt emergence of ST4378 (closely related to the notorious ST1407) since 2009 is a cause for alarm. Changes in sexual behavior, including an increase in sexual activity without the use of condoms, may have contributed to the peak in gonorrhea in 2010. Further molecular epidemiological investigations are required. Copyright © 2014. Published by Elsevier B.V.

  2. Antimicrobial resistance in Neisseria gonorrhoeae in the UK: surveillance and management.

    PubMed

    Ison, Catherine A; Alexander, Sarah

    2011-10-01

    Successful antimicrobial therapy is fundamental to the public health control of gonorrhea, in the absence of a protective immune response. Neisseria gonorrhoeae, the causative agent, has presented a constant challenge for the provision of such therapy as it has demonstrated the ability to become resistant to successive highly active agents chosen for first-line treatment. Acquisition of plasmids from other bacteria and long-term use of a single agent has selected both single step high-level and low-level resistance due to multiple mutations. While therapeutic failure of the current recommended agents cefixime and ceftriaxone begins to emerge, choice of alternative therapies is limited. Guidelines for therapy will be dependent on surveillance programs but individual patient management will require a viable organism to detect emerging resistance. Advances in molecular detection, while advantageous for the diagnosis of gonorrhea, fail to provide a viable organism, posing even greater challenges for the definition of treatment failure, and appropriate end points for test of cure. Innovative and collaborative approaches will be essential to maintain gonorrhea as a treatable infection.

  3. Neisseria gonorrhoeae Challenge Increases Matrix Metalloproteinase-8 Expression in Fallopian Tube Explants.

    PubMed

    Juica, Natalia E; Rodas, Paula I; Solar, Paula; Borda, Paula; Vargas, Renato; Muñoz, Cristobal; Paredes, Rodolfo; Christodoulides, Myron; Velasquez, Luis A

    2017-01-01

    Background:Neisseria gonorrhoeae (Ngo) is the etiological agent of gonorrhea, a sexually transmitted infection that initially infects the female lower genital tract. In untreated women, the bacteria can ascend to the upper genital reproductive tract and infect the fallopian tube (FTs), which is associated with salpingitis and can lead to impaired FT function and infertility. The extracellular matrix (ECM) plays an important role in cell migration and differentiation in the female genital tract, and some pathogens modify the ECM to establish successful infections. The ECM is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), their endogenous inhibitors; MMP deregulation causes pathological conditions in a variety of tissues. Results: The aim of this work was to analyze the expression and localization of MMP-3, MMP-8, MMP-9, and TIMP-1 in FT explants during Ngo infection using real-time PCR, immunohistochemistry, zymography and ELISA. No significant variations in MMP-3, MMP-9, and TIMP-1 transcript levels were observed. In contrast, a significant increase (p < 0.05) was observed for MMP-8 expression and was accompanied by stromal immunoreactivity in infected explants. ELISA results supported these findings and showed that MMP-8 release increased upon gonococcal infection. Conclusions: Our results indicate that gonococcal infection induces increased MMP-8 expression, which might contribute to FT damage during infection.

  4. Neisseria gonorrhoeae Phagosomes Delay Fusion with Primary Granules to Enhance Bacterial Survival Inside Human Neutrophils

    PubMed Central

    Johnson, M. Brittany; Criss, Alison K.

    2013-01-01

    Summary Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leukocytes (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 gonorrhea is by delaying primary granule-phagosome fusion, thus preventing formation of a degradative phagolysosome. PMID:23374609

  5. Neisseria gonorrhoeae Challenge Increases Matrix Metalloproteinase-8 Expression in Fallopian Tube Explants

    PubMed Central

    Juica, Natalia E.; Rodas, Paula I.; Solar, Paula; Borda, Paula; Vargas, Renato; Muñoz, Cristobal; Paredes, Rodolfo; Christodoulides, Myron; Velasquez, Luis A.

    2017-01-01

    Background: Neisseria gonorrhoeae (Ngo) is the etiological agent of gonorrhea, a sexually transmitted infection that initially infects the female lower genital tract. In untreated women, the bacteria can ascend to the upper genital reproductive tract and infect the fallopian tube (FTs), which is associated with salpingitis and can lead to impaired FT function and infertility. The extracellular matrix (ECM) plays an important role in cell migration and differentiation in the female genital tract, and some pathogens modify the ECM to establish successful infections. The ECM is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), their endogenous inhibitors; MMP deregulation causes pathological conditions in a variety of tissues. Results: The aim of this work was to analyze the expression and localization of MMP-3, MMP-8, MMP-9, and TIMP-1 in FT explants during Ngo infection using real-time PCR, immunohistochemistry, zymography and ELISA. No significant variations in MMP-3, MMP-9, and TIMP-1 transcript levels were observed. In contrast, a significant increase (p < 0.05) was observed for MMP-8 expression and was accompanied by stromal immunoreactivity in infected explants. ELISA results supported these findings and showed that MMP-8 release increased upon gonococcal infection. Conclusions: Our results indicate that gonococcal infection induces increased MMP-8 expression, which might contribute to FT damage during infection. PMID:28932707

  6. 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

  7. 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.

  8. Fur-Mediated Activation of Gene Transcription in the Human Pathogen Neisseria gonorrhoeae

    PubMed Central

    Yu, Chunxiao

    2012-01-01

    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

  9. Cell envelope of Neisseria gonorrhoeae: relationship between autolysis in buffer and the hydrolysis of peptidoglycan.

    PubMed Central

    Wegener, W S; Hebeler, B H; Morse, S A

    1977-01-01

    Neisseria gonorrhoeae readily underwent autolysis when suspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at alkaline pH values. Autolysis was inhibited by the addition of Mg2+ or other divalent cations. Autolysis was also suppressed at acid pH (pH 6.0). Suspension of cells in buffer was accompanied by the hydrolysis of peptidoglycan. The rate of peptidoglycan hydrolysis in HEPES buffer was maximal at pH 8.5 and was similar in the presence or absence of Mg2+. Therefore, divalent cation stabilization against autolysis is not mediated by inhibition of peptidoglycan hydrolysis. Peptidoglycan hydrolysis occurred in HEPES buffer (pH 6.0), but at a rate that was 50% of the maximum. Incubation of cells with chloramphenicol or rifampin before suspension in HEPES buffer (pH 8.5) partially prevented autolysis; under these conditions, peptidoglycan hydrolysis still occurred, but at a reduced rate. Old and new peptidoglycans were hydrolyzed at similar rates. Peptidoglycan hydrolysis results in solubilization of both the peptide and glycan moieties. Images PMID:20406

  10. 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

  11. 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.

  12. Urethral exudates of men with Neisseria gonorrhoeae infections select a restricted lipooligosaccharide phenotype during transmission.

    PubMed

    McLaughlin, Stephanie E; Cheng, Hui; Ghanem, Khalil G; Yang, Zhijie; Melendez, Johan; Zenilman, Jonathan; Griffiss, J McLeod

    2012-10-01

    Neisseria gonorrhoeae lipooligosaccharides (LOSs) induce immunoglobulin G that protects men from experimental infection. This raises the possibility that an LOS vaccine might prevent gonorrhea. Gonococci make different LOS molecules, depending on whether 3 genes, lgtA, lgtC, and lgtD, are in frame (IF) or out of frame (OOF). Mispairing of polymeric guanine (polyG) tracts within each gene determines its frame during replication. We amplified lgtA, lgtC, and lgtD from diagnostic slides of urethral exudates and sequenced their polyG tracts. We found that lgtA in exudative bacteria is IF and that lgtC is OOF. The frame of lgtD varied widely: it was OOF in most but not all cases. This genotype would result in synthesis of polylactosamine α chains that could be sialylated. Polylactosamine α chains would enhance virulence, and their sialylation would enable gonococci to survive within polymorphonuclear cells; however, an active LgtD in a few bacteria could provide a survival advantage in other sites of infection.

  13. Secretory Leukocyte Protease Inhibitor Binds to Neisseria gonorrhoeae Outer Membrane Opacity Protein and is Bactericidal

    PubMed Central

    Cooper, Morris D.; Roberts, Melissa H.; Barauskas, Ona L.; Jarvis, Gary A.

    2012-01-01

    Problem Secretory leukocyte protease inhibitor (SLPI) is an innate immune peptide present on the genitourinary tract mucosa which has antimicrobial activity. In this study, we investigated the interaction of SLPI with Neisseria gonorrhoeae. Method of study ELISA and far-western blots were used to analyze binding of SLPI to gonococci. The binding site for SLPI was identified by tryptic digests and mass spectrometry. Antimicrobial activity of SLPI for gonococci was determined using bactericidal assays. SLPI protein levels in cell supernatants were measured by ELISA, and SLPI mRNA levels were assessed by quantitative RT-PCR. Results SLPI bound directly to the gonococcal Opa protein and was bactericidal. Epithelial cells from the reproductive tract constitutively expressed SLPI at different levels. Gonococcal infection of cells did not affect SLPI expression. Conclusion We conclude that SLPI is bactericidal for gonococci and is expressed by reproductive tract epithelial cells and thus is likely to play a role in the pathogenesis of gonococcal infection. PMID:22537232

  14. Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae

    PubMed Central

    Vik, Åshild; Aas, Finn Erik; Anonsen, Jan Haug; Bilsborough, Shaun; Schneider, Andrea; Egge-Jacobsen, Wolfgang; Koomey, Michael

    2009-01-01

    Protein glycosylation is an important element of biologic systems because of its significant effects on protein properties and functions. Although prominent within all domains of life, O-linked glycosylation systems modifying serine and threonine residues within bacteria and eukaryotes differ substantially in target protein selectivity. In particular, well-characterized bacterial systems have been invariably dedicated to modification of individual proteins or related subsets thereof. Here we characterize a general O-linked glycosylation system that targets structurally and functionally diverse groups of membrane-associated proteins in the Gram-negative bacterium Neisseria gonorrhoeae, the etiologic agent of the human disease gonorrhea. The 11 glycoproteins identified here are implicated in activities as varied as protein folding, disulfide bond formation, and solute uptake, as well as both aerobic and anaerobic respiration. Along with their common trafficking within the periplasmic compartment, the protein substrates share quasi-related domains bearing signatures of low complexity that were demonstrated to encompass sites of glycan occupancy. Thus, as in eukaryotes, the broad scope of this system is dictated by the relaxed specificity of the glycan transferase as well as the bulk properties and context of the protein-targeting signal rather than by a strict amino acid consensus sequence. Together, these findings reveal previously unrecognized commonalities linking O-linked protein glycosylation in distantly related life forms. PMID:19251655

  15. Neisseria gonorrhoeae breaches the apical junction of polarized epithelial cells for transmigration by activating EGFR

    PubMed Central

    Edwards, Vonetta L.; Wang, Liang-Chun; Dawson, Valerie; Stein, Daniel C.; Song, Wenxia

    2017-01-01

    Summary Neisseria gonorrhoeae initiates infection at the apical surface of columnar endocervical epithelial cells in the female reproductive tract. These cells provide a physical barrier against pathogens by forming continuous apical junctional complexes between neighbouring cells. This study examines the interaction of gonococci (GC) with polarized epithelial cells. We show that viable GC preferentially localize at the apical side of the cell–cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84. In GC-infected cells, continuous apical junctional complexes are disrupted, and the junction-associated protein β-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites; however, overall cellular levels remain unchanged. This redistribution of junctional proteins is associated with a decrease in the ‘fence’ function of the apical junction but not its ‘gate’ function. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and β-catenin, while inhibition of EGFR kinase activity significantly reduces both GC-induced β-catenin redistribution and GC transmigration. Therefore, the gonococcus is capable of weakening the apical junction and polarity of epithelial cells by activating EGFR, which facilitates GC transmigration across the epithelium. PMID:23279089

  16. Neisseria gonorrhoeae breaches the apical junction of polarized epithelial cells for transmigration by activating EGFR.

    PubMed

    Edwards, Vonetta L; Wang, Liang-Chun; Dawson, Valerie; Stein, Daniel C; Song, Wenxia

    2013-06-01

    Neisseria gonorrhoeae initiates infection at the apical surface of columnar endocervical epithelial cells in the female reproductive tract. These cells provide a physical barrier against pathogens by forming continuous apical junctional complexes between neighbouring cells. This study examines the interaction of gonococci (GC) with polarized epithelial cells. We show that viable GC preferentially localize at the apical side of the cell-cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84. In GC-infected cells, continuous apical junctional complexes are disrupted, and the junction-associated protein β-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites; however, overall cellular levels remain unchanged. This redistribution of junctional proteins is associated with a decrease in the 'fence' function of the apical junction but not its 'gate' function. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and β-catenin, while inhibition of EGFR kinase activity significantly reduces both GC-induced β-catenin redistribution and GC transmigration. Therefore, the gonococcus is capable of weakening the apical junction and polarity of epithelial cells by activating EGFR, which facilitates GC transmigration across the epithelium.

  17. 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

  18. Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

    PubMed Central

    Aas, Finn Erik; Vik, Åshild; Vedde, John; Koomey, Michael; Egge-Jacobsen, Wolfgang

    2007-01-01

    Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a ‘top-down’ mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies. PMID:17608667

  19. Quantitative Proteomics of the Neisseria Gonorrhoeae Cell Envelope and Membrane Vesicles for the Discovery of Potential Therapeutic Targets*

    PubMed Central

    Zielke, Ryszard A.; Wierzbicki, Igor H.; Weber, Jacob V.; Gafken, Philip R.; Sikora, Aleksandra E.

    2014-01-01

    Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC

  20. In vivo selection for Neisseria gonorrhoeae opacity protein expression in the absence of human carcinoembryonic antigen cell adhesion molecules.

    PubMed

    Simms, Amy N; Jerse, Ann E

    2006-05-01

    The neisserial opacity (Opa) proteins are phase-variable, antigenically distinct outer membrane proteins that mediate adherence to and invasion of human cells. We previously reported that Neisseria gonorrhoeae Opa protein expression appeared to be selected for or induced during experimental murine genital tract infection. Here we further defined the kinetics of recovery of Opa variants from the lower genital tracts of female mice and investigated the basis for this initial observation. We found that the recovery of different Opa phenotypes from mice appears cyclical. Three phases of infection were defined. Following intravaginal inoculation with primarily Opa- gonococci, the majority of isolates recovered were Opa+ (early phase). A subsequent decline in the percentage of Opa+ isolates occurred in a majority of mice (middle phase) and was followed by a reemergence of Opa+ variants in mice that were infected for longer than 8 days (late phase). We showed the early phase was due to selection for preexisting Opa+ variants in the inoculum by constructing a chloramphenicol-resistant (Cm(r)) strain and following Cm(r) Opa+ populations mixed with a higher percentage of Opa- variants of the wild-type (Cm(s)) strain. Reciprocal experiments (Opa- Cm(r) gonococci spiked with Opa+ Cm(s) bacteria) were consistent with selection of Opa+ variants. Based on the absence in mice of human carcinoembryonic antigen cell adhesion molecules, the major class of Opa protein adherence receptors, we conclude the observed selection for Opa+ variants early in infection is not likely due to a specific adherence advantage and may be due to Opa-mediated evasion of innate defenses.

  1. Treatment of infection due to penicillinase-producing Neisseria gonorrhoeae with oral thiamphenicol and with oral lymecycline.

    PubMed

    Latif, A S; Marowa, E; Mason, P R; Sithole, J; Tambo, J; Dhamu, F; Paraiwa, E

    1986-01-01

    Seventy-five men with gonococcal urethritis were treated with a single oral dose of thiamphenicol, and 88 men with this infection were treated with two 1.5-g oral doses of lymecycline taken 12 hr apart. Of the 75 subjects treated with thiamphenicol, 72 (96%) were cured, as compared with 80 (91%) treated with lymecycline. Sixty subjects (37%) were infected with penicillinase-producing Neisseria gonorrhoeae. In this group, 28 (97%) of 29 subjects treated with thiamphenicol were cured, as compared with 29 (94%) of 31 subjects treated with lymecycline. Patient compliance with the two-dose regimen was excellent, and no adverse effects occurred with either drug. Lymecycline may therefore be an effective alternative to thiamphenicol in those countries where strains of N. gonorrhoeae remain sensitive to the tetracyclines.

  2. Factor H facilitates adherence of Neisseria gonorrhoeae to complement receptor 3 on eukaryotic cells1

    PubMed Central

    Agarwal, Sarika; Ram, Sanjay; Ngampasutadol, Jutamas; Gulati, Sunita; Zipfel, Peter F.; Rice, Peter A.

    2010-01-01

    Neisseria gonorrhoeae can engage human complement receptor 3 (CR3) directly or through surface-bound iC3b. Factor H (fH) that binds to bacteria facilitates conversion of C3b to iC3b. fH also binds directly to CR3 on professional phagocytes. Certain non-professional phagocytes such as primary cervical epithelial cells also express CR3. We hypothesized that fH could bridge bacteria to CR3 and facilitate gonococcal association with host cells. Specificity of the fH-CR3 interaction was confirmed using human CR3-transfected CHO (CHO/CR3) cells. Using recombinant proteins that comprised contiguous fH domains (fH contains 20 short consensus repeat (SCR) domains) fused to murine Fc, we observed strong binding through SCRs 18–20, while weaker binding occurred through SCRs 6–10. Both regions also bound to unsialylated porin (Por) B.1A-expressing N. gonorrhoeae. Accordingly, fH-related protein 1 (CFHR1) (three of its five SCRs are highly homologous to fH SCRs 18–20) bound to CHO-CR3 and to unsialylated PorB.1A gonococci. An alternatively spliced variant of fH called fH-like protein-1 (FHL-1) (contains fH SCRs 1–7) bound to gonococci but minimally to CHO/CR3. A fH SCR 6–20 construct enhanced binding of unsialylated PorB.1A gonococci to CHO/CR3. However, a construct that contained only the apparently relevant SCRs (6–7 and 18–20) bound to CHO/CR3 and to gonococci separately, but did not enhance bacteria-CR3 interactions, suggesting that the intervening SCRs (8–17) may impart a configurational and spatial requirement for fH to bridge gonococci to CR3. These results indicate adherence between fH-coated gonococci and CR3 and may provide a means for gonococci to gain sanctuary into non-professional phagocytes. PMID:20826755

  3. Severe conjunctivitis due to multidrug-resistant Neisseria gonorrhoeae and adenovirus 53 coinfection in a traveler returning from Thailand.

    PubMed

    Tappe, Dennis; Mueller, Andreas; Weißbrich, Benedikt; Schubert, Jörg; Schargus, Marc; Stich, August

    2013-01-01

    A male traveler returning from Thailand with severe bilateral conjunctivitis was tested for causative pathogens by culture and polymerase chain reaction in late 2010. The culturally grown Neisseria gonorrhoeae strain was resistant against penicillin, ciprofloxacin, and tetracycline. The patient was also found to have an eye infection with the unusual and likely recombinant adenovirus type 53. Besides multidrug-resistant gonococcal strains the unusual adenovirus strain is found circulating in Asia and both pathogens may be a risk for travelers. © 2013 International Society of Travel Medicine.

  4. [Detection of Neisseria gonorrhoeae(NG), Chlamydia trachomatis(CT) and Ureaplasma urealyticum(UU) by multiple primer PCR].

    PubMed

    Lu, Y; Li, J; Chen, Y

    2000-04-01

    For the detection of Neisseria gonorrhoeae(NG), Chlamydia trachomat is(CT) and Ureaplasma urealyticum(UU) in urogenital infection in one specimen, multiple primer PCR was used in 156 sex transmitted disease patients, it was shown that in 156 specimens there were 40 NG positive and 31 CT positive and 27 UU positive, 10 NG + CT positive, 4 NG + UU positive, 3 CT + UU positive and 2 NG + CT + UU positive respectively. The results were in correspondence with each other. It is suggested that multiple primer PCR is a quick, sensitive, simple and specific technique which can be applied in clinical test.

  5. Mosaic-like organization of IgA protease genes in Neisseria gonorrhoeae generated by horizontal genetic exchange in vivo.

    PubMed Central

    Halter, R; Pohlner, J; Meyer, T F

    1989-01-01

    IgA protease is a putative virulence factor that exists in several allelic forms in Neisseria gonorrhoeae. However, extracellular secretion of these variant IgA proteases occurs by the same pathway involving three steps of autoproteolytic maturation from a large precursor. Two principal precursor types (H1 and H2) can be distinguished with respect to the location of autoproteolytic sites and the sizes of the mature products. By partial DNA sequence analysis, additional variations have been detected which are not unique to one particular gene; rather, otherwise unrelated iga genes often share homology, thus revealing a composite organization. In the context of other gonococcal features, this observation implies that recombination has occurred in vivo between iga genes of different strains, probably via the route of species-specific DNA transformation. This process may be of general significance for the modulation and the natural exchange of virulence properties among pathogenic Neisseriae. Images PMID:2511009

  6. 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.

  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. [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.

  9. Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae

    PubMed Central

    2011-01-01

    Background Maintenance of an anaerobic denitrification system in the obligate human pathogen, Neisseria gonorrhoeae, suggests that an anaerobic lifestyle may be important during the course of infection. Furthermore, mounting evidence suggests that reduction of host-produced nitric oxide has several immunomodulary effects on the host. However, at this point there have been no studies analyzing the complete gonococcal transcriptome response to anaerobiosis. Here we performed deep sequencing to compare the gonococcal transcriptomes of aerobically and anaerobically grown cells. Using the information derived from this sequencing, we discuss the implications of the robust transcriptional response to anaerobic growth. Results We determined that 198 chromosomal genes were differentially expressed (~10% of the genome) in response to anaerobic conditions. We also observed a large induction of genes encoded within the cryptic plasmid, pJD1. Validation of RNA-seq data using translational-lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. We also confirmed expression and regulation of a small RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family. We also determined that many genes found to be responsive to anaerobiosis have also been shown to be responsive to iron and/or oxidative stress. Conclusions Gonococci will be subject to many forms of environmental stress, including oxygen-limitation, during the course of infection. Here we determined that the anaerobic stimulon in gonococci was larger than previous studies would suggest. Many new targets for future research have been uncovered, and the results derived from this study may have helped to elucidate factors or mechanisms of virulence that may have otherwise been overlooked. PMID:21251255

  10. Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae.

    PubMed

    Isabella, Vincent M; Clark, Virginia L

    2011-01-20

    Maintenance of an anaerobic denitrification system in the obligate human pathogen, Neisseria gonorrhoeae, suggests that an anaerobic lifestyle may be important during the course of infection. Furthermore, mounting evidence suggests that reduction of host-produced nitric oxide has several immunomodulary effects on the host. However, at this point there have been no studies analyzing the complete gonococcal transcriptome response to anaerobiosis. Here we performed deep sequencing to compare the gonococcal transcriptomes of aerobically and anaerobically grown cells. Using the information derived from this sequencing, we discuss the implications of the robust transcriptional response to anaerobic growth. We determined that 198 chromosomal genes were differentially expressed (~10% of the genome) in response to anaerobic conditions. We also observed a large induction of genes encoded within the cryptic plasmid, pJD1. Validation of RNA-seq data using translational-lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. We also confirmed expression and regulation of a small RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family. We also determined that many genes found to be responsive to anaerobiosis have also been shown to be responsive to iron and/or oxidative stress. Gonococci will be subject to many forms of environmental stress, including oxygen-limitation, during the course of infection. Here we determined that the anaerobic stimulon in gonococci was larger than previous studies would suggest. Many new targets for future research have been uncovered, and the results derived from this study may have helped to elucidate factors or mechanisms of virulence that may have otherwise been overlooked.

  11. Reaching homeless youths for Chlamydia trachomatis and Neisseria gonorrhoeae screening in Denver, Colorado.

    PubMed

    Van Leeuwen, J M; Rietmeijer, C A; LeRoux, T; White, R; Petersen, J

    2002-10-01

    The availability of urine based testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) provides a unique opportunity to screen for these pathogens outside traditional clinical settings. The researchers investigated the feasibility of street based CT/GC screening in the context of an outreach programme among street/homeless adolescents in Denver. To describe the integration of urine based CT/GC screening into an existing outreach programme among street/homeless youths and the yield of CT/GC testing in this setting. The Denver Department of Public Health (DPH) collaborated with outreach staff from Urban Peak (a community based organisation serving street/homeless youths in Denver) to offer urine based CT/GC testing to males and females in street settings. Tests were conducted on the street in areas where street/homeless youths congregate. Urban Peak outreach staff were trained by DPH staff to conduct CT testing, process urine specimens, and provide test results to participating youths. DPH remained responsible for treatment of people with CT or GC infection as well as CT/GC case reporting. CT testing started in January 2000; GC testing was added in July 2000. Throughout April 2002 a total of 414 CT and 302 GC tests were conducted, respectively 11.6% and 2.7% of which were positive. Among first testers, 13.0% were positive for CT and 3.7% for GC. CT/GC urine testing can be incorporated into existing outreach programmes without considerable extra effort. Overall CT rates were high and suggest the need for ongoing screening in this manner. To understand the role of CT/GC screening in the context of services provided to street/homeless youths in outreach settings.

  12. 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.

  13. [Neisseria gonorrhoeae: antimicrobial resistance and study of population dynamics. Situation in Barcelona in 2011].

    PubMed

    Serra-Pladevall, Judit; Barberá-Gracia, María Jesús; Roig-Carbajosa, Glòria; Juvé-Saumell, Rosa; Gonzalez-Lopez, Juan José; Bartolomé-Comas, Rosa; Andreu-Domingo, Antònia

    2013-11-01

    Due to the high rates of antimicrobial resistance to certain antibiotics, together with the emergence of Neisseria gonorrhoeae (NG) with reduced susceptibility and resistance to third-generation cephalosporins, gonococcal infection is becoming a public health problem. The objectives of the study were: To keep track of the antimicrobial susceptibility of NG strains obtained from January to August 2011. To study the population dynamics. The antimicrobial susceptibility was studied by disk-diffusion and E-test. The genotyping was performed by NG-MAST method. Of a total of 100strains studied, 59% showed intermediate sensitivity to penicillin and 9% were resistant. According to EUCAST, we detected 3gonococci with reduced susceptibility to ceftriaxone, 10 to cefixime and one with high-level resistance to both antibiotics (MIC 1.5μg/ml). MIC50 and MIC90 to cefixime were 0.016 and 0.125μg/ml, respectively, whereas to ceftriaxone they were <0.016 and 0.064μg/ml, respectively. Almost all (99%) of the strains were resistant to doxycycline, 53% to ciprofloxacin, 3% to azithromycin, and 1% to spectinomycin. The most prevalent ST was ST1407, predominantly associated to resistance or reduced sensitivity to cephalosporins or macrolides. NG has developed significant rates of resistance to various antibiotics. One strain has been detected with high level resistance to third generation cephalosporins, and several strains with reduced susceptibility. An increase in MIC50 and MIC90 to these antibiotics has also been observed. NG population structure remains stable and common to the rest of Europe, although two new ST (ST7226 and ST7227) have been identified that could be selected and acquire high levels of resistance to cephalosporins. Copyright © 2012 Elsevier España, S.L. All rights reserved.

  14. Identification of regulatory elements that control expression of the tbpBA operon in Neisseria gonorrhoeae.

    PubMed

    Vélez Acevedo, Rosuany N; Ronpirin, Chalinee; Kandler, Justin L; Shafer, William M; Cornelissen, Cynthia Nau

    2014-08-01

    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.

  15. 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. Copyright © 2011. Published by Elsevier Masson SAS.

  16. High-Level Cefixime- and Ceftriaxone-Resistant Neisseria gonorrhoeae in France: Novel penA Mosaic Allele in a Successful International Clone Causes Treatment Failure

    PubMed Central

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

    2012-01-01

    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

  17. New treatment options for infections caused by increasingly antimicrobial-resistant Neisseria gonorrhoeae.

    PubMed

    Lee, Hyukmin; Lee, Kyungwon; Chong, Yunsop

    2016-01-01

    The emergence of high-level resistance to ceftriaxone is giving rise to serious concern about absence of effective treatment options to cure gonococcal infections. Increasing the dosage regimen can be applied to ceftriaxone and azithromycin, but the emergence of high-level resistance has already been reported. Spectinomycin is another active drug but has low efficacy in the treatment of pharyngeal gonorrhoea. Conventional antibiotics could be introduced for gonococcal treatment, but they have some limitations, such as the absence of clinical trials and breakpoint. Combining antibiotics is another promising method to cure patients and to prevent the emergence of resistance. The most important strategy to maintain the efficacy of antibiotics is rapid detection and dissemination control of novel resistant isolate.

  18. Multiplexed nanoplasmonic biosensor for one-step simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

    PubMed

    Soler, Maria; Belushkin, Alexander; Cavallini, Andrea; Kebbi-Beghdadi, Carole; Greub, Gilbert; Altug, Hatice

    2017-08-15

    Development of rapid and multiplexed diagnostic tools is a top priority to address the current epidemic problem of sexually transmitted diseases. Here we introduce a novel nanoplasmonic biosensor for simultaneous detection of the two most common bacterial infections: Chlamydia trachomatis and Neisseria gonorrhoeae. Our plasmonic microarray is composed of gold nanohole sensor arrays that exhibit the extraordinary optical transmission (EOT), providing highly sensitive analysis in a label-free configuration. The integration in a microfluidic system and the precise immobilization of specific antibodies on the individual sensor arrays allow for selective detection and quantification of the bacteria in real-time. We achieved outstanding sensitivities for direct immunoassay of urine samples, with a limit of detection of 300 colony forming units (CFU)/mL for C. trachomatis and 1500CFU/mL for N. gonorrhoeae. The multiplexing capability of our biosensor was demonstrated by analyzing different urine samples spiked with either C. trachomatis or N. gonorrhoeae, and also containing both bacteria. We could successfully detect, identify and quantify the levels of the two bacteria in a one-step assay, without the need for DNA extraction or amplification techniques. This work opens up new possibilities for the implementation of point-of-care biosensors that enable fast, simple and efficient diagnosis of sexually transmitted infections. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Chlamydia trachomatis and Neisseria gonorrhoeae in HIV-infected Pregnant Women and Adverse Infant Outcomes

    PubMed Central

    Adachi, Kristina; Klausner, Jeffrey D.; Xu, Jiahong; Ank, Bonnie; Bristow, Claire C.; Morgado, Mariza G.; Watts, D. Heather; Weir, Fred; Persing, David; Mofenson, Lynne M.; Veloso, Valdilea G.; Pilotto, Jose Henrique; Joao, Esau; Gray, Glenda; Theron, Gerhard; Santos, Breno; Fonseca, Rosana; Kreitchmann, Regis; Pinto, Jorge; Mussi-Pinhata, Marisa M.; Ceriotto, Mariana; Machado, Daisy Maria; Bryson, Yvonne J.; Grinsztejn, Beatriz; Bastos, Francisco I.; Siberry, George; Nielsen-Saines, Karin

    2016-01-01

    BACKGROUND Sexually transmitted infections (STIs) in pregnancy such as Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) may lead to adverse infant outcomes. METHODOLOGY Individual urine specimens from HIV-infected pregnant women diagnosed with HIV during labor were collected at the time of infant birth and tested by polymerase chain reaction for CT and NG. Infant HIV infection was determined at 3 months with morbidity/mortality assessed through 6 months. RESULTS Of 1373 maternal urines, 277 (20.2%) were positive for CT and/or NG; 249 (18.1%) for CT, 63 (4.6%) for NG, and 35 (2.5%) for both CT and NG. HIV infection was diagnosed in 117 (8.5%) infants. Highest rates of adverse outcomes (sepsis, pneumonia, congenital syphilis, septic arthritis, conjunctivitis, low birth weight, preterm delivery, death) were noted in infants of women with CT and NG (23/35, 65.7%) compared to NG (16/28, 57.1%), CT (84/214, 39.3%), and no STI (405/1096, 37%, p=0.001). Death (11.4% vs. 3%, p=0.02), low birth weight (42.9% vs. 16.9%, p=0.001), and preterm delivery (28.6% vs. 10.2%, p=0.008) were higher among infants of CT and NG co-infected women. Infants who had any adverse outcome and were born to women with CT and/or NG were 3.5 times more likely to be HIV-infected after controlling for maternal syphilis (OR 3.5, 95% CI 1.4-8.3). By adjusted multivariate logistic regression, infants born to mothers with any CT and/or NG were 1.35 times more likely to have an adverse outcome (OR 1.35, 95% CI 1.03-1.76). CONCLUSION STIs in HIV-infected pregnant women are associated with adverse outcomes in HIV-exposed infected and uninfected infants. PMID:27164464

  20. [Screening of Neisseria gonorrhoeae and Chlamydia trachomatis using techniques of self collected vaginal sample in young women].

    PubMed

    Conejero, Carolina; Cannoni, Gigliola; Merino, Paulina M; Bollmann, Josefina; Hidalgo, Camila; Castro, Magdalena; Schulin-Zeuthen, Carolina

    2013-10-01

    Chlamydia trachomatis and Neisseria gonorrhoeae are responsible for 3-10% of sexually transmitted diseases in adolescents. 75% are asymptomatic. International standards recommend annual screening for C. trachomatis in sexually active women under 26 years. Self-collected vaginal swab is one of the less invasive screening methods, it is well accepted by patients and rarely used in our country. To determine the frequency of C. trachomatis and n. gonorrhoeae by a self-sampling method of vaginal swab and its acceptability in a group of adolescents and young adults. Women 18 to 25 years old. Vaginal samples were processed by nucleic acid amplification tests, Gen Probe APTIMA Combo2. Data were collected on sexual behavior and perception of self-sampling by survey. We studied 344 patients with an average age of 21.7 years. Detection of C. trachomatis was positive in 7.9% women and it was not found in any of the patients studied for N. gonorrhoeae. 98% considered self-sampling instructions easy to understand, 87.5% felt comfortable taking the sample. Prevalence of C. trachomatis in the study population was similar to that described in other national and international studies. N. gonorrhoeae was not found in this series, which is consistent with literature reports. The self-sampling technique of vaginal sample was well accepted by the patients. However, they were anxious about the quality of the sample. According to our results, it is important to emphasize the importance of annual detection of these pathogens and that self-sampling technique is a valid alternative.

  1. A novel mechanism of high-level, broad-spectrum antibiotic resistance caused by a single base pair change in Neisseria gonorrhoeae.

    PubMed

    Ohneck, Elizabeth A; Zalucki, Yaramah M; Johnson, Paul J T; Dhulipala, Vijaya; Golparian, Daniel; Unemo, Magnus; Jerse, Ann E; Shafer, William M

    2011-01-01

    The MtrC-MtrD-MtrE multidrug efflux pump of Neisseria gonorrhoeae confers resistance to a diverse array of antimicrobial agents by transporting these toxic compounds out of the gonococcus. Frequently in gonococcal strains, the expression of the mtrCDE operon is differentially regulated by both a repressor, MtrR, and an activator, MtrA. The mtrR gene lies 250 bp upstream of and is transcribed divergently from the mtrCDE operon. Previous research has shown that mutations in the mtrR coding region and in the mtrR-mtrCDE intergenic region increase levels of gonococcal antibiotic resistance and in vivo fitness. Recently, a C-to-T transition mutation 120 bp upstream of the mtrC start codon, termed mtr₁₂₀, was identified in strain MS11 and shown to be sufficient to confer high levels of antimicrobial resistance when introduced into strain FA19. Here we report that this mutation results in a consensus -10 element and that its presence generates a novel promoter for mtrCDE transcription. This newly generated promoter was found to be stronger than the wild-type promoter and does not appear to be subject to MtrR repression or MtrA activation. Although rare, the mtr₁₂₀ mutation was identified in an additional clinical isolate during sequence analysis of antibiotic-resistant strains cultured from patients with gonococcal infections. We propose that cis-acting mutations can develop in gonococci that significantly alter the regulation of the mtrCDE operon and result in increased resistance to antimicrobials. Gonorrhea is the second most prevalent sexually transmitted bacterial infection and a worldwide public health concern. As there is currently no vaccine against Neisseria gonorrhoeae, appropriate diagnostics and subsequent antibiotic therapy remain the primary means of infection control. However, the effectiveness of antibiotic treatment is constantly challenged by the emergence of resistant strains, mandating a thorough understanding of resistance mechanisms to

  2. 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

  3. Genomic fingerprinting of penicillinase-producing strains of Neisseria gonorrhoeae in Valencia, Spain.

    PubMed Central

    Dasi, M A; Nogueira, J M; Camarena, J J; Gil, C; García-Verdú, R; Barberá, J L; Barberá, J

    1992-01-01

    OBJECTIVE--To compare the value of different markers and their combinations with the restriction enzyme technique in the differentiations of penicillinase-producing N. gonorrhoeae (PPNG) strains. MATERIALS AND METHODS--17 PPNG strains isolated from symptomatic, untreated male patients with urethritis were characterised by antibiotic sensitivity testing, auxotyping, serotyping, plasmid profile, and restriction endonuclease fingerprinting (Hind III digestion). Cluster analysis with the method of unweighted pair-group average (UPGMA) linkage was used to calculate similarity or dissimilarity for PPNG strains. MAIN RESULTS--Either auxotyping or plasmid profile alone differentiated three groups of PPNG strains, whereas the combination auxotyping/serotyping identified 10. Although the combination auxotyping/serotyping/plasmid profile and the restriction enzyme technique showed a similar discrimination ability (differentiation of 11 PPNG strains), genomic fingerprinting gave highly specific restriction patterns on individual gonococcal isolates. CONCLUSIONS--The combination of different markers gave more epidemiological information than the use of only one. The sequence of discriminating ability for PPNG strains was: auxotyping/serotyping less than auxotyping/serotyping/plasmid profile less than restriction patterns of genomic DNA. Images PMID:1607193

  4. Penicillinase producing Neisseria gonorrhoeae from St Thomas' Hospital 1976-1990--the first fifteen years.

    PubMed

    Warren, C; Phillips, I

    1993-06-01

    To examine the penicillinase producing N. gonorrhoeae (PPNG) collected at St Thomas' Hospital from 1976-1990 and, by determination of antibiotic susceptibility pattern and application of three typing methods, examine the prevalence of different gonococcal types. Also to determine whether there is any relationship between antibiotic sensitivity, plasmid profile, auxotype and serovar. A total of 665 isolates of PPNG from patients attending the Department of Genitourinary Medicine at St Thomas' Hospital were characterised by antibiotic MIC, plasmid profile, auxotyping and serotyping. Penicillin MICs for 85% of all isolates were between 0.25 and 32 mg/l. The MIC of tetracycline for 60-80% of the isolates was < 1 mg/l. A small number of isolates had tetracycline MICs of 32 mg/l but MICs > 32 mg/l were not seen. Over 90% of the isolates were sensitive to the remaining three antibiotics tested, erythromycin, cefuroxime and spectinomycin. The 3.2 or 4.4 MDa plasmid with or without the 24.5 MDa conjugal plasmid was seen in all isolates until 1989/90 when a 2.9 MDa beta-lactamase encoding plasmid and the 25.2 MDa plasmid mediating tetracycline resistance were also recognised. Ninety-nine percent of all isolates belonged to one of four auxotypes, prototrophic, arginine, proline or proline/arginine requiring. An initial predominance of isolates with the 1A outer membrane protein was reversed in 1982 and 1B has remained predominant. Thirty two different serovars were identified among the 665 isolates, 14 belonged to serogroup 1A and 18 to 1B, and the eight most common accounted for 83.9% (554) of all isolates. Analysis of the results of combined typing methods showed there was an association between antibiotic resistance, plasmid profile and serogroup. The number of auxotypes and serovars detected in the collection, indicates the possibility that PPNG have been introduced from abroad or outside our local population. Temporal trends in the distribution of auxotype

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

    PubMed Central

    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

  6. Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics.

    PubMed

    Gulati, Sunita; Schoenhofen, Ian C; Whitfield, Dennis M; Cox, Andrew D; Li, Jianjun; St Michael, Frank; Vinogradov, Evgeny V; Stupak, Jacek; Zheng, Bo; Ohnishi, Makoto; Unemo, Magnus; Lewis, Lisa A; Taylor, Rachel E; Landig, Corinna S; Diaz, Sandra; Reed, George W; Varki, Ajit; Rice, Peter A; Ram, Sanjay

    2015-12-01

    Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5'-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic acid (Neu5Ac), a sialic acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation ("serum-resistance"). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, legionaminic acid (Leg5Ac7Ac) and pseudaminic acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP

  7. Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics

    PubMed Central

    Gulati, Sunita; Schoenhofen, Ian C.; Whitfield, Dennis M.; Cox, Andrew D.; Li, Jianjun; St. Michael, Frank; Vinogradov, Evgeny V.; Stupak, Jacek; Zheng, Bo; Ohnishi, Makoto; Unemo, Magnus; Lewis, Lisa A.; Taylor, Rachel E.; Landig, Corinna S.; Diaz, Sandra; Reed, George W.; Varki, Ajit; Rice, Peter A.; Ram, Sanjay

    2015-01-01

    Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5’-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic acid (Neu5Ac), a sialic acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation (“serum-resistance”). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, legionaminic acid (Leg5Ac7Ac) and pseudaminic acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from

  8. Expression of proinflammatory cytokines and receptors by human fallopian tubes in organ culture following challenge with Neisseria gonorrhoeae.

    PubMed

    Maisey, Kevin; Nardocci, Gino; Imarai, Monica; Cardenas, Hugo; Rios, Miguel; Croxatto, Horacio B; Heckels, John E; Christodoulides, Myron; Velasquez, Luis A

    2003-01-01

    Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an inflammatory condition, which is a major cause of infertility. Challenge of explants of human FT with gonococci induced mRNA expression and protein secretion for the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor alpha (TNF-alpha) but not for granulocyte-macrophage colony-stimulating factor. In contrast, FT expression of IL-6 and of the cytokine receptors IL-6R, TNF receptor I (TNF-RI), and TNF-RII was constitutive and was not increased by gonococcal challenge. These studies suggest that several proinflammatory cytokines are likely to contribute to the cell and tissue damage observed in gonococcal salpingitis.

  9. Expression of Proinflammatory Cytokines and Receptors by Human Fallopian Tubes in Organ Culture following Challenge with Neisseria gonorrhoeae

    PubMed Central

    Maisey, Kevin; Nardocci, Gino; Imarai, Monica; Cardenas, Hugo; Rios, Miguel; Croxatto, Horacio B.; Heckels, John E.; Christodoulides, Myron; Velasquez, Luis A.

    2003-01-01

    Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an inflammatory condition, which is a major cause of infertility. Challenge of explants of human FT with gonococci induced mRNA expression and protein secretion for the proinflammatory cytokines interleukin (IL)-1α, IL-1β, and tumor necrosis factor alpha (TNF-α) but not for granulocyte-macrophage colony-stimulating factor. In contrast, FT expression of IL-6 and of the cytokine receptors IL-6R, TNF receptor I (TNF-RI), and TNF-RII was constitutive and was not increased by gonococcal challenge. These studies suggest that several proinflammatory cytokines are likely to contribute to the cell and tissue damage observed in gonococcal salpingitis. PMID:12496205

  10. An insight into the drug resistance profile & mechanism of drug resistance in Neisseria gonorrhoeae

    PubMed Central

    Patel, Achchhe Lal; Chaudhry, Uma; Sachdev, Divya; Sachdeva, Poonam Nagpal; Bala, Manju; Saluja, Daman

    2011-01-01

    Among the aetiological agents of treatable sexually transmitted diseases (STDs), Neissseria gonorrhoeae is considered to be most important because of emerging antibiotic resistant strains that compromise the effectiveness of treatment of the disease - gonorrhoea. In most of the developing countries, treatment of gonorrhoea relies mainly on syndromic management rather than the aetiological based therapy. Gonococcal infections are usually treated with single-dose therapy with an agent found to cure > 95 per cent of cases. Unfortunately during the last few decades, N. gonorrhoeae has developed resistance not only to less expensive antimicrobials such as sulphonamides, penicillin and tetracyclines but also to fluoroquinolones. The resistance trend of N. gonorrhoeae towards these antimicrobials can be categorised into pre-quinolone, quinolone and post-quinolone era. Among the antimicrobials available so far, only the third-generation cephalosporins could be safely recommended as first-line therapy for gonorrhoea globally. However, resistance to oral third-generation cephalosporins has also started emerging in some countries. Therefore, it has become imperative to initiate sustained national and international efforts to reduce infection and misuse of antibiotics so as to prevent further emergence and spread of antimicrobial resistance. It is necessary not only to monitor drug resistance and optimise treatment regimens, but also to gain insight into how gonococcus develops drug resistance. Knowledge of mechanism of resistance would help us to devise methods to prevent the occurrence of drug resistance against existing and new drugs. Such studies could also help in finding out new drug targets in N. gonorrhoeae and also a possibility of identification of new drugs for treating gonorrhoea. PMID:22089602

  11. An insight into the drug resistance profile & mechanism of drug resistance in Neisseria gonorrhoeae.

    PubMed

    Patel, Achchhe Lal; Chaudhry, Uma; Sachdev, Divya; Sachdeva, Poonam Nagpal; Bala, Manju; Saluja, Daman

    2011-10-01

    Among the aetiological agents of treatable sexually transmitted diseases (STDs), Neissseria gonorrhoeae is considered to be most important because of emerging antibiotic resistant strains that compromise the effectiveness of treatment of the disease - gonorrhoea. In most of the developing countries, treatment of gonorrhoea relies mainly on syndromic management rather than the aetiological based therapy. Gonococcal infections are usually treated with single-dose therapy with an agent found to cure > 95 per cent of cases. Unfortunately during the last few decades, N. gonorrhoeae has developed resistance not only to less expensive antimicrobials such as sulphonamides, penicillin and tetracyclines but also to fluoroquinolones. The resistance trend of N. gonorrhoeae towards these antimicrobials can be categorised into pre-quinolone, quinolone and post-quinolone era. Among the antimicrobials available so far, only the third-generation cephalosporins could be safely recommended as first-line therapy for gonorrhoea globally. However, resistance to oral third-generation cephalosporins has also started emerging in some countries. Therefore, it has become imperative to initiate sustained national and international efforts to reduce infection and misuse of antibiotics so as to prevent further emergence and spread of antimicrobial resistance. It is necessary not only to monitor drug resistance and optimise treatment regimens, but also to gain insight into how gonococcus develops drug resistance. Knowledge of mechanism of resistance would help us to devise methods to prevent the occurrence of drug resistance against existing and new drugs. Such studies could also help in finding out new drug targets in N. gonorrhoeae and also a possibility of identification of new drugs for treating gonorrhoea.

  12. Modified profile of matrix metalloproteinase-2 and -9 production by human Fallopian tube epithelial cells following infection in vitro with Neisseria gonorrhoeae.

    PubMed

    Rodas, Paula I; Pérez, Doris; Jaffret, Claudia; González, Yaquelin; Carreño, Carolina; Tapia, Cecilia V; Osorio, Eduardo; Velasquez, Luis A; Christodoulides, Myron

    2016-12-08

    Epithelial shedding and scarring of Fallopian tube mucosa are the main consequences of sexually transmitted Neisseria gonorrhoeae infection and likely involves an imbalance of host extracellular matrix components (ECM) and their regulators such as matrix metalloproteinases (MMPs). In this brief report, primary human Fallopian tube epithelial cells were infected with N. gonorrhoeae and MMP patterns examined. Gonococcal infection induced a significant increase in secreted MMP-9 and an accumulation of cytoplasmic MMP-2 over time, but no significant MMP-3 or MMP-8 production was observed. Thus, MMP-9 in particular could play a role in tubal scarring in response to gonococcal infection.

  13. Infection of Human Fallopian Tube Epithelial Cells with Neisseria gonorrhoeae Protects Cells from Tumor Necrosis Factor Alpha-Induced Apoptosis

    PubMed Central

    Morales, Priscilla; Reyes, Paz; Vargas, Macarena; Rios, Miguel; Imarai, Mónica; Cardenas, Hugo; Croxatto, Horacio; Orihuela, Pedro; Vargas, Renato; Fuhrer, Juan; Heckels, John E.; Christodoulides, Myron; Velasquez, Luis

    2006-01-01

    Following infection with Neisseria gonorrhoeae, bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil+ Opa+ gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-α). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-α was a key contributor to apoptosis, since (i) culture supernatants from cells infected with gonococci (MOI = 1) induced apoptosis in naïve cultures, suggesting that a soluble factor was responsible; (ii) gonococcal infection-induced apoptosis was inhibited with anti-TNF-α antibodies; and (iii) the addition of exogenous TNF-α induced apoptosis, which was inhibited by the presence of increasing numbers of bacteria (MOI = 10 to 100). These data suggest that TNF-α-mediated apoptosis of FT epithelial cells is likely a primary host defense mechanism to prevent pathogen colonization. However, epithelial cell-associated gonococci have evolved a mechanism to protect the cells from undergoing TNF-α-mediated apoptosis, and this modulation of the host innate response may contribute to establishment of infection. Understanding the antiapoptotic mechanisms used by Neisseria gonorrhoeae will inform the pathogenesis of salpingitis and could suggest new intervention strategies for prevention and treatment of the disease. PMID:16714596

  14. [Incidence of Neisseria gonorrhoeae infections in Belgium: trends 2000-2006].

    PubMed

    Defraye, A; Crucitti, T; Ducoffre, G; Mak, R; Sasse, A

    2009-01-01

    In Belgium, three registration systems collect epidemiological information on N. gonorrhoeae infections. The descriptive analysis of the data presented in this article allows describing the epidemiology of N. gonorrhoeae infections in Belgium in terms of trends in time, describing the characteristics of the patients, and providing information on resistance to antibiotics. The results on the incidence of N. gonorrhoeae infections show an important increase since the year 2000, and this increase is even more pronounced between 2005 and 2006. The majority of the patients reside in big cities, mainly in the district of Antwerp and in the Brussels-Capital region. Among the N. gonorrhoeae specimens that were sent to the reference laboratory, the proportion of specimens resistant to ciprofloxacine increases each year; this proportion reaches 61.4% in 2006. The increase in the incidence of N. gonorrhoeae infections and in antimicrobial resistance is also observed in other European countries. The increase in incidence may be partly related to the important increase of resistance to ciprofloxacine. It is very important to continue the surveillance of antimicrobial resistance, to adapt treatment in function of the recent evolutions and to inform physicians at a regular basis. The results show that homo- and bisexual men are most at risk for N. gonorrhoeae infections. The prevention campaigns for sexually transmitted infections and screening policy have to be reinforced, particularly among homo- and bisexual men.

  15. Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness.

    PubMed

    Tomberg, Joshua; Fedarovich, Alena; Vincent, Leah R; Jerse, Ann E; Unemo, Magnus; Davies, Christopher; Nicholas, Robert A

    2017-02-28

    Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upward of 60 amino acid changes relative to wild-type PBP2, or by nonmosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates that harbored a mosaic penA allele with an A501P mutation were identified. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon 501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, and A501S) that increased resistance and preserved essential transpeptidase function were isolated. To understand their structural implications, these mutations were introduced into the nonmosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was determined and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.

  16. High in vitro activity of the novel spiropyrimidinetrione AZD0914, a DNA gyrase inhibitor, against multidrug-resistant Neisseria gonorrhoeae isolates suggests a new effective option for oral treatment of gonorrhea.

    PubMed

    Jacobsson, Susanne; Golparian, Daniel; Alm, Richard A; Huband, Michael; Mueller, John; Jensen, Jorgen Skov; Ohnishi, Makoto; Unemo, Magnus

    2014-09-01

    We evaluated the activity of the novel spiropyrimidinetrione AZD0914 (DNA gyrase inhibitor) against clinical gonococcal isolates and international reference strains (n=250), including strains with diverse multidrug resistance and extensive drug resistance. The AZD0914 MICs were substantially lower than those of most other currently or previously recommended antimicrobials. AZD0914 should be further evaluated, including in vitro selection, in vivo emergence and mechanisms of resistance, pharmacokinetics/pharmacodynamics in humans, optimal dosing, and performance, in appropriate randomized and controlled clinical trials.

  17. The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

    SciTech Connect

    Dong, Jinlan; George, Nicholas P.; Duckett, Katrina L.; DeBeer, Madeleine A.P.; Lopper, Matthew E.

    2010-05-25

    Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 {angstrom} resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeae and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.

  18. Post-transcriptional regulation of target genes by the sRNA FnrS in Neisseria gonorrhoeae.

    PubMed

    Tanwer, Pooja; Bauer, Susanne; Heinrichs, Elisabeth; Panda, Gurudutta; Saluja, Daman; Rudel, Thomas; Beier, Dagmar

    2017-07-01

    Small non-coding RNAs (sRNAs) are well-established post-transcriptional regulators of gene expression in bacteria that respond to a variety of environmental stimuli. They usually act by base-pairing with their target mRNAs, which is commonly facilitated by the RNA chaperone Hfq. In this study we initiated the analysis of the sRNA FnrS of Neisseria gonorrhoeae, which is induced under anaerobic conditions. We identified four putative FnrS target genes using bioinformatics approaches and validated these target genes using translational reporter gene fusions in both Escherichia coli and N. gonorrhoeae, thereby demonstrating their downregulation by direct base-pairing between the respective mRNA and FnrS. We demonstrate deregulation of target mRNAs upon deletion of fnrS and provide evidence that the isc gene cluster required for iron-sulfur cluster biosynthesis, which harbours iscS, which is a direct target of FnrS, is coordinately downregulated by the sRNA. By mutational analysis we show that, surprisingly, three distinct regions of FnrS are employed for interaction with different target genes.

  19. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae

    SciTech Connect

    Su, Chih-Chia; Long, Feng; McDermott, Gerry; Shafer, William M.; Yu, Edward W.

    2008-04-01

    The multidrug efflux transporter NorM from N. gonorrhoeae has been crystallized and X-ray diffraction data have been collected to a resolution of 6.5 Å. The crystallization and preliminary X-ray data analysis of the NorM multidrug efflux pump produced by Neisseria gonorrhoeae are reported. NorM is a cytoplasmic membrane protein that consists of 459 amino-acid residues. It is a member of the recently classified multidrug and toxic compound extrusion (MATE) family of transporters and recognizes a number of cationic toxic compounds such as ethidium bromide, acriflavin, 2-N-methylellipticinium and ciprofloxacin. Recombinant NorM protein was expressed in Escherichia coli and purified by metal-affinity and gel-filtration chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted anisotropically to 3.8 Å and diffraction data were complete to 6.5 Å resolution. The space group was determined to be C2, with unit-cell parameters a = 81.5, b = 164.4, c = 111.5 Å.

  20. Experimental Vaccine Induces Th1-driven Immune Responses and Resistance to Neisseria gonorrhoeae Infection in a Murine Model

    PubMed Central

    Liu, Yingru; Hammer, Laura A.; Liu, Wensheng; Hobbs, Marcia M.; Zielke, Ryszard A.; Sikora, Aleksandra E.; Jerse, Ann E.; Egilmez, Nejat K.; Russell, Michael W.

    2017-01-01

    Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMV) plus microencapsulated IL-12, and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10–13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6–9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal IgG and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4+ T cells secreted IFNγ, but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as EF-Tu and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a non-living gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate Th1-driven immune responses in the genital tract. PMID:28272393

  1. High In Vitro Activity of the Novel Spiropyrimidinetrione AZD0914, a DNA Gyrase Inhibitor, against Multidrug-Resistant Neisseria gonorrhoeae Isolates Suggests a New Effective Option for Oral Treatment of Gonorrhea

    PubMed Central

    Jacobsson, Susanne; Golparian, Daniel; Alm, Richard A.; Huband, Michael; Mueller, John; Jensen, Jorgen Skov; Ohnishi, Makoto

    2014-01-01

    We evaluated the activity of the novel spiropyrimidinetrione AZD0914 (DNA gyrase inhibitor) against clinical gonococcal isolates and international reference strains (n = 250), including strains with diverse multidrug resistance and extensive drug resistance. The AZD0914 MICs were substantially lower than those of most other currently or previously recommended antimicrobials. AZD0914 should be further evaluated, including in vitro selection, in vivo emergence and mechanisms of resistance, pharmacokinetics/pharmacodynamics in humans, optimal dosing, and performance, in appropriate randomized and controlled clinical trials. PMID:24982070

  2. Sampling technique is important for optimal isolation of pharyngeal gonorrhoea.

    PubMed

    Mitchell, M; Rane, V; Fairley, C K; Whiley, D M; Bradshaw, C S; Bissessor, M; Chen, M Y

    2013-11-01

    Culture is insensitive for the detection of pharyngeal gonorrhoea but isolation is pivotal to antimicrobial resistance surveillance. The aim of this study was to ascertain whether recommendations provided to clinicians (doctors and nurses) on pharyngeal swabbing technique could improve gonorrhoea detection rates and to determine which aspects of swabbing technique are important for optimal isolation. This study was undertaken at the Melbourne Sexual Health Centre, Australia. Detection rates among clinicians for pharyngeal gonorrhoea were compared before (June 2006-May 2009) and after (June 2009-June 2012) recommendations on swabbing technique were provided. Associations between detection rates and reported swabbing technique obtained via a clinician questionnaire were examined. The overall yield from testing before and after provision of the recommendations among 28 clinicians was 1.6% (134/8586) and 1.8% (264/15,046) respectively (p=0.17). Significantly higher detection rates were seen following the recommendations among clinicians who reported a change in their swabbing technique in response to the recommendations (2.1% vs. 1.5%; p=0.004), swabbing a larger surface area (2.0% vs. 1.5%; p=0.02), applying more swab pressure (2.5% vs. 1.5%; p<0.001) and a change in the anatomical sites they swabbed (2.2% vs. 1.5%; p=0.002). The predominant change in sites swabbed was an increase in swabbing of the oropharynx: from a median of 0% to 80% of the time. More thorough swabbing improves the isolation of pharyngeal gonorrhoea using culture. Clinicians should receive training to ensure swabbing is performed with sufficient pressure and that it covers an adequate area that includes the oropharynx.

  3. Phenotypic and Genotypic Analyses of Neisseria gonorrhoeae Isolates That Express Frequently Recovered PorB PIA Variable Region Types Suggest that Certain P1a Porin Sequences Confer a Selective Advantage for Urogenital Tract Infection▿

    PubMed Central

    Garvin, Lotisha E.; Bash, Margaret C.; Keys, Christine; Warner, Douglas M.; Ram, Sanjay; Shafer, William M.; Jerse, Ann E.

    2008-01-01

    Typing of the porB variable region (VR) is an epidemiological tool that classifies gonococcal strains based on sequence differences in regions of the porB gene that encode surface-exposed loops. The frequent isolation of certain porB VR types suggests that some porin sequences confer a selective advantage during infection and/or transmission. Alternatively, certain porin types may be markers of strains that are successful due to factors unrelated to porin. In support of the first hypothesis, here we show urogenital tract isolates representing the most common PIA VR types identified in an urban clinic in Baltimore, MD, over a 10-year period belonged to several different clonal types, as determined by pulsed-field gel electrophoresis (PFGE). Serum resistance, which was confirmed by factor H and C4b-binding protein binding studies, was more often associated with gonococcal the most common VR types. In contrast, three porin-independent phenotypes, namely, lactoferrin utilization, β-lactamase production, and multiple transferable resistance (Mtr), were segregated with the PFGE cluster and not with the VR type. Data combined with another PIA strain collection showed a strong correlation between serum resistance and the most common VR types. A comparison of VR typing hybridization patterns and nucleotide sequences of 12 porB1a genes suggests that certain porin loop 1, 3, 6, and/or 7 sequences may play a role in the serum resistance phenotype. We conclude that some PorB PIA sequences confer a survival or transmission advantage in the urogenital tract, perhaps via increased resistance to complement-mediated killing. The capacity of some porin types to evade a porin-specific adaptive immune response must also be considered. PMID:18541655

  4. Differences in nitric oxide steady states between arginine, hypoxanthine, uracil auxotrophs (AHU) and non-AHU strains of Neisseria gonorrhoeae during anaerobic respiration in the presence of nitrite.

    PubMed

    Barth, Kenneth; Clark, Virginia L

    2008-08-01

    Neisseria gonorrhoeae can grow by anaerobic respiration using nitrite as an alternative electron acceptor. Under these growth conditions, N. gonorrhoeae produces and degrades nitric oxide (NO), an important host defense molecule. Laboratory strain F62 has been shown to establish and maintain a NO steady-state level that is a function of the nitrite reductase/NO reductase ratio and is independent of cell number. The nitrite reductase activities (122-197 nmol NO2 reduced x min(-1) x OD600(-1)) and NO reductase activities (88-155 nmol NO reduced x min(-1) x OD600(-1)) in a variety of gonococcal clinical isolates were similar to the specific activities seen in F62 (241 nmol NO2 reduced x min(-1) x OD600(-1) and 88 nmol NO reduced x min(-1) x OD600(-1), respectively). In seven gonococcal strains, the NO steady-state levels established in the presence of nitrite were similar to that of F62 (801-2121 nmol x L-1 NO), while six of the strains, identified as arginine, hypoxanthine, and uracil auxotrophs (AHU), that cause asymptomatic infection in men had either two- to threefold (373-579 nmol x L-1 NO) or about 100-fold (13-24 nmol x L-1 NO) lower NO steady-state concentrations. All tested strains in the presence of a NO donor, 2,2'-(hydroxynitrosohydrazono)bis-ethanimine/NO, quickly lowered and maintained NO levels in the noninflammatory range of NO (<300 nmol x L-1). The generation of a NO steady-state concentration was directly affected by alterations in respiratory control in both F62 and an AHU strain, although differences in membrane function are suspected to be responsible for NO steady-state level differences in AHU strains.

  5. Contribution of Neisseria Gonorrhoeae Catalase in Defense Against Toxic Oxygen Radicals and Neutrophils, and Its Role During Experimental Genital Tract Infection of Mice

    DTIC Science & Technology

    2002-01-01

    the disturbed vaginal ecosystem that is characteristic of bacterial vaginosis (Eschenbach, 1993; Fredricsson et al., 1993). The consequence of...bacterial vaginosis is an increased risk in acquiring some genital tract infections and their 19 adverse effects given that the antagonism normally exerted by...bacterial vaginosis . On the other hand, Faro et al. (1993) could not establish the same relationship between bacterial vaginosis and Neisseria gonorrhoeae

  6. Evaluation of the new AmpliSens multiplex real-time PCR assay for simultaneous detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis.

    PubMed

    Rumyantseva, Tatiana; Golparian, Daniel; Nilsson, Christian S; Johansson, Emma; Falk, My; Fredlund, Hans; Van Dam, Alje; Guschin, Alexander; Unemo, Magnus

    2015-10-01

    In this study, we performed an evaluation of the new CE-marked multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay compared to APTIMA tests, i.e., APTIMA COMBO 2 assay, APTIMA Trichomonas vaginalis assay (FDA-approved), and two different APTIMA Mycoplasma genitalium assays (research use only; one of them only used for discrepancy analysis). Vaginal swabs (n = 209) and first-void urine (FVU) specimens from females (n = 498) and males (n = 554), consecutive attendees (n = 1261) at a dermatovenerological clinic in Sweden, were examined. The sensitivity of the AmpliSens PCR assay for detection of C. trachomatis (6.3% prevalence), M. genitalium (5.7% prevalence), N. gonorrhoeae (0.3% prevalence), and T. vaginalis (0.08% prevalence) was 97.5% (95% confidence interval (CI): 91.2-99.6%), 81.9% (95% CI: 70.7-89.7%), 100% (95% CI: 40.2-100%) and 100% (95% CI: 16.5-100%), respectively. The specificity of the AmpliSens PCR assay was 100% (95% CI: 99.6-100%) for all agents. The analytical sensitivity and specificity for N. gonorrhoeae detection was excellent, i.e., 55 international gonococcal strains detected and 135 isolates of 13 non-gonococcal Neisseria species were negative. In conclusion, the multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay demonstrated high sensitivity and excellent specificity for the detection of C. trachomatis, N. gonorrhoeae, and T. vaginalis, and excellent specificity but suboptimal sensitivity for M. genitalium detection.

  7. Genetic Studies of Sulfadiazine-resistant and Methionine-requiring Neisseria Isolated From Clinical Material

    PubMed Central

    Catlin, B. Wesley

    1967-01-01

    Deoxyribonucleate (DNA) preparations were extracted from Neisseria meningitidis (four isolates from spinal fluid and blood) and N. gonorrhoeae strains, all of which were resistant to sulfadiazine upon primary isolation. These DNA preparations, together with others from in vitro mutants of N. meningitidis and N. perflava, were examined in transformation tests by using as recipient a drug-susceptible strain of N. meningitidis (Ne 15 Sul-s Met+) which was able to grow in a methionine-free defined medium. The sulfadiazine resistance typical of each donor was introduced into the uniform constitution of this recipient. Production of p-aminobenzoic acid was not significantly altered thereby. Transformants elicited by DNA from the N. meningitidis clinical isolates were resistant to at least 200 μg of sulfadiazine/ml, and did not show a requirement for methionine (Sul-r Met+). DNA from six strains of N. gonorrhoeae, which were isolated during the period of therapeutic use of sulfonamides, conveyed lower degrees of resistance and, invariably, a concurrent methionine requirement (Sul-r/Met−). The requirement of these transformants, and that of in vitro mutants selected on sulfadiazine-agar, was satisfied by methionine, but not by vitamin B12, homocysteine, cystathionine, homoserine, or cysteine. Sul-r Met+ and Sul-r/Met− loci could coexist in the same genome, but were segregated during transformation. On the other hand, the dual Sul-r/Met− properties were not separated by recombination, but were eliminated together. DNA from various Sul-r/Met− clones tested against recipients having nonidentical Sul-r/Met− mutant sites yielded Sul-s Met+ transformants. The met locus involved is genetically complex, and will be a valuable tool for studies of genetic fine structure of members of Neisseria, and of genetic homology between species. Images PMID:4962305

  8. Prevalence and persistence of Neisseria cinerea and other Neisseria spp. in adults.

    PubMed

    Knapp, J S; Hook, E W

    1988-05-01

    Neisseria cinerea is a commensal Neisseria sp. which was first described in 1906 but was subsequently misclassified as a subtype of Branhamella catarrhalis. N. cinerea resembles Neisseria gonorrhoeae in both cultural and biochemical characteristics and, thus, may also have been misidentified as N. gonorrhoeae. Of 202 patients whose oropharynges were colonized by Neisseria spp., N. cinerea was isolated in 57 (28.2%) patients, including 25 (30.1%) of 83 women, 22 (23.9%) of 92 heterosexual men, and 10 (37.0%) of 27 homosexual men in Seattle, Wash., in 1983 to 1984. N. cinerea was isolated from the urethra of only one (1.1%) patient. The oropharynges of many individuals were colonized persistently by strains of N. cinerea and other Neisseria spp.

  9. Neisseria gonorrhoeae prevalence, incidence and associated risk factors among female sex workers in a high HIV-prevalence area of China

    PubMed Central

    Luo, Li; Li, Xin; Zhang, Lu-lu

    2015-01-01

    Background Neisseria gonorrhoeae (N. gonorrhoeae) is one of the most prevalent non-ulcerative sexually transmitted infections (STIs) in China, however, the data about N. gonorrhoeae infections are limited in this population. The objective of this study is to determine N. gonorrheae incidence and associated risk factors among female sex workers (FSWs) in China. Methods This serial cross-sectional study was conducted semi-annually among FSWs in a City of Yunnan Province, which constituted an open cohort study. Participants were interviewed and tested for N. gonorrhoeae every 6 months. Results During 3 years of follow-up, 64 incident cases of N. gonorrhoeae infection were diagnosed, yielding an overall incidence of 5.9 per 100 person years (PY) (95% confidence interval (CI), 4.53–7.41). Working in higher risk commercial sex venue (adjusted hazard ratio (AHR)=2.7, 95% CI, 1.56–4.55), inconsistently use condom with clients in previous week (AHR=1.9, 95% CI, 1.07–3.35) and being infected with C. trachomatis (AHR=1.9, 95% CI, 1.06–3.26) were independent risk factors for incident N. gonorrhoeae infection. Conclusions A relatively high prevalence and incidence of N. gonorrhoeae among a prospective cohort of FSWs underscore the urgency for traditional HIV/STIs prevention methods among FSWs, such as condom promotion, screening and treatment of STIs, considering the high prevalence of STIs. Significant attention should focus on FSWs working in higher risk commercial sex venues as they are at higher risk for N. gonorrhoeae and transmission than those in lower risk commercial sex venues. PMID:26255890

  10. CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway.

    PubMed

    Hauck, C R; Meyer, T F; Lang, F; Gulbins, E

    1998-01-15

    The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52-expressing gonococci on human neutrophils. Here we show that Opa52-expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src-like protein tyrosine kinases, Rac1 and PAK to Jun-N-terminal kinase. The induced signal is distinct from Fcgamma-receptor-mediated signalling and is specific for Opa52, since piliated Opa- gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src-like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin-independent, Opa52/CD66-mediated phagocytosis of pathogenic Neisseria.

  11. Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae

    PubMed Central

    Cahoon, Laty A.; Seifert, H. Steven

    2013-01-01

    The strict human pathogen Neisseria gonorrhoeae can utilize homologous recombination to generate antigenic variability in targets of immune surveillance. To evade the host immune response, N. gonorrhoeae promotes high frequency gene conversion events between many silent pilin copies and the expressed pilin locus (pilE), resulting in the production of variant pilin proteins. Previously, we identified a guanine quartet (G4) structure localized near pilE that is required for the homologous recombination reactions leading to pilin antigenic variation (Av). In this work, we demonstrate that inactivating the promoter of a small non-coding RNA (sRNA) that initiates within the G4 forming sequence blocks pilin Av. The sRNA promoter is conserved in all sequenced gonococcal strains, and mutations in the predicted transcript downstream of the G4 forming sequence do not alter pilin Av. A mutation that produces a stronger promoter or substitution of the pilE G4-associated sRNA promoter with a phage promoter (when the phage polymerase was expressed) produced wild-type levels of pilin Av. Altering the direction and orientation of the pilE G4-associated sRNA disrupted pilin Av. In addition, expression of the sRNA at a distal site on the gonococcal chromosome in the context of a promoter mutant did not support pilin Av. We conclude that the DNA containing the G-rich sequence can only form the G4 structure during transcription of this sRNA, thus providing a unique molecular step for the initiation of programmed recombination events. PMID:23349628

  12. Neisseria gonorrhoeae Evades Calprotectin-Mediated Nutritional Immunity and Survives Neutrophil Extracellular Traps by Production of TdfH.

    PubMed

    Jean, Sophonie; Juneau, Richard A; Criss, Alison K; Cornelissen, Cynthia N

    2016-10-01

    Neisseria gonorrhoeae successfully overcomes host strategies to limit essential nutrients, termed nutritional immunity, by production of TonB-dependent transporters (TdTs)-outer membrane proteins that facilitate nutrient transport in an energy-dependent manner. Four gonococcal TdTs facilitate utilization of iron or iron chelates from host-derived proteins, including transferrin (TbpA), lactoferrin (LbpA), and hemoglobin (HpuB), in addition to xenosiderophores from other bacteria (FetA). The roles of the remaining four uncharacterized TdTs (TdfF, TdfG, TdfH, and TdfJ) remain elusive. Regulatory data demonstrating that production of gonococcal TdfH and TdfJ are unresponsive to or upregulated under iron-replete conditions led us to evaluate the role of these TdTs in the acquisition of nutrients other than iron. In this study, we found that production of gonococcal TdfH is both Zn and Zur repressed. We also found that TdfH confers resistance to calprotectin, an immune effector protein highly produced in neutrophils that has antimicrobial activity due to its ability to sequester Zn and Mn. We found that TdfH directly binds calprotectin, which enables gonococcal Zn accumulation in a TdfH-dependent manner and enhances bacterial survival after exposure to neutrophil extracellular traps (NETs). These studies highlight Zn sequestration by calprotectin as a key functional arm of NET-mediated killing of gonococci. We demonstrate for the first time that N. gonorrhoeae exploits this host strategy in a novel defense mechanism, in which TdfH production hijacks and directly utilizes the host protein calprotectin as a zinc source and thereby evades nutritional immunity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Multiple Origins and Replication Proteins Influence Biological Properties of β-Lactamase-Producing Plasmids from Neisseria gonorrhoeae

    PubMed Central

    Pagotto, Franco; Dillon, Jo-Anne R.

    2001-01-01

    The β-lactamase-producing Asia-type plasmid pJD4 of Neisseria gonorrhoeae is a 7.4-kb, broad-host-range plasmid. It is part of a family of plasmids which are structurally related yet vary in size, found in both N. gonorrhoeae and Haemophilus ducreyi. Branch-point analysis by electron microscopy indicates that pJD4 carries three clustered but distinguishable origins of replication, which we named ori1, ori2, and ori3. Although pJD4 belongs to incompatibility (Inc) group W, it also carries a silent IncFII determinant which is expressed when ori2 and ori3 are absent. The Africa-type plasmid pJD5, a naturally occurring deletion derivative of pJD4, carries only ori1, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I (Pol I) for replication. Plasmids constructed from pJD4 which lack ori1 but carry ori2 and ori3 do not require Pol I and are incompatible with IncW plasmids, suggesting that the ori2 or ori3 region contains the IncW determinant. We have cloned a replication initiation protein (RepB) that is necessary for ori2 and ori3 to function. This Rep protein is distinct from RepA, which is necessary for ori1. Thus, pJD4 is unique because it is the smallest plasmid characterized containing three origins of replication and two unique Rep proteins. PMID:11544207

  14. Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins.

    PubMed

    Phillips, Nancy J; Steichen, Christopher T; Schilling, Birgit; Post, Deborah M B; Niles, Richard K; Bair, Thomas B; Falsetta, Megan L; Apicella, Michael A; Gibson, Bradford W

    2012-01-01

    Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with (13)C(6)-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related

  15. Neisseria gonorrhoeae Evades Calprotectin-Mediated Nutritional Immunity and Survives Neutrophil Extracellular Traps by Production of TdfH

    PubMed Central

    Jean, Sophonie; Juneau, Richard A.; Criss, Alison K.

    2016-01-01

    Neisseria gonorrhoeae successfully overcomes host strategies to limit essential nutrients, termed nutritional immunity, by production of TonB-dependent transporters (TdTs)—outer membrane proteins that facilitate nutrient transport in an energy-dependent manner. Four gonococcal TdTs facilitate utilization of iron or iron chelates from host-derived proteins, including transferrin (TbpA), lactoferrin (LbpA), and hemoglobin (HpuB), in addition to xenosiderophores from other bacteria (FetA). The roles of the remaining four uncharacterized TdTs (TdfF, TdfG, TdfH, and TdfJ) remain elusive. Regulatory data demonstrating that production of gonococcal TdfH and TdfJ are unresponsive to or upregulated under iron-replete conditions led us to evaluate the role of these TdTs in the acquisition of nutrients other than iron. In this study, we found that production of gonococcal TdfH is both Zn and Zur repressed. We also found that TdfH confers resistance to calprotectin, an immune effector protein highly produced in neutrophils that has antimicrobial activity due to its ability to sequester Zn and Mn. We found that TdfH directly binds calprotectin, which enables gonococcal Zn accumulation in a TdfH-dependent manner and enhances bacterial survival after exposure to neutrophil extracellular traps (NETs). These studies highlight Zn sequestration by calprotectin as a key functional arm of NET-mediated killing of gonococci. We demonstrate for the first time that N. gonorrhoeae exploits this host strategy in a novel defense mechanism, in which TdfH production hijacks and directly utilizes the host protein calprotectin as a zinc source and thereby evades nutritional immunity. PMID:27481245

  16. Phosphoethanolamine decoration of Neisseria gonorrhoeae lipid A plays a dual immunostimulatory and protective role during experimental genital tract infection.

    PubMed

    Packiam, Mathanraj; Yedery, Roshan D; Begum, Afrin A; Carlson, Russell W; Ganguly, Jhuma; Sempowski, Gregory D; Ventevogel, Melissa S; Shafer, William M; Jerse, Ann E

    2014-06-01

    The induction of an intense inflammatory response by Neisseria gonorrhoeae and the persistence of this pathogen in the presence of innate effectors is a fascinating aspect of gonorrhea. Phosphoethanolamine (PEA) decoration of lipid A increases gonococcal resistance to complement-mediated bacteriolysis and cationic antimicrobial peptides (CAMPs), and recently we reported that wild-type N. gonorrhoeae strain FA1090 has a survival advantage relative to a PEA transferase A (lptA) mutant in the human urethral-challenge and murine lower genital tract infection models. Here we tested the immunostimulatory role of this lipid A modification. Purified lipooligosaccharide (LOS) containing lipid A devoid of the PEA modification and an lptA mutant of strain FA19 induced significantly lower levels of NF-κB in human embryonic kidney Toll-like receptor 4 (TLR4) cells and murine embryonic fibroblasts than wild-type LOS of the parent strain. Moreover, vaginal proinflammatory cytokines and chemokines were not elevated in female mice infected with the isogenic lptA mutant, in contrast to mice infected with the wild-type and complemented lptA mutant bacteria. We also demonstrated that lptA mutant bacteria were more susceptible to human and murine cathelicidins due to increased binding by these peptides and that the differential induction of NF-κB by wild-type and unmodified lipid A was more pronounced in the presence of CAMPs. This work demonstrates that PEA decoration of lipid A plays both protective and immunostimulatory roles and that host-derived CAMPs may further reduce the capacity of PEA-deficient lipid A to interact with TLR4 during infection.

  17. Rapid Detection of the Mosaic Structure of the Neisseria gonorrhoeae penA Gene, Which Is Associated with Decreased Susceptibilities to Oral Cephalosporins▿

    PubMed Central

    Ochiai, Susumu; Ishiko, Hiroaki; Yasuda, Mitsuru; Deguchi, Takashi

    2008-01-01

    In Neisseria gonorrhoeae, the mosaic structure of the penA gene (encoding penicillin-binding protein 2 [PBP 2]), which is composed of fragments of the penA genes from Neisseria cinerea and Neisseria perflava, has been significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to develop a rapid assay for the detection of mosaic PBP 2 of N. gonorrhoeae by real-time PCR. This assay successfully detected the mosaic penA gene of N. gonorrhoeae, and its sensitivity was ≥101 copies/reaction. Six hundred twenty-one clinical strains were examined by this assay for the presence of mosaic PBP 2, which was detected in 85 (39.4%) of 216 strains from 2002, 69 (40.6%) of 170 strains from 2003, 71 (44.4%) of 160 strains from 2004, and 31 (41.3%) of 75 strains from 2005. The MICs of cephalosporins for strains with the mosaic PBP 2 detected by the assay were statistically higher than those for strains without the mosaic PBP 2. One hundred sixty-six (64.8%) of 256 strains with the mosaic PBP 2 exhibited cefixime MICs of ≥0.5 μg/ml. The emergence and spread of strains with mosaic PBP 2 could be a threat to the cefixime treatment of gonorrhea. This real-time PCR assay for the detection of mosaic PBP 2 of N. gonorrhoeae is thus useful in the prediction of decreased susceptibilities to oral cephalosporins. PMID:18367575

  18. Rapid detection of the mosaic structure of the Neisseria gonorrhoeae penA Gene, which is associated with decreased susceptibilities to oral cephalosporins.

    PubMed

    Ochiai, Susumu; Ishiko, Hiroaki; Yasuda, Mitsuru; Deguchi, Takashi

    2008-05-01

    In Neisseria gonorrhoeae, the mosaic structure of the penA gene (encoding penicillin-binding protein 2 [PBP 2]), which is composed of fragments of the penA genes from Neisseria cinerea and Neisseria perflava, has been significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to develop a rapid assay for the detection of mosaic PBP 2 of N. gonorrhoeae by real-time PCR. This assay successfully detected the mosaic penA gene of N. gonorrhoeae, and its sensitivity was >or=10(1) copies/reaction. Six hundred twenty-one clinical strains were examined by this assay for the presence of mosaic PBP 2, which was detected in 85 (39.4%) of 216 strains from 2002, 69 (40.6%) of 170 strains from 2003, 71 (44.4%) of 160 strains from 2004, and 31 (41.3%) of 75 strains from 2005. The MICs of cephalosporins for strains with the mosaic PBP 2 detected by the assay were statistically higher than those for strains without the mosaic PBP 2. One hundred sixty-six (64.8%) of 256 strains with the mosaic PBP 2 exhibited cefixime MICs of >or=0.5 microg/ml. The emergence and spread of strains with mosaic PBP 2 could be a threat to the cefixime treatment of gonorrhea. This real-time PCR assay for the detection of mosaic PBP 2 of N. gonorrhoeae is thus useful in the prediction of decreased susceptibilities to oral cephalosporins.

  19. The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae

    PubMed Central

    Kandler, Justin L.; Holley, Concerta L.; Reimche, Jennifer L.; Dhulipala, Vijaya; Balthazar, Jacqueline T.; Muszyński, Artur; Carlson, Russell W.

    2016-01-01

    During infection, the sexually transmitted pathogen Neisseria gonorrhoeae (the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogen Neisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility of misR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression of lptA and mtrCDE was not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity. PMID:27216061

  20. Type III Methyltransferase M.NgoAX from Neisseria gonorrhoeae FA1090 Regulates Biofilm Formation and Interactions with Human Cells

    PubMed Central

    Kwiatek, Agnieszka; Mrozek, Agnieszka; Bacal, Pawel; Piekarowicz, Andrzej; Adamczyk-Popławska, Monika

    2015-01-01

    Neisseria gonorrhoeae is the etiological factor of the sexually transmitted gonorrhea disease that may lead, under specific conditions, to systemic infections. The gonococcal genome encodes many restriction modification (RM) systems, which main biological role is to defend the pathogen from potentially harmful foreign DNA. However, RM systems seem also to be involved in several other functions. In this study, we examined the effect of inactivation the N. gonorrhoeae FA1090 ngoAXmod gene encoding M.NgoAX methyltransferase on the global gene expression, biofilm formation, interactions with human epithelial host cells and overall bacterial growth. Expression microarrays showed at least a twofold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wild-type (wt) strain under standard grow conditions. Genes with changed expression levels encoded mostly proteins involved in cell metabolism, DNA replication and repair or regulating cellular processes and signaling (such as cell wall/envelop biogenesis). As determined by the assay with crystal violet, the NgoAX knock-out strain formed a slightly larger biofilm biomass per cell than the wt strain. Live biofilm observations showed that the biofilm formed by the gonococcal ngoAXmod gene mutant is more relaxed, dispersed and thicker than the one formed by the wt strain. This more relaxed feature of the biofilm, in respect to adhesion and bacterial interactions, can be involved in pathogenesis. Moreover, the overall adhesion of mutant bacterial cells to human cells was lower than adhesion of the wt gonococci [adhesion index = 0.672 (±0.2) and 2.15 (±1.53), respectively]; yet, a higher number of mutant than wt bacteria were found inside the Hec-1-B epithelial cells [invasion index = 3.38 (±0.93) × 105 for mutant and 4.67 (±3.09) × 104 for the wt strain]. These results indicate that NgoAX knock-out cells have lower ability to attach to human cells, but more easily penetrate inside the host

  1. Prevalence of bla TEM-220 gene in Penicillinase-producing Neisseria gonorrhoeae strains carrying Toronto/Rio plasmid in Argentina, 2002 - 2011.

    PubMed

    Gianecini, Ricardo; Oviedo, Claudia; Guantay, Cristina; Piccoli, Laura; Stafforini, Graciela; Galarza, Patricia

    2015-12-16

    Penicillinase-producing Neisseria gonorroheae (PPNG) was first isolated in 1976. PPNG strains carrying bla TEM-1 and bla TEM-135 gene have been described in different countries. Recently, a novel bla TEM-220 allele was detected in PPNG isolates carrying Toronto/Rio plasmid. The prevalence and characteristics of TEM-220 strains worldwide are unknown, and therefore, it needs to be studied. The purpose of this study was to detect bla TEM-220 gene in PPNG strains possessing Toronto/Rio plasmid over a period of ten years in Argentina, and to evaluate the proportion of isolates producing non-TEM-220 containing the T539C substitution in the bla TEM allele. One hundred and fifty one PPNG isolates carrying Toronto/Rio plasmid were studied between 2002 and 2011. A mismatch amplification mutation assay (MAMA) PCR was used to identify the T539C substitution in the bla TEM allele and a MAMA-PCR protocol was developed to detect the G547A substitution in the bla TEM-220. The reference agar dilution method of the Clinical and Laboratory Standard Institute (CLSI) was used for susceptibility testing to five β-lactams antibiotics, ciprofloxacin, tetracycline and azithromycin. In all TEM-220-producing isolates, the whole bla TEM gene was sequenced and the isolates were typed using N. gonorroheae multiantigen sequence typing (NG-MAST). MAMA PCR successfully identified the G547A substitution in the bla TEM-220 allele. The proportion of isolates that possessed the bla TEM-220 allele was 2.6 %, and 93.2 % MAMA TEM-220 PCR-negative isolates showed the T539C substitution in the bla TEM gene. No differences in the susceptibility to five beta-lactam antibiotics tested were observed in PPNG isolates TEM-220-producing and PPNG isolates carrying the T539C substitution in the bla TEM gene. All TEM-220 isolates were indistinguishable by NG-MAST. This is the first study which shows the prevalence of bla TEM-220 in N. gonorrhoeae isolates carrying Toronto/Rio plasmid in Argentina. Although the bla

  2. Neisseria-Avoiding the Jump to Conclusions

    ERIC Educational Resources Information Center

    Spivey, Maria I.; Paschall, Robert T.; Ferrett, Rhonda; Alexander, Randell

    2011-01-01

    "Neisseria gonorrhoeae" infection in a prepubertal child is virtually diagnostic of sexual abuse, provided perinatal infection has been excluded. Therefore, it is imperative that "Neisseria gonorrhoeae" be correctly identified. We present two cases of false positive "Neisseria gonorrhoeae" meningitis encountered at two different children's…

  3. Neisseria-Avoiding the Jump to Conclusions

    ERIC Educational Resources Information Center

    Spivey, Maria I.; Paschall, Robert T.; Ferrett, Rhonda; Alexander, Randell

    2011-01-01

    "Neisseria gonorrhoeae" infection in a prepubertal child is virtually diagnostic of sexual abuse, provided perinatal infection has been excluded. Therefore, it is imperative that "Neisseria gonorrhoeae" be correctly identified. We present two cases of false positive "Neisseria gonorrhoeae" meningitis encountered at two different children's…

  4. Neisseria gonorrhoeae infects the human endocervix by activating non-muscle myosin II-mediated epithelial exfoliation

    PubMed Central

    Yu, Qian; Lin, Brian; Qiu, Jessica; Stein, Daniel C.

    2017-01-01

    Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation. PMID:28406994

  5. Cervical Infection with Herpes simplex Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae among Symptomatic Women, Dubai, UAE: A Molecular Approach.

    PubMed

    Mehrabani, Davood; Behzadi, Mohammad Amin; Azizi, Saeed; Payombarnia, Hamid; Vahdani, Ali; Namayandeh, Mandana; Ziyaeyan, Mazyar

    2014-01-01

    Tragically, genital tract infections are still a major public health problem in many regions. This study was undertaken to determine the prevalence of cervical infection with Herpes simplex virus (HSV), Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (NG) among married women referring to Iranian Hospital, Dubai, UAE. In a retrospective cross-sectional survey, 201 female patients aged 16-80 years who referred to the Obstetrics and Gynecology Department of Iranian Hospital, Dubai, UAE, in 2010 were enrolled. The patients were categorized into three age groups: 15-30 (group I), 31-40 (group II), and ≥41 years old (group III). A cervical swab sample was collected from each woman and the prevalence of cervical infection with HSV, CT, and NG was determined by PCR method. HSV, CT, and NG were detected in 6.5%, 10.4%, and 5.5% of swab samples, respectively. Regarding age, a significant difference was noticed for prevalence of NG and HSV between groups I and III. Because of public health importance of sexual transmitted diseases (STDs), their long-lasting impact on quality of life, and their economic burden, preventing measures and education of women seem necessary.

  6. Loop structures in the 5' untranslated region and antisense RNA mediate pilE gene expression in Neisseria gonorrhoeae.

    PubMed

    Masters, Thao L; Wachter, Jenny; Hill, Stuart A

    2016-11-01

    Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem-loop structures with favourable free energies within the 5' untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem-loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5' untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.

  7. Cervical Infection with Herpes simplex Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae among Symptomatic Women, Dubai, UAE: A Molecular Approach

    PubMed Central

    Behzadi, Mohammad Amin; Azizi, Saeed; Payombarnia, Hamid; Vahdani, Ali; Namayandeh, Mandana; Ziyaeyan, Mazyar

    2014-01-01

    Tragically, genital tract infections are still a major public health problem in many regions. This study was undertaken to determine the prevalence of cervical infection with Herpes simplex virus (HSV), Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (NG) among married women referring to Iranian Hospital, Dubai, UAE. In a retrospective cross-sectional survey, 201 female patients aged 16–80 years who referred to the Obstetrics and Gynecology Department of Iranian Hospital, Dubai, UAE, in 2010 were enrolled. The patients were categorized into three age groups: 15–30 (group I), 31–40 (group II), and ≥41 years old (group III). A cervical swab sample was collected from each woman and the prevalence of cervical infection with HSV, CT, and NG was determined by PCR method. HSV, CT, and NG were detected in 6.5%, 10.4%, and 5.5% of swab samples, respectively. Regarding age, a significant difference was noticed for prevalence of NG and HSV between groups I and III. Because of public health importance of sexual transmitted diseases (STDs), their long-lasting impact on quality of life, and their economic burden, preventing measures and education of women seem necessary. PMID:24982675

  8. Constitutively Opa-Expressing and Opa-Deficient Neisseria gonorrhoeae Strains Differentially Stimulate and Survive Exposure to Human Neutrophils

    PubMed Central

    Ball, Louise M.

    2013-01-01

    The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in frame, termed Opaless. Opaless Gc remained uniformly Opa negative (Opa−), whereas cultures of predominantly Opa− parental Gc and an intermediate lacking the “translucent” subset of opa genes (ΔopaBEGK) stochastically gave rise to Opa-positive (Opa+) bacterial colonies. Loss of Opa expression did not affect Gc growth. Opaless Gc survived exposure to primary human PMNs and suppressed the PMN oxidative burst akin to parental, Opa− bacteria. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. When a non-phase-variable, in-frame allele of FA1090 opaD was reintroduced into Opaless Gc, the bacteria induced the PMN oxidative burst, and OpaD+ Gc survived less well after exposure to PMNs compared to Opa− bacteria. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology. PMID:23625842

  9. Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    PubMed Central

    Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

    2014-01-01

    The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies. PMID:25460012

  10. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae

    SciTech Connect

    Su, C.C.; Long, F.; McDermott, G.; Shafer, W.M.; Yu, E.W.

    2008-06-03

    The crystallization and preliminary X-ray data analysis of the NorM multidrug efflux pump produced by Neisseria gonorrhoeae are reported. NorM is a cytoplasmic membrane protein that consists of 459 amino-acid residues. It is a member of the recently classified multidrug and toxic compound extrusion (MATE) family of transporters and recognizes a number of cationic toxic compounds such as ethidium bromide, acriflavin, 2-N-methylellipticinium and ciprofloxacin. Recombinant NorM protein was expressed in Escherichia coli and purified by metal-affinity and gel-filtration chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted anisotropically to 3.8 {angstrom} and diffraction data were complete to 6.5 {angstrom} resolution. The space group was determined to be C2, with unit-cell parameters a = 81.5, b = 164.4, c = 111.5 {angstrom}.

  11. MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking Antibodies

    PubMed Central

    Semchenko, Evgeny A.; Day, Christopher J.

    2016-01-01

    ABSTRACT Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection (STI) gonorrhea, is a growing public health threat for which a vaccine is urgently needed. We characterized the functional role of the gonococcal MetQ protein, which is the methionine binding component of an ABC transporter system, and assessed its potential as a candidate antigen for inclusion in a gonococcal vaccine. MetQ has been found to be highly conserved in all strains investigated to date, it is localized on the bacterial surface, and it binds l-methionine with a high affinity. MetQ is also involved in gonococcal adherence to cervical epithelial cells. Mutants lacking MetQ have impaired survival in human monocytes, macrophages, and serum. Furthermore, antibodies raised against MetQ are bactericidal and are able to block gonococcal adherence to epithelial cells. These data suggest that MetQ elicits both bactericidal and functional blocking antibodies and is a valid candidate antigen for additional investigation and possible inclusion in a vaccine for prevention of gonorrhea. PMID:27895130

  12. Recent advances in the development and use of molecular tests to predict antimicrobial resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Low, Nicola; Golparian, Daniel; Unemo, Magnus

    2017-09-01

    The number of genetic tests, mostly real-time PCRs, to detect antimicrobial resistance (AMR) determinants and predict AMR in Neisseria gonorrhoeae is increasing. Several of these assays are promising, but there are important shortcomings and few assays have been adequately validated and quality assured. Areas covered: Recent advances, focusing on publications since 2012, in the development and use of molecular tests to predict gonococcal AMR for surveillance and for clinical use, advantages and disadvantages of these tests and of molecular AMR prediction compared with phenotypic AMR testing, and future perspectives for effective use of molecular AMR tests for different purposes. Expert commentary: Several challenges for direct testing of clinical, especially extra-genital, specimens remain. The choice of molecular assay needs to consider the assay target, quality controls, sample types, limitations intrinsic to molecular technologies, and specific to the chosen methodology, and the intended use of the test. Improved molecular- and particularly genome-sequencing-based methods will supplement AMR testing for surveillance purposes, and translate into point-of-care tests that will lead to personalized treatments, while sparing the last available empiric treatment option (ceftriaxone). However, genetic AMR prediction will never completely replace phenotypic AMR testing, which detects also AMR due to unknown AMR determinants.

  13. Opa+ Neisseria gonorrhoeae Exhibits Reduced Survival in Human Neutrophils Via Src Family Kinase-Mediated Bacterial Trafficking Into Mature Phagolysosomes

    PubMed Central

    Johnson, M. Brittany; Ball, Louise M.; Daily, Kylene P.; Martin, Jennifer N.; Columbus, Linda; Criss, Alison K.

    2015-01-01

    Summary During gonorrheal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial content. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signaling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signaling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils’ full antimicrobial arsenal. PMID:25346239

  14. Porphyrin-based compounds exert antibacterial action against the sexually transmitted pathogens Neisseria gonorrhoeae and Haemophilus ducreyi.

    PubMed

    Bozja, J; Yi, K; Shafer, W M; Stojiljkovic, I

    2004-12-01

    A series of porphyrin based compounds without (nMP) or with (MP) metals were found to have potent bactericidal action in vitro against the sexually transmitted pathogens Neisseria gonorrhoeae and Haemophilus ducreyi. nMP and MP did not show bactericidal activity against five species of lactobacilli. An MP containing gallium had the capacity to block a gonococcal infection in a murine vaginal model, indicating that its development as a topical microbicide to block sexually transmitted bacterial infections is warranted. In contrast to other bacterial species, loss of the gonococcal haemoglobin uptake system encoded by hpuB or energy supplied through the TonB-ExbB-ExbD system did not significantly affect levels of MP-susceptibility in gonococci. In contrast, mutations in gonococci that inactivate the mtrCDE-encoded efflux pump were found to enhance gonococcal susceptibility to nMPs and MPs while over-production of this efflux pump decreased levels of gonococcal susceptibility to these compounds.

  15. Performance of the Cepheid CT/NG Xpert Rapid PCR Test for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

    PubMed

    Gaydos, Charlotte A; Van Der Pol, Barbara; Jett-Goheen, Mary; Barnes, Mathilda; Quinn, Nicole; Clark, Carey; Daniel, Grace E; Dixon, Paula B; Hook, Edward W

    2013-06-01

    Tests for Chlamydia trachomatis and Neisseria gonorrhoeae, which can provide results rapidly to guide therapeutic decision-making, offer patient care advantages over laboratory-based tests that require several days to provide results. We compared results from the Cepheid GeneXpert CT/NG (Xpert) assay to results from two currently approved nucleic acid amplification assays in 1,722 female and 1,387 male volunteers. Results for chlamydia in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 97.4%, 98.7%, and 97.6%, respectively, and for urine samples from males, a sensitivity of 97.5%, with all specificity estimates being ≥ 99.4%. Results for gonorrhea in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 100.0%, 100.0%, and 95.6%, respectively, and for urine samples from males, a sensitivity of 98.0%, with all estimates of specificity being ≥ 99.8%. These results indicate that this short-turnaround-time test can be used to accurately test patients and to possibly do so at the site of care, thus potentially improving chlamydia and gonorrhea control efforts.

  16. Positive Testing for Neisseria gonorrhoeae and Chlamydia trachomatis and the Risk of Pelvic Inflammatory Disease in IUD Users

    PubMed Central

    Birgisson, Natalia E.; Zhao, Qiuhong; Secura, Gina M.; Madden, Tessa

    2015-01-01

    Abstract Background: Unintended pregnancies are a major public health problem in the United States, and intrauterine devices (IUDs) are among the most effective reversible birth control methods available. Historically, there have been concerns about IUD use and infection among young and/or high-risk women that may increase the risk of pelvic inflammatory disease (PID) and subsequent infertility. Methods: The Contraceptive CHOICE Project (CHOICE) was a prospective cohort study of over 9,000 women 14–45 years of age residing in the St. Louis area who were interested in initiating a new form of reversible contraception. At enrollment, participants were counseled regarding long-acting contraceptive methods with the goal of increasing awareness of all reversible methods available. Participants were also tested for Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) during enrollment and were provided with contraception at no cost for 2–3 years. Results: We estimate the frequency of self-reported PID in new IUD users compared with women using other contraceptive methods. Among both new IUD users who tested positive for GC and/or CT and those who tested negative, the PID rate was 1% or below. Conclusions: Our results demonstrate that IUD use is safe for all women, including women at high risk for sexually transmitted infections. PMID:25836384

  17. Prevalence of Chlamydia trachomatis, Trichomonas vaginalis and Neisseria gonorrhoeae Based on Data Collected by a Network of Clinical Microbiology Laboratories, in Italy.

    PubMed

    Salfa, Maria Cristina; Suligoi, Barbara

    Bacterial and protozoal sexually transmitted infections (STIs), such as Chlamydia trachomatis, Trichomonas vaginalis and Neisseria gonorrhoeae, may cause acute symptoms, chronic infections and severe long-term complications. The complications of these infections in women include pelvic inflammatory disease, chronic pelvic pain, tubal infertility, ectopic pregnancy, and infertility. Moreover, infection during pregnancy is associated with premature rupture of the membranes, low birth weight and miscarriage.In Italy, Chlamydia trachomatis and Trichomonas vaginalis infections are not subject to mandatory reporting; while gonorrhoea is subject to mandatory reporting.To extend surveillance to STIs that are widespread yet often asymptomatic and to improve the knowledge on the epidemiology of these infections in Italy, in 2009 the "Centro Operativo AIDS of the Istituto Superiore di SanitÁ", in collaboration with the Association of Italian Clinical Microbiologists (AMCLI, Associazione Microbiologi Clinici Italiani), launched the sentinel STIs surveillance system based on a network of 13 clinical microbiology laboratories.The main objective of the surveillance was to assess the prevalence and risk factors associated with Chlamydia trachomatis, Trichomonas vaginalis and Neisseria gonorrhoea infections among individuals attending microbiology laboratories in Italy.

  18. Rapid change in the ciprofloxacin resistance pattern among Neisseria gonorrhoeae strains in Nuuk, Greenland: time to reconsider preventive and treatment strategies.

    PubMed

    Skjerbæk Rolskov, Anne; Bjorn-Mortensen, Karen; Mulvad, Gert; Poulsen, Peter; Skov Jensen, Jørgen; Lynge Pedersen, Michael

    2015-01-01

    Objectives Sexually transmitted infections (STIs), including infections with Neisseria gonorrhoeae (GC), are highly incident in Greenland. Since January 2011, GC testing has been performed on urine with nucleic acid amplification tests (NAATs) by strand displacement amplification (Becton Dickinson ProbeTec). Monitoring of GC antibiotic susceptibility by culture was introduced in Nuuk in 2012. Until 2014, no cases of ciprofloxacin-resistant GC strains were reported. In this paper, we report the finding of ciprofloxacin-resistant GC and describe the most recent incidence of GC infections in Greenland. Methods The number of urine NAATs and culture-positive swabs from January to October 2014 were obtained from the Central Laboratory at Queens Ingrid's Hospital in Nuuk and stratified on gender, place and period of testing. Incidence rates were estimated as number of urine NAAT * (12/10) per 100,000 inhabitants. Men in Nuuk with a positive NAAT for GC were encouraged to provide a urethral swab for culture and susceptibility testing. Results From January to October 2014, a total of 5,436 urine GC NAATs were performed on patients from Nuuk and 9,031 from the rest of Greenland. Of these, 422 (8%) and 820 (9%) were positive, respectively. From January to August, 6 (15%) cultures from Nuuk were ciprofloxacin resistant while in September and October, 26 (59%) were ciprofloxacin resistant (p<0.01). In total, 35 (40%) of 88 culture-positive isolates showed ciprofloxacin resistance. GC incidence in Nuuk was 3,017 per 100,000 inhabitants per year, compared to 2,491 per 100,000 inhabitants per year in the rest of Greenland. Conclusion Within a short period, a rapid and dramatic change in ciprofloxacin susceptibility among GC strains isolated in Nuuk was documented and recommendation for first line treatments has changed. Continued monitoring and rethinking of primary and secondary preventive initiatives is highly recommended in this high GC incidence setting.

  19. Rapid change in the ciprofloxacin resistance pattern among Neisseria gonorrhoeae strains in Nuuk, Greenland: time to reconsider preventive and treatment strategies.

    PubMed

    Rolskov, Anne Skjerbæk; Bjorn-Mortensen, Karen; Mulvad, Gert; Poulsen, Peter; Jensen, Jørgen Skov; Pedersen, Michael Lynge

    2015-01-01

    Sexually transmitted infections (STIs), including infections with Neisseria gonorrhoeae (GC), are highly incident in Greenland. Since January 2011, GC testing has been performed on urine with nucleic acid amplification tests (NAATs) by strand displacement amplification (Becton Dickinson ProbeTec). Monitoring of GC antibiotic susceptibility by culture was introduced in Nuuk in 2012. Until 2014, no cases of ciprofloxacin-resistant GC strains were reported. In this paper, we report the finding of ciprofloxacin-resistant GC and describe the most recent incidence of GC infections in Greenland. The number of urine NAATs and culture-positive swabs from January to October 2014 were obtained from the Central Laboratory at Queens Ingrid's Hospital in Nuuk and stratified on gender, place and period of testing. Incidence rates were estimated as number of urine NAAT * (12/10) per 100,000 inhabitants. Men in Nuuk with a positive NAAT for GC were encouraged to provide a urethral swab for culture and susceptibility testing. From January to October 2014, a total of 5,436 urine GC NAATs were performed on patients from Nuuk and 9,031 from the rest of Greenland. Of these, 422 (8%) and 820 (9%) were positive, respectively. From January to August, 6 (15%) cultures from Nuuk were ciprofloxacin resistant while in September and October, 26 (59%) were ciprofloxacin resistant (p<0.01). In total, 35 (40%) of 88 culture-positive isolates showed ciprofloxacin resistance. GC incidence in Nuuk was 3,017 per 100,000 inhabitants per year, compared to 2,491 per 100,000 inhabitants per year in the rest of Greenland. Within a short period, a rapid and dramatic change in ciprofloxacin susceptibility among GC strains isolated in Nuuk was documented and recommendation for first line treatments has changed. Continued monitoring and rethinking of primary and secondary preventive initiatives is highly recommended in this high GC incidence setting.

  20. Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae

    PubMed Central

    Foerster, Sunniva; Golparian, Daniel; Jacobsson, Susanne; Hathaway, Lucy J.; Low, Nicola; Shafer, William M.; Althaus, Christian L.; Unemo, Magnus

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants) were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914). ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections), should be considered for phase III clinical trials and future gonorrhea treatment. PMID:26696986

  1. Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae.

    PubMed

    Foerster, Sunniva; Golparian, Daniel; Jacobsson, Susanne; Hathaway, Lucy J; Low, Nicola; Shafer, William M; Althaus, Christian L; Unemo, Magnus

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants) were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914). ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections), should be considered for phase III clinical trials and future gonorrhea treatment.

  2. Slide Agglutination Method for the Serological Identification of Neisseria gonorrhoeae with Anti-Gonococcal Antibodies Adsorbed to Protein A-Containing Staphylococci

    PubMed Central

    Danielsson, Dan; Kronvall, Göran

    1974-01-01

    A rapid slide agglutination test has been developed for the identification of Neisseria gonorrhoeae that are primarily detected as oxidase-positive colonies in gonococcal cultures. The technique is based on the specific nonimmune reactivity between the Fc portion of immunoglobulin (Ig)G and staphylococcal protein A. IgG molecules adsorbed to stabilized staphylococci will thereby become oriented with their antigen-reactive sites that are directed outwards. Protein A-containing staphylococci with unabsorbed anti-gonococcal antibodies gave positive co-agglutination reactions with gonococci but also with meningococci, some Moraxella, Haemophilus, and Pseudomonas strains. These crossreactions were eliminated by absorption of the anti-gonococcal antiserum with meningococcal and Moraxella organisms prior to the coating of reagent staphylococci. In the routine culture diagnosis of N. gonorrhoeae the use of specific gonococcal reagent staphylococci gave concordant results with fermentation procedures and immunofluorescent techniques. Images PMID:4207280

  3. [Survey of the sensitivity of the strains Neisseria gonorrhoeae to antibioticas in the CSSR during 1957-1973 (authors transl)].

    PubMed

    Hejzlar, M; Výmola, F; Trojanová, M

    1975-01-01

    Although the preponderant majority of gonococcal strains is belonging to the most susceptible micro-organisms in respect of the action of antibiotics, certain variations of sensitivity which might be of importance not only for the genetics of microbes, but also for biochemistry and, above all, for therapy may occur also among them, perhaps as a consequence of along-time pressure by antibiotics. In a preceding study (J. Hyg. Epidem. Microb. Immunol. (Prague) 12,296, 1968), the authors analyzed the situation in Czechoslovakia from 1957 to 1967 by means of 911 strains of N. gonorrhoeae isolated from male patients suffering from gonorrhoea. It was also pointed out that there was a certain tendency towards reduced efficacy of penicillin G and a presence of streptomycin-resistant strains. In later years, the studies were extended to include also strains isolated from female patients and sensitivity to some more modern antibiotics was tested. In the present report, a survey is given of the state of antibiotics resistance among gonococci in Czechoslovakia in a chronological order of 17 years which is intended to serve as a basic information for those who are dealing with this subject.

  4. Comparison of GMT presto assay and Roche cobas® 4800 CT/NG assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in dry swabs.

    PubMed

    de Waaij, Dewi J; Dubbink, Jan Henk; Peters, Remco P H; Ouburg, Sander; Morré, Servaas A

    2015-11-01

    Urogenital Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the most prevalent bacterial STIs worldwide. Molecular tests are the standard for the detection of CT and NG, as these are difficult to culture. The recently introduced CE-IVD marked GMT Presto assay promises to be a valuable addition in CT and NG diagnostics. The advantage of the Presto assay is that it works on many PCR systems and the DNA can be isolated by any system.We compared the Presto assay to the widely used Roche cobas® 4800 CT/NG test for the detection of CT and NG in 612 vaginal and rectal dry collected swabs. Discrepant samples were tested by the TIB MOLBIOL Lightmix Kit 480 HT CT/NG assay. The alloyed gold standard was defined as two concurring Presto and cobas® 4800 results, or, with discrepant Presto and cobas® results, two concurring results of either test together with the Lightmix Kit 480 HT CT/NG assay. For the Presto assay,we observed 77 CT positive (13%) and 22 NG positive (3,6%) vaginal samples, and 41 CT positive (6,7%) and 11 NG positive (1,8%) rectal samples. For the cobas® 4800 assay,we observed 77 CT positive (13%) and 21NG positive (3,4%) vaginal samples, and 39 CT positive (6,4%) and 11 NG positive (1,8%) rectal samples. Ten CT samples were discrepant between Presto and cobas® 4800 CT/NG assays, while two NG samples were discrepant. CT sensitivity in both assays was 100% compared to the alloyed gold standard. The sensitivity was 100% for both vaginal and rectal dry swabs, underlining the suitability of these sample types for detection of CT and NG. The Presto assay is therefore valuable for molecular detection of CT and NG in dry vaginal and rectal swabs.

  5. Neisseria gonorrhoeae Bacterial DNA Load in the Pharynges and Saliva of Men Who Have Sex with Men.

    PubMed

    Chow, Eric P F; Tabrizi, Sepehr N; Phillips, Samuel; Lee, David; Bradshaw, Catriona S; Chen, Marcus Y; Fairley, Christopher K

    2016-10-01

    Neisseria gonorrhoeae can be cultured in the saliva of individuals with pharyngeal gonorrhea. The aim of this study was to quantify the gonococcal bacterial DNA loads in the pharynges and saliva among men who have sex with men (MSM) with untreated pharyngeal gonorrhea. Untreated MSM who tested positive for pharyngeal gonorrhea by culture and returned for antibiotic treatment within 14 days at the Melbourne Sexual Health Centre between October 2014 and March 2015 were eligible for this study. The gonococcal bacterial DNA load was measured using real-time quantitative PCR. The median gonococcal bacterial DNA loads in the pharynges and saliva were calculated and compared to culture positivity using the Mann-Whitney U test. A total of 33 men were included in this study. The median gonococcal bacterial DNA load did not differ between the pharynges in men who were culture positive (2.5 × 10(5) copies/swab) and culture negative (2.9 × 10(4) copies/swab) (P = 0.166) and the saliva (culture positive, 2.2 × 10(5) copies/ml; culture negative, 2.7 × 10(5) copies/ml) (P = 0.499). The bacterial DNA load in the pharynges (P = 0.695) and saliva (P = 0.969) did not differ between who men returned for treatment within 7 days and those who returned 8 to 14 days later. Substantial gonococcal bacterial DNA loads were detected in both saliva and pharynges among MSM with pharyngeal gonorrhea. These findings suggest that gonorrhea can be transmitted via sexual practices involving exposure to saliva, such as oroanal practices (rimming) and saliva use as a lubricant for anal sex.

  6. Neisseria gonorrhoeae Bacterial DNA Load in the Pharynges and Saliva of Men Who Have Sex with Men

    PubMed Central

    Tabrizi, Sepehr N.; Phillips, Samuel; Lee, David; Bradshaw, Catriona S.; Chen, Marcus Y.; Fairley, Christopher K.

    2016-01-01

    Neisseria gonorrhoeae can be cultured in the saliva of individuals with pharyngeal gonorrhea. The aim of this study was to quantify the gonococcal bacterial DNA loads in the pharynges and saliva among men who have sex with men (MSM) with untreated pharyngeal gonorrhea. Untreated MSM who tested positive for pharyngeal gonorrhea by culture and returned for antibiotic treatment within 14 days at the Melbourne Sexual Health Centre between October 2014 and March 2015 were eligible for this study. The gonococcal bacterial DNA load was measured using real-time quantitative PCR. The median gonococcal bacterial DNA loads in the pharynges and saliva were calculated and compared to culture positivity using the Mann-Whitney U test. A total of 33 men were included in this study. The median gonococcal bacterial DNA load did not differ between the pharynges in men who were culture positive (2.5 × 105 copies/swab) and culture negative (2.9 × 104 copies/swab) (P = 0.166) and the saliva (culture positive, 2.2 × 105 copies/ml; culture negative, 2.7 × 105 copies/ml) (P = 0.499). The bacterial DNA load in the pharynges (P = 0.695) and saliva (P = 0.969) did not differ between who men returned for treatment within 7 days and those who returned 8 to 14 days later. Substantial gonococcal bacterial DNA loads were detected in both saliva and pharynges among MSM with pharyngeal gonorrhea. These findings suggest that gonorrhea can be transmitted via sexual practices involving exposure to saliva, such as oroanal practices (rimming) and saliva use as a lubricant for anal sex. PMID:27413195

  7. Opt-out screening for Chlamydia trachomatis and Neisseria gonorrhoeae in female detainees at Cook County jail in Chicago, IL.

    PubMed

    Cole, Joanna; Hotton, Anna; Zawitz, Chad; Kessler, Harold

    2014-03-01

    In April of 2011, the Cook County Jail initiated opt-out screening of all women for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) at the time of intake. In this retrospective review, we assess the impact of opt-out protocol on rates of testing, diagnoses, and successful treatment. We collected the results of all CT and GC tests ordered during intake from April 2011 through December 2012 and reviewed the medical chart of every patient with a positive result for documentation of treatment. Univariable and multivariable analyses were performed to examine the factors associated with receipt of treatment. Opt-out screening increased the number of diagnoses by more than 4-fold (from 9.3 to 40.8 cases/mo). Among 17,065 women eligible for screening, 3729 (22%) women opted out of screening, and screening was completed in 9265 (54.2%). There were 235 (2.5%) gonococcal infections and 702 (7.6%) chlamydial infections. Of 866 women with a positive test result, 602 (69.5%) received treatment while in jail. In multivariable analysis, older age (adjusted odds ratio [aOR], 1.73; 95% confidence interval [CI], 1.14-2.63), pregnancy (aOR, 2.51; 95% CI, 1.22-5.18), and longer length of stay in jail (aOR, 18.1; 95% CI, 11.7-28.1) were associated with greater likelihood of treatment. Women entering the Cook County Jail have high prevalence of GC/CT infections. Opt-out screening increased the number of GC and CT diagnoses made, and a high proportion of women were treated while incarcerated. Significant challenges remain in ensuring that screening is completed for all women who do not opt out and in providing treatment to women with short duration of incarceration.

  8. Structure of the Vibrio cholerae Type IVb Pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus.

    PubMed

    Li, Juliana; Egelman, Edward H; Craig, Lisa

    2012-04-20

    Type IV pili are multifunctional filaments displayed on many bacterial pathogens. Members of the Type IVa pilus subclass are found on a diverse group of human pathogens, whereas Type IVb pili are found almost exclusively on enteric bacteria. The Type IVa and IVb subclasses are distinguished by differences in the pilin subunits, including the fold of the globular domain. To understand the implications of the distinct pilin folds, we compared the stabilities of pilin subunits and pilus filaments for the Type IVa GC pilus from Neisseria gonorrhoeae and the Type IVb toxin-coregulated pilus (TCP) from Vibrio cholerae. We show that while recombinant TCP pilin is more stable than GC pilin, the GC pili are more resistant to proteolysis, heat and chemical denaturation than TCP, remaining intact in 8 M urea. To understand these differences, we determined the TCP structure by electron microscopy and three-dimensional image reconstruction. TCP have an architecture similar to that of GC pili, with subunits arranged in a right-handed 1-start helix and related by an 8.4-Å axial rise and a 96.8° azimuthal rotation. However, the TCP subunits are not as tightly packed as GC pilins, and the distinct Type IVb pilin fold exposes a segment of the α-helical core of TCP. Hydrophobic interactions dominate for both pilus subtypes, but base stacking by aromatic residues conserved among the Type IVa pilins may contribute to GC pilus stability. The extraordinary stability of GC pili may represent an adaptation of the Type IVa pili to harsh environments and the need to retract against external forces. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Adverse Birth Outcomes and Maternal Neisseria gonorrhoeae Infection: A Population-Based Cohort Study in Washington State.

    PubMed

    Heumann, Christine L; Quilter, Laura A S; Eastment, McKenna C; Heffron, Renee; Hawes, Stephen E

    2017-05-01

    Neisseria gonorrhoeae (gonorrhea) remains an important cause of reproductive and obstetric complications. There has been limited population-based research to evaluate the association between maternal gonorrhea and adverse birth outcomes. A population-based retrospective cohort study was conducted of women with singleton pregnancies in Washington State from 2003 to 2014 using linked birth certificate and birth hospitalization discharge data. The exposed cohort consisted of women with gonorrhea diagnosed during pregnancy. The unexposed group, defined as pregnant women without gonorrhea, was selected by frequency-matching by birth year in a 4:1 ratio. Logistic regression was used to determine crude and adjusted odds ratios (OR) for the association of maternal gonorrhea and adverse birth outcomes. Women with gonorrhea during pregnancy (N = 819) were more likely to be younger, black, single, less educated, multiparous, and smokers compared with women without gonorrhea (N = 3276). Maternal gonorrhea was significantly associated with a 40% increased odds (adjusted OR, 1.4; 95% confidence interval [CI], 1.0-1.8) of low birth weight infants compared with women without gonorrhea when adjusted for marital and smoking status. Maternal gonorrhea was associated with a 60% increased odds (OR, 1.6; 95% CI, 1.3-2.0) of small for gestational age infants compared with women without gonorrhea. This analysis showed that pregnant women with gonorrhea were more likely to have low birth weight infants, consistent with prior literature, and provided new evidence that maternal gonorrhea is associated with small for gestational age infants. These findings support increased public health efforts to prevent, identify, and treat gonorrhea infection during pregnancy.

  10. Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae

    PubMed Central

    Cash, Devin R.; Noinaj, Nicholas; Buchanan, Susan K.

    2015-01-01

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. However, the transferrin (Tf) receptor system, composed of TbpA and TbpB, is an ideal target for novel therapeutics and vaccine development. Using a three-dimensional structure of gonococcal TbpA, we investigated two hypotheses, i.e., that loop-derived antibodies can interrupt ligand-receptor interactions in the native bacterium and that the loop 3 helix is a critical functional domain. Preliminary loop-derived antibodies, as well as optimized second-generation antibodies, demonstrated similar modest ligand-blocking effects on the gonococcal surface but different effects in Escherichia coli. Mutagenesis of loop 3 helix residues was employed, generating 11 mutants. We separately analyzed the mutants' abilities to (i) bind Tf and (ii) internalize Tf-bound iron in the absence of the coreceptor TbpB. Single residue mutations resulted in up to 60% reductions in ligand binding and up to 85% reductions in iron utilization. All strains were capable of growing on Tf as the sole iron source. Interestingly, in the presence of TbpB, only a 30% reduction in Tf-iron utilization was observed, indicating that the coreceptor can compensate for TbpA impairment. Complete deletion of the loop 3 helix of TbpA eliminated the abilities to bind Tf, internalize iron, and grow with Tf as the sole iron source. Our studies demonstrate that while the loop 3 helix is a key functional domain, its function does not exclusively rely on any single residue. PMID:26351283

  11. [Prevalence of infections by Chlamydia trachomatis and Neisseria gonorrhoeae among high school students in the Sabana Central area of Cundinamarca, Colombia].

    PubMed

    Paredes, María Cecilia; Gómez, Yenny Milena; Torres, Ana Marcela; Fernández, Marcela; Tovar, María Belén

    2015-01-01

    Infections by Chlamydia trachomatis and Neisseria gonorrhoeae are the two most common bacterial sexually transmitted infections in the world. These infections affect principally less than 25-year-old individuals with sexual, reproductive and maternal and child health consequences. This study sought to estimate the prevalence of C. trachomatis and N. gonorrhoeae among high school students in the 11 municipalities of the Sabana Central area of Cundinamarca, Colombia. A cross-sectional study was conducted among 14 to 19-year old adolescents attending schools in the study area. After signing their consent, participants completed a questionnaire and provided a urine sample for the detection of C. trachomatis and N. gonorrhoeae by real-time polymerase chain reaction. In the sample of 972 sexually active adolescents, the prevalence of C. trachomatis and N. gonorrhoeae was 2.24% and 0.10%, respectively. No co-infections were detected. Overall, 21.4% of women who tested positive for C. trachomatis were asymptomatic. Significant relationships were found between C. trachomatis infection and female gender, the consumption of alcohol or other substances during the previous sexual relationship, having one or more partners and the presence of sexually transmitted infections. The prevalence of C. trachomatis detected in this study was lower than the prevalence reported for similar groups; besides, contrary to the general trends, fewer asymptomatic cases were identified in this study. To prioritize preventive strategies in high-risk populations, an epidemiological surveillance system for C. trachomatis and N. gonorrhoeae should be implemented in Colombia, with screening performed using non-invasive molecular techniques.

  12. [Usefulness of real-time PCR in detecting Chlamydia trachomatis and Neisseria gonorrhoeae in endocervical swabs and first-voided urine specimens].

    PubMed

    Hamasuna, Ryoichi; Kawai, Shuichi; Ando, Yukiko; Ito, Kenji; Kurashima, Motoko; Nishimura, Hirohumi; Yamaguchi, Takamasa; Yoshimura, Makoto; Kobayashi, Tomoko; Muratani, Tetsuro; Matsumoto, Tetsuro

    2013-09-01

    We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detectig N. gonorrhoeae was 100% in all 3 specimen types. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.

  13. [Usefulness of real-time PCR in detecting Chlamydia trachomatis and Neisseria gonorrhoeae in endocervical swabs and first-voided urine specimens].

    PubMed

    Hamasuna, Ryoichi; Kawai, Shuichi; Ando, Yukiko; Ito, Kenji; Kurashima, Motoko; Nishimura, Hirohumi; Yamaguchi, Takamasa; Yoshimura, Makoto; Kobayashi, Tomoko; Muratani, Tetsuro; Matsumoto, Tetsuro

    2011-01-01

    We evaluated performance of Abbott RealTime CT/NG assay (real-time PCR, Abbott Japan) for detect Chlamydia trachomatis and Neisseria gonorrhoeae by real-time PCR in 88 female patients with cervicitis symptoms seen at gynecological clinics and 100 male patients with urethritis symptoms seen at urological or dermatology clinics in Kitakyushu, Japan. Endocervical swab and first-voided urine (FVU) specimens were then collected from women and FVU specimens from men. Detection rates of C. trachomatis and N. gonorrhoeae by real-time PCR in the 3 types of specimens were compared to those by ProbeTec ET assay (ProbeTec, BD Diagnostic System). The overall positive concordance between real-time PCR and ProbTec were 97.1% (66/68) for C. trachomatis and 100% (33/33) for N. gonorrhoeae, C. trachomatis detection yielded 3 discordant results in endocervical specimens and 1 discordant result in male FVU by real-time PCR and ProbTec. Three of 4 reexamined using Aptime Combo 2 Assay (Fuji Rebio Inc.) were positive for C. trachomatis. Endocervical swab and FVU specimen results for C. trachomatis were discordant in 3 cases in real-time PCR and 4 in ProbeTec. Subjects with 2 or more positive endocervical awab results in female or male FVU specimens were assumed to be "true positive" for C. trachomatis. The sensitivities of real-time PCR for detecting C. trachomatis was 94.4% in endocervical swabs, 77.8% in female FVU and 97.4% in the male FVU. The sensitivities for real-time PCR for detecting N. gonorrhoeae was 100% in all 3 specimentypes. Abbott RealTime CT/NG assay was useful for detecting C. trachomatis using endocervical swabs or male FVU specimens and for detecting N. gonorrhoeae using endocervical swabs and all FVU specimens.

  14. Community-wide outbreak of Neisseria gonorrhoeae conjunctivitis in Konso district, North Omo administrative region.

    PubMed

    Mikru, F S; Molla, T; Ersumo, M; Henriksen, T H; Klungseyr, P; Hudson, P J; Kindan, T T

    1991-01-01

    We describe a large outbreak of severe pustular conjunctivitis due to Neiserria gonorrhoeae. Over 9,000 cases occurred during 8 months in 1987-1988 in one district in North Omo, Ethiopia. Both sexes and all age groups were affected, particularly children under 5 years of age; only a small minority were neonates. Despite a highly successful cure rate for individual cases, the outbreak continued for a number of months. Several epidemiologic approaches were used to monitor the outbreak and identify the mechanism of transmission. The epidemic curve suggested person-to-person transmission. Routine surveillance data showed that there was no concurrent genital gonorrhea outbreak and genital transmission could not explain a community-wide outbreak. In the setting of intense crowding and relative lack of water, peak transmission of illness coincided with two periods following the rains, suggesting that flies were important in transmission. A case-control study identified lack of face-washing as a household risk factor. Eighty seven of one-hundred and forty six (59%) control houses with children were judged to contain children with clean faces, while only 102 of 216 (47%) case households contained children with clean faces (p less than .05). Our recommendations include measures to improve personal hygiene.

  15. Production of 14C-labeled gas in BACTEC Neisseria Differentiation kits by Neisseria cinerea.

    PubMed

    Boyce, J M; Mitchell, E B; Knapp, J S; Buttke, T M

    1985-09-01

    Six strains of Neisseria cinerea were tested in BACTEC Neisseria Differentiation kits (Johnston Laboratories, Inc., Towson, Md.), and all yielded positive glucose growth indices and negative maltose and fructose growth indices. These results were similar to those achieved with Neisseria gonorrhoeae. However, most of the N. cinerea isolates tested yielded 3-h glucose growth indices that were lower than those obtained with gonococci. 14C-labeled gas was produced significantly faster (P less than 0.02) by N. gonorrhoeae than by N. cinerea. Additional studies suggested that the 14C-labeled gas produced by N. cinerea was carbon dioxide. N. cinerea strains were similar to Branhamella catarrhalis strains because both species failed to produce detectable acid from glucose, maltose, sucrose, fructose, and lactose in cysteine-tryptic agar media. However, in contrast to N. cinerea strains, B. catarrhalis strains did not metabolize glucose in BACTEC Neisseria Differentiation kits.

  16. Production of UC-labeled gas in BACTEC Neisseria Differentiation kits by Neisseria cinerea

    SciTech Connect

    Boyce, J.M.; Mitchell, E.B. Jr.; Knapp, J.S.; Buttke, T.M.

    1985-09-01

    Six strains of Neisseria cinerea were tested in BACTEC Neisseria Differentiation kits (Johnston Laboratories, Inc., Towson, Md.), and all yielded positive glucose growth indices and negative maltose and fructose growth indices. These results were similar to those achieved with Neisseria gonorrhoeae. However, most of the N. cinerea isolates tested yielded 3-h glucose growth indices that were lower than those obtained with gonococci. UC-labeled gas was produced significantly faster by N. gonorrhoeae than by N. cinerea. Additional studies suggested that the UC-labeled gas produced by N. cinerea was carbon dioxide. N. cinerea strains were similar to Branhamella catarrhalis strains because both species failed to produce detectable acid from glucose, maltose, sucrose, fructose, and lactose in cysteine-tryptic agar media. However, in contrast to N. cinerea strains, B. catarrhalis strains did not metabolize glucose in BACTEC Neisseria Differentiation kits.

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

    PubMed

    Rodas, Paula I; Álamos-Musre, A Said; Álvarez, Francisca P; Escobar, Alejandro; Tapia, Cecilia V; Osorio, Eduardo; Otero, Carolina; Calderón, Iván L; Fuentes, Juan A; Gil, Fernando; Paredes-Sabja, Daniel; Christodoulides, Myron

    2016-09-01

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

  18. Phosphoethanolamine substitution of lipid A and resistance of Neisseria gonorrhoeae to cationic antimicrobial peptides and complement-mediated killing by normal human serum.

    PubMed

    Lewis, Lisa A; Choudhury, Biswa; Balthazar, Jacqueline T; Martin, Larry E; Ram, Sanjay; Rice, Peter A; Stephens, David S; Carlson, Russell; Shafer, William M

    2009-03-01

    The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide beta-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.

  19. Evaluation of pooled ocular and vaginal swabs by the Cepheid GeneXpert CT/NG assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae compared to the GenProbe Aptima Combo 2 Assay

    PubMed Central

    Dize, L.; West, S.K.; Mkocha, H.; Quinn, T.C.; Gaydos, C.A.

    2015-01-01

    Ocular swabs from Tanzania were tested for Chlamydia trachomatis (CT), and self-collected vaginal swabs collected through a home collection program, iwantthekit.org, were tested for Neisseria gonorrhoeae and CT to evaluate Cepheid GeneXpert for the use of pooling multiple specimens before testing. GeneXpert shows to be a promising test for pooling. PMID:25497459

  20. Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

    PubMed Central

    Golparian, Daniel; Boräng, Stina; Sundqvist, Martin

    2015-01-01

    The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection of Neisseria gonorrhoeae. PMID:26468501

  1. Multicenter Evaluation of the BDProbeTec ET System for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine Specimens, Female Endocervical Swabs, and Male Urethral Swabs

    PubMed Central

    Van Der Pol, Barbara; Ferrero, Dennis V.; Buck-Barrington, Linda; Hook, Edward; Lenderman, Connie; Quinn, Thomas; Gaydos, Charlotte A.; Lovchik, Judith; Schachter, Julius; Moncada, Jeanne; Hall, Geraldine; Tuohy, Marion J.; Jones, Robert B.

    2001-01-01

    The performance of the Becton Dickinson BDProbe Tec ET System Chlamydia trachomatis and Neisseria gonorrhoeae Amplified DNA Assays (BD Biosciences, Sparks, Md.) was evaluated in a multicenter study. Specimens were collected from 2,109 men and women, with or without symptoms, attending sexually transmitted disease, family planning, and obstetrics and gynecology clinics. Both swab and urine samples were collected, and the results obtained from 4,131 specimens were compared to those from culture and the LCx nucleic acid amplification test (Abbott Industries, Abbott Park, Ill.). PCR and cytospin of the culture transport medium with chlamydia direct fluorescent antibody staining were used to adjudicate chlamydia culture-negative results. Sensitivity and specificity were calculated both with and without use of the amplification control (AC), with little apparent difference in the results. Without the AC result, sensitivity for C. trachomatis and N. gonorrhoeae were 92.8 and 96.6%, respectively, for cervical swabs and 80.5 and 84.9% for urine from women. C. trachomatis and N. gonorrhoeae sensitivities were 92.5 and 98.5%, respectively, for male urethral swabs and 93.1 and 97.9% for urine from men. This amplified DNA system for simultaneous detection of chlamydial and gonococcal infections demonstrated superior sensitivity compared to chlamydia culture and has performance characteristics comparable to those of other commercially available nucleic acid-based assays for these organisms. PMID:11230419

  2. Use of the UriSwab collection device for testing of Chlamydia trachomatis and Neisseria gonorrhoeae: implications for a postal testing service.

    PubMed

    McNicol, J; Debattista, J

    2013-06-01

    In order to demonstrate the reliability of UriSwab, a trial was conducted using urine samples that had previously returned a detected result for Chlamydia trachomatis and/or Neisseria gonorrhoeae. Urine specimens (115 samples) were received from sexual health clinics and tested using the Roche Cobas 4800 CT/NG method. Concurrently, the urine samples were pipetted directly on to the sponge applicator of the UriSwab, simulating micturition, and the urine harvested from the UriSwab was tested using the Roche Cobas 4800 method. Of the 87 standard urine specimens that were C. trachomatis detected, 85 (98%) were also detected in the corresponding UriSwab specimen (sensitivity 97.7%, specificity 95.7%). Of the 34 standard specimens that were N. gonorrhoeae detected, 33 (97%) were also detected in the corresponding UriSwab specimen (sensitivity 97.1%, specificity 100%). The performance of the UriSwab in this trial was comparable with the testing of neat first-catch urine specimens for both C. trachomatis and N. gonorrhoeae.

  3. Control of gdhR Expression in Neisseria gonorrhoeae via Autoregulation and a Master Repressor (MtrR) of a Drug Efflux Pump Operon

    PubMed Central

    Rouquette-Loughlin, Corinne E.; Zalucki, Yaramah M.; Dhulipala, Vijaya L.; Balthazar, Jacqueline T.; Doyle, Raúl G.; Nicholas, Robert A.; Begum, Afrin A.; Raterman, Erica L.; Jerse, Ann E.

    2017-01-01

    ABSTRACT The MtrCDE efflux pump of Neisseria gonorrhoeae contributes to gonococcal resistance to a number of antibiotics used previously or currently in treatment of gonorrhea, as well as to host-derived antimicrobials that participate in innate defense. Overexpression of the MtrCDE efflux pump increases gonococcal survival and fitness during experimental lower genital tract infection of female mice. Transcription of mtrCDE can be repressed by the DNA-binding protein MtrR, which also acts as a global regulator of genes involved in important metabolic, physiologic, or regulatory processes. Here, we investigated whether a gene downstream of mtrCDE, previously annotated gdhR in Neisseria meningitidis, is a target for regulation by MtrR. In meningococci, GdhR serves as a regulator of genes involved in glucose catabolism, amino acid transport, and biosynthesis, including gdhA, which encodes an l-glutamate dehydrogenase and is located next to gdhR but is transcriptionally divergent. We report here that in N. gonorrhoeae, expression of gdhR is subject to autoregulation by GdhR and direct repression by MtrR. Importantly, loss of GdhR significantly increased gonococcal fitness compared to a complemented mutant strain during experimental murine infection. Interestingly, loss of GdhR did not influence expression of gdhA, as reported for meningococci. This variance is most likely due to differences in promoter localization and utilization between gonococci and meningococci. We propose that transcriptional control of gonococcal genes through the action of MtrR and GdhR contributes to fitness of N. gonorrhoeae during infection. PMID:28400529

  4. Should screening for Chlamydia trachomatis and Neisseria gonorrhoeae in HIV-men who have sex with men be recommended?

    PubMed Central

    Pérez-Hernández, Isabel; Palacios, Rosario; González-Doménech, Carmen; García, Victoria; Márquez, Manuel; Clavijo, Encarnación; Santos, Jesús

    2014-01-01

    Introduction Sexually transmitted infections (STI) like Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) have been associated with increased risk of HIV acquisition [1]. It has been also described as a high prevalence of asymptomatic CT and NG infections in men who have sex with men (MSM) [2]. The aim of this study was to know the prevalence of CT and/or NG infections in asymptomatic HIV-MSM and the related factors. Materials and Methods Prospective study of a cohort of asymptomatic HIV-MSM with follow-up in Malaga (southern Spain) during October 2012–May 2014. Patients with an opportunistic event or who received active antibiotic therapy for CT and/or NG in the previous month were excluded. All of them completed a questionnaire about sexual behaviour, barrier methods and recreational drugs use. Demographical, epidemiological, clinical, analytical and therapeutic data were also collected. Pharyngeal and rectal swabs, and urine samples were collected to be tested for CT and NG by nucleic acid amplification test (c4800 CT/NG. Roche Diagnostics, Mannheim, Germany) [3]. Statistics analysis: SPSS 17.0. Results 255 patients were asked to participate and 248 of them accepted. Median age was 37.7 (30.6–46.3) years, median time since HIV diagnosis was 47.7 (10.5–104.1) months, and median CD4 cells count was 607 (440–824) cell/µL. There were 195 (78.6%) patients on antiretroviral therapy; 81.5% of them had undetectable viral load. 80.5% of the patients had a past history of STI. Infection by CT and/or NG was diagnosed in 24 (9.7%) patients. Overall four urine samples, two pharyngeal, and 15 rectal ones were positive for CT, and five pharyngeal and five rectal swabs were positive for NG. Two patients were co-infected by CT and NG: one with CT in urine and both in rectum, another with CT in urine and rectum and NG in pharynx. One patient presented CT in pharynx and rectum, and two patients NG in pharynx and rectum. Positive CT and/or NG tests were only

  5. Should screening for Chlamydia trachomatis and Neisseria gonorrhoeae in HIV-men who have sex with men be recommended?

    PubMed

    Pérez-Hernández, Isabel; Palacios, Rosario; González-Doménech, Carmen; García, Victoria; Márquez, Manuel; Clavijo, Encarnación; Santos, Jesús

    2014-01-01

    Sexually transmitted infections (STI) like Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) have been associated with increased risk of HIV acquisition (1). It has been also described as a high prevalence of asymptomatic CT and NG infections in men who have sex with men (MSM) (2). The aim of this study was to know the prevalence of CT and/or NG infections in asymptomatic HIV-MSM and the related factors. Prospective study of a cohort of asymptomatic HIV-MSM with follow-up in Malaga (southern Spain) during October 2012-May 2014. Patients with an opportunistic event or who received active antibiotic therapy for CT and/or NG in the previous month were excluded. All of them completed a questionnaire about sexual behaviour, barrier methods and recreational drugs use. Demographical, epidemiological, clinical, analytical and therapeutic data were also collected. Pharyngeal and rectal swabs, and urine samples were collected to be tested for CT and NG by nucleic acid amplification test (c4800 CT/NG. Roche Diagnostics, Mannheim, Germany) (3). SPSS 17.0. 255 patients were asked to participate and 248 of them accepted. Median age was 37.7 (30.6-46.3) years, median time since HIV diagnosis was 47.7 (10.5-104.1) months, and median CD4 cells count was 607 (440-824) cell/µL. There were 195 (78.6%) patients on antiretroviral therapy; 81.5% of them had undetectable viral load. 80.5% of the patients had a past history of STI. Infection by CT and/or NG was diagnosed in 24 (9.7%) patients. Overall four urine samples, two pharyngeal, and 15 rectal ones were positive for CT, and five pharyngeal and five rectal swabs were positive for NG. Two patients were co-infected by CT and NG: one with CT in urine and both in rectum, another with CT in urine and rectum and NG in pharynx. One patient presented CT in pharynx and rectum, and two patients NG in pharynx and rectum. Positive CT and/or NG tests were only related with detectable HIV viral load (OR 3.08, 95% CI 1.2-7.4; p=0.01). It

  6. The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization.

    PubMed

    Unemo, Magnus; Golparian, Daniel; Sánchez-Busó, Leonor; Grad, Yonatan; Jacobsson, Susanne; Ohnishi, Makoto; Lahra, Monica M; Limnios, Athena; Sikora, Aleksandra E; Wi, Teodora; Harris, Simon R

    2016-11-01

    Gonorrhoea and MDR Neisseria gonorrhoeae remain public health concerns globally. Enhanced, quality-assured, gonococcal antimicrobial resistance (AMR) surveillance is essential worldwide. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009. We describe the phenotypic, genetic and reference genome characteristics of the 2016 WHO gonococcal reference strains intended for quality assurance in the WHO global GASP, other GASPs, diagnostics and research worldwide. The 2016 WHO reference strains (n = 14) constitute the eight 2008 WHO reference strains and six novel strains. The novel strains represent low-level to high-level cephalosporin resistance, high-level azithromycin resistance and a porA mutant. All strains were comprehensively characterized for antibiogram (n = 23), serovar, prolyliminopeptidase, plasmid types, molecular AMR determinants, N. gonorrhoeae multiantigen sequence typing STs and MLST STs. Complete reference genomes were produced using single-molecule PacBio sequencing. The reference strains represented all available phenotypes, susceptible and resistant, to antimicrobials previously and currently used or considered for future use in gonorrhoea treatment. All corresponding resistance genotypes and molecular epidemiological types were described. Fully characterized, annotated and finished references genomes (n = 14) were presented. The 2016 WHO gonococcal reference strains are intended for internal and external quality assurance and quality control in laboratory investigations, particularly in the WHO global GASP and other GASPs, but also in phenotypic (e.g. culture, species determination) and molecular diagnostics, molecular AMR detection, molecular epidemiology and as fully characterized, annotated and finished reference genomes in WGS analysis, transcriptomics, proteomics and other molecular technologies and data analysis. © The Author 2016. Published by Oxford University Press on behalf of the

  7. Community-Based Sexually Transmitted Infection Screening and Increased Detection of Pharyngeal and Urogenital Chlamydia trachomatis and Neisseria gonorrhoeae Infections in Female Sex Workers in Hong Kong

    PubMed Central

    Wong, Horas T.H.; Lee, Krystal C.K.; Chan, Denise P.C.

    2015-01-01

    Background Female sex workers (FSWs) are vulnerable to sexually transmitted infections (STIs) and are one of the key populations being infected most by Chlamydia trachomatis and Neisseria gonorrhoeae infections. In Hong Kong, limited data on the burden of chlamydial and gonococcal infections exist because regular screenings are not offered. This study aimed to investigate the prevalence of C. trachomatis and N. gonorrhoeae in FSWs and to assess predictors associated with unprotected fellatio. Methods A cross-sectional study was conduct on 340 FSWs attending a community organization for HIV/STI screening, and a questionnaire addressing sociodemographic and behavioral characteristics was administered to all FSWs. Results The prevalence of syphilis infection was 2.1%, and none was tested positive for HIV. The positivity for pharyngeal C. trachomatis and N. gonorrhoeae was 3.2% and 4.4%, respectively, whereas that for urogenital chlamydial and gonococcal infection was 10.6% and 0.9%, respectively. Of 313 FSWs offering fellatio, having unprotected fellatio with clients was significantly associated with the perceived low risk of contracting STI via fellatio (adjusted odds ratio [OR], 1.88), working in clubs (adjusted OR, 11.14), working on streets (adjusted OR, 3.28), recently started working in the sex industry for 1 year or less (adjusted OR, 3.05), and reporting group sex in the previous year (adjusted OR, 11.03). Conclusions The prevalence of HIV and syphilis infection remains low. This study reveals a relatively high prevalence of N. gonorrhoeae detected mostly in the pharynx. Offering pharyngeal screening for STI would facilitate early diagnosis and treatment of gonococcal infection in FSWs in Hong Kong. PMID:25768859

  8. Public health measures to control the spread of antimicrobial resistance in Neisseria gonorrhoeae in men who have sex with men.

    PubMed

    Xiridou, M; Soetens, L C; Koedijk, F D H; VAN DER Sande, M A B; Wallinga, J

    2015-06-01

    Gonorrhoea is one of the most common sexually transmitted infections. The control of gonorrhoea is extremely challenging because of the repeated development of resistance to the antibiotics used for its treatment. We explored different strategies to control the spread of antimicrobial resistance and prevent increases in gonorrhoea prevalence. We used a mathematical model that describes gonorrhoea transmission among men who have sex with men and distinguishes gonorrhoea strains sensitive or resistant to three antibiotics. We investigated the impact of combination therapy, switching first-line antibiotics according to resistance thresholds, and other control efforts (reduced sexual risk behaviour, increased treatment rate). Combination therapy can delay the spread of resistance better than using the 5% resistance threshold. Increased treatment rates, expected to enhance gonorrhoea control, may reduce gonorrhoea prevalence only in the short term, but could lead to more resistance and higher prevalence in the long term. Re-treatment of resistant cases with alternative antibiotics can substantially delay the spread of resistance. In conclusion, combination therapy and re-treatment of resistant cases with alternative antibiotics could be the most effective strategies to prevent increases in gonorrhoea prevalence due to antimicrobial resistance.

  9. Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species.

    PubMed

    Mechergui, Arij; Achour, Wafa; Hassen, Assia Ben

    2014-05-01

    In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains. © 2013 APMIS. Published by John Wiley & Sons Ltd.

  10. The usefulness of Neisseria gonorrhoeae strain typing by Pulse-Field Gel Electrophoresis (PFGE) and DNA detection as the