The ability of human monocytes to phagocytize and kill nonpiliated opaque (T3) and transparent (T4) gonococci was investigated in a tumbling tube suspension assay. A serum-sensitive strain, F62, and a serum-resistant strain, FA19, were studied. Viable colony-forming units remaining after incubation with monocytes were used to assess the extent of killing. The data show that 50% of T3 and T4 gonococci of both strains were killed by monocytes over a 2 hour period. Serum was necessary for the phagocytic killing of transparent gonococci of both strains as well as for FA19 T3. Concentrations of serum ranging from 0.5% to 10% were equally effective, and heat-labile components were required. Killing of opaque F62 T3 organisms, however, occurred in the absence of serum. Increased ratios of bacteria-to-monocytes decreased the efficiency of monocyte phagocytic killing, while a 30 minute pre-opsonization of gonococci in serum enhanced the rate of killing. Monocytes were able to kill plate grown bacteria, but not log phase organisms. A limited duration of phagocytic capability was demonstrated. The absence of lysosomal enzyme release and the Cytochalasin B inhibition of colony reduction indicated that killing was the result of intracellular bactericidal activity. Disruption of the monocytes by sonication to release internalized bacteria did not alter the number of viable colony-forming units recovered indicating that significant numbers of internalized gonococci fail to survive. Additional experiments with adherent monocyte monolayers suggested that all internalized gonococci are indeed killed immediately on ingestion. Similar results obtained from several modifications of the tumbling tube assay supported these observations. Human monocytes responded to contact by opsonized gonococci with an enhanced oxidative metabolism as measured by chemiluminescence. Non-opsonized gonococci also stimulated chemiluminescence although the magnitude of the response was 10-fold less. Both strains and types of gonococci evoked similar chemiluminescent responses which were unrelated to the extent of bactericidal activity. Inhibition of monocyte chemiluminescence by sodium azide, catalase and aminotriazole had no effect on the ability of the cells to phagocytize and kill gonococci, whereas, incubation in iodoacetic acid and phenylbutazone inhibited phagocytic killing, probably as the result of impaired phagocytosis. These data indicate that freshly isolated and in vitro cultured adherent monocytes are capable of phagocytizing and killing N. gonorrhoeae, and that the mechanism of bactericidal activity is not solely dependent on oxidative metabolism.

No description available.

Ein Proteintransportsystem genannt Zwei-Partner-Sekretionssystem ist bei gram-negativen Bakterien weit verbreitet. In B. pertussis ist es bereits ausführlich untersucht. Diese Arbeit widmet sich dem Zwei-Partner-Sekretionssystem in Meningokokken. / Characterisation of a protein transoport system in meningococci. This system, called two-partner-secretion-system is allready well examined in B. pertussis.

Thesis (PhD Med.(Microbiology))--University of Limpopo, 2010. / Studies were performed on local isolates of Neisseria gonorrhoeae to assess their aetiological role in different clinical populations, to determine the evolution of antimicrobial resistance and characterisation of quinolone resistance. In the study performed on women presenting for termination of pregnancy (TOP) the prevalence of common sexually transmitted pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis) was much higher than reported from other studies in TOP populations. The commonest organism isolated was C. trachomatis. There were no significant differences in infection rates in women with or without signs and symptoms of vaginal discharge. Therefore all women presenting for TOP need to be screened and treated for sexually transmitted pathogens. In the study to determine the co-infection rates in men presenting with urethritis (discharge and/or burning on micturition (BOM)), the overall infection rate was 65% with co-infections (more than one pathogen detected) in 8%. N. gonorrhoeae was found in 45%, C. trachomatis in 15% and T. vaginalis in 6% of the men. T. vaginalis was found in higher percentages in men with BOM only, in the absence of visible discharge. There may be a need to add an anti-trichomonicidal agent in men presenting with BOM only. When comparing sexual partners and the pathogens isolated, significantly fewer pathogens were detected in males that had their wives as sole contact when comparing them to men who had sex with casual contacts, reflecting high sexual risk behaviour. M DE JONGH Page xi PhD Med Microbiology N. gonorrhoeae isolates were obtained from men presenting to a general practitioner in Pretoria with urethral discharge. The antimicrobial susceptibility profile was determined to currently used, previously used and potential antimicrobial agents by the disk diffusion, Etest and agar dilution methods. High-level ciprofloxacin resistance emerged in the Pretoria region at 7%. No isolate showed a MIC value of intermediate resistance, suggesting importation of resistant strains, rather than a step-wise incremental increase. Penicillin-resistant gonococcal isolates are entrenched in the community; overall there was 32% resistance (MIC≥2μg/mℓ), with 16% due to penicillinase-producing (PPNG) isolates. Tetracycline-resistant isolates has increased dramatically at 54% and with 36% showing high-level (plasmid-mediated) resistance. All isolates remained susceptible to ceftriaxone, cefoxitin and cefpodoxime. Local gonococcal isolates were sequenced using Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST). In this study a total of 18 isolates resolved into 13 different sequence types (STs). A cluster of four isolates of known sequence type, ST217, was found. Two other known sequence types (ST189 & ST523) have previously been seen in Durban. The ten quinolone-resistant isolates resolved into six STs, five of which were new STs and one cluster of four isolates of a known sequence type. This demonstrates the heterogeneity of ciprofloxacin-resistant strains suggesting introduction of strains from multiple sources rather than clonal spread of a single strain.

Neisseria meningitidis ist ein wichtiger Erreger von Meningitis und Sepsis insbesondere bei jungen Menschen, gleichzeitig sind hohe Raten asymptomatischen Trägertums bekannt. Als die Virulenz begünstigende Faktoren wurden unter anderem die Kapsel, Pili, äußere Membranvesikel (OMV) und Lipopolysaccharid (LPS) identifiziert, die es dem Erreger erleichtern, das menschliche Immunsystem zu überwinden. Dabei war bisher die Rolle von Neutrophil Extracellular Traps (NETs) als neu beschriebene Komponente der angeborenen Immunantwort nicht untersucht worden. NETs stellen spinnennetzartige DNA-Strukturen mit globulären Proteindomänen dar, die aus neutrophilen Granulozyten entstehen und als antimikrobiell gelten. Ziel dieser Arbeit war es, die Wirkung von NETs auf Meningokokken zu charakterisieren und mögliche Resistenzmechanismen der Bakterien zu identifizieren. In den vorliegenden Versuchen konnte gezeigt werden, dass Meningokokken an NETs binden und durch diese in ihrer Proliferation gehemmt werden. Eine Lokalisation der Bakterien an die NETs konnte dargestellt werden, LPS und Pili wurden als wichtige Strukturen für die Vermittlung der NET-Bindung identifiziert. OMVs zeigten sich als protektiv gegenüber dem Einfluss der NETs, indem sie die Bindung der Erreger an die NETs blockierten. Wenig empfindlich zeigten sich die Bakterien gegenüber Histonen als den quantitativ bedeutsamsten NET-Proteinen. Meningokokken schützen sich gegenüber dem Einfluss der NETs durch Ausbildung von Kapsel und LPS mit intakter Phosphoethanolamin-Modifikation. Ebenso vermitteln zwei Cathelicidin-Resistenzgene den Bakterien einen Überlebensvorteil. Keine Rolle bei der NET-Resistenz spielten die untersuchten Effluxmechanismen. Neuere Untersuchungen von Lappann et al. indentifizierten Meningokokken und OMVs als potente NET-Induktoren. Damit könnten durch die relativ NET-resistenten Mikroorganismen andere Abwehrmechanismen der Neutrophilen konterkariert werden und eine Immunevasion begünstigt werden. Genauere Untersuchungen diesbezüglich stehen noch aus. / Neisseria meningitidis is an important pathogenic agent of meningitis and sepsis especially in young adults, at the same time high rates of asymptomatic carriage are well-established. Known factors promoting virulence are, among others, the capsule, pili, outer membrane vesicles (OMV) and the lipopolysaccharide (LPS). In host defense against meningococcal disease, the role of Neutrophil Extracellular Traps (NETs), a recently described component of the innate immune response, had not yet been investigated. NETs are web like structures with globular protein domains that arise from neutrophil granulocytes and are considered being antimicrobial. The aim of the present study was to further investigate interactions between N. meningitidis and NETs and to identify possible resistance mechanisms of meningococci. In this thesis I could demonstrate that meningococci bind to NETs and are therewith being restricted in proliferation. A localization of bacteria to NETs was illustrated, and the mediating effect of LPS and pili on binding was identified. OMVs were shown to be protective against the properties of NETs by blocking the binding of pathogens to NETs. Bacteria were minor sensitive to histones which represent the quantitatively most significant group of proteins in NETs. Meningococci are protected against the effect of NETs by the formation of capsule and LPS when correctly modified with phosphoethanolamine. Two genes involved in cathelicidin resistance were shown to be beneficial on the survival of the bacteria. The investigated efflux mechanisms did not affect resistance to NETs though. Recent data by Lappann et al. identified the role of meningococci and OMVs as potent inducers of NET-formation. This might be a strategy of the NET-resilient microorganism to thwart neutrophil phagocytosis or degranulation and to facilitate immune escape, which is yet to be investigated.

Chromosomal mediated antimicrobial resistance in Neisseria gonorrhoeae can develop as a result of three main processes including the alteration of target enzymes, changes in transmembrane transport channels and active efflux pump function. Single nucleotide polymorphisms (SNPs) of target genes such as DNA gyrase (gyrA) and topoisomerase (parC), together with mutations in the promoter regions of the efflux pumps norM and mtr can confer resistance to the macrolides, penicillins and fluoroquinolones. These SNPs were analyzed using the SNaPshot method to allow for rapid detection of resistant isolates. Oligonucleotides were developed in the 5’ to the 3’ direction, ending one nucleotide adjacent to the specific SNP of interest. Single base extension reactions were performed and were detected using capillary electrophoresis. The SNaPshot procedure from Applied Biosystems employed in this study adds a single fluorescently-labelled nucleotide complementary to this SNP at the 3’ end by a primer extension polymerase reaction. Then using capillary electrophoresis, the labelled nucleotide is detected, enabling differentiation between A, C, T, or G. SNP results obtained were verified using DNA sequencing and both single and multiplexed reactions were carried out to increase the efficiency of the procedure. Spiked urine samples were also observed to determine if SNPs could be detected clinically. Single reactions enabled the characterization of all confirmed and relevant SNPs. With multiplex primer extension, multiple peaks were observed, each corresponding to one of the SNPs in the gene. This technique was explored for its applicability to detect SNPs of gyrA and parC mutations. Observable SNP detection limits were seen in spiked urine samples at 108 cells/mL in as early as 4 hours. DNA sequencing results confirmed the SNPs identity in each case. Thus, capillary electrophoresis using the SNaPshot protocol is another way to rapidly identify clinically resistant strains of Neisseria gonorrhoeae. This technique has also been shown to reduce analysis time compared to DNA sequencing and produces the same results. / February 2005

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Microbiology and Immunology. Title from title-page of electronic thesis. Bibliography: leaves 146-172.

No description available.

No description available.

Bacterial species of the genus Neisseria include pathogens that are responsible for diseases of humans including bacterial meningitis (Neisseria meningitidis) and the sexually transmitted disease gonorrhea (Neisseria gonorrhoeae). These diseases are often characterized by a massive influx and activation of neutrophils, white blood cells involved in the early/innate immune response to pathogens, at the infection site. Neisseria spp. bind to and activate neutrophils via their Opacity-associated (Opa) outer membrane proteins, which interact with some members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. One of these CEACAMs, CEACAM3 (CD66d), is unique in its restriction to neutrophils and its expression of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM; YxxL/Ix6-8YxxL/I). In the course of my thesis work, I have shown that this motif is critically dependent for the activation of the neutrophil response to Neisseria, through the coupling of neisserial binding to activation of the tyrosine kinase, Syk, which initiates downstream signaling responsible for the antimicrobial responses of neutrophils. These data contribute to the knowledge of how seemingly unrelated receptors of neutrophils (such as the IgG-binding Fcγ receptors, the fungal receptor Dectin-1, and the bacterial-binding CEACAM3) converge functionally on the presence of the ITAM.