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Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 89-93).

Thesis (M.Med.Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 89-93). Also available in print.

Thesis--Copenhagen. / "Litteraturfortegnelse": p. [230]-239.

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.

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.

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.

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