Rozprawy doktorskie na temat „Gonorrhoea”

Doctor of Philosophy(PhD)<br>From Federation in 1901 through the first three decades of the twentieth century there was a perceptible shift in modes of rule in New South Wales (NSW) related to the management of venereal diseases. At the beginning of the twentieth century a medicopenal approach was central. By 1925, persuasion and ‘responsibilisation’ were becoming important modes, and young people rather than ‘case-hardened prostitutes' were assessed as being a ‘venereal’ risk. Framing this period were three important legislative developments which informed, and were informed by, these shifts: the NSW Prisoners Detention Act 1909, the NSW Select Committee into the Prevalence of Venereal Diseases 1915 and the NSW Venereal Diseases Act 1918. At its core this thesis is concerned with examining shifting modes of rule. This thesis closely examines each. I suggest that these modes of rule can be viewed through the lens of biopolitics, and following Foucault, deploy the ‘medical gaze’ and the ‘watchful eye’ as constructs to examine the relationship between the government of self, government of others and government of the state. I use the medical gaze to describe not only the individual venereal patient attending a hospital and the body of the patient diagnosed with syphilis and/or gonorrhoea, but most importantly to describe the power relationship between the medical practitioner, the teaching hospital and the patient. I use the watchful eye in a more overarching way to suggest the suite of techniques and apparatus deployed by government to monitor and regulate the venereal body politic, both the populations perceived to be posing a venereal risk, and populations at risk of venereal infection. In relation to the venereal body and the venereal body politic, I analyse three fundamental aspects of the management of venereal diseases: treatment, prevention and epidemiology. Treatment: Over this period, treatment moved from lock institutions to outpatient clinics. Embodied in this change was a widespread institutional ambivalence towards treating venereal patients. I contend that treatment of venereal diseases was painful, prolonged and punitive precisely because of the moral sickness perceived to be at the iv heart of venereal infection. I track this ambivalence to a systemic fear of institutional ‘venerealisation’, which decreased perceptibly across the period. Closely analysing surviving patient records, I argue that in their conduct, venereal patients were often compliant, conscientious and responsible. Prevention: I argue that preventative approaches to venereal diseases became increasingly complex, and operated in three domains – preventative medicine (diagnosis, treatment and vaccination); public health prevention (notification, isolation and disinfection); and prevention education (social purity campaigns and sex hygiene). An emerging plethora of community-based organisations and campaigns began to shift the sites and practices of power. Epidemiology: I suggest that there was a shift from danger to risk in the conceptualisation of venereal diseases. This shift necessitated a focus on factors affecting populations, as opposed to factors affecting individuals. This in turn led to the deployment of various techniques to monitor the conduct of venereal populations. The NSW Venereal Diseases Act 1918 created two important new venereal categories: the ‘notified person’ and the ‘defaulter,’ both of which came to permeate renditions of venereal patients throughout the 20th century.

This thesis presents data on two separate areas relevant to the prevention of HIV and sexually transmitted infection (STI) transmission in homosexual men. These data arise from the community-based Health in Men (HIM) cohort of HIV-negative homosexual men in Sydney. First, the association of circumcision status with HIV and STIs was examined. Older age, ethnicity and country of birth were demographic factors independently associated with circumcision status. Self-report was a valid measure of circumcision status in this population. Overall, being circumcised was associated with a non-significant reduced risk of HIV seroconversion in the HIM cohort (HR 0.76, 95% CI 0.41-1.41, p=0.381). Among the one-third of participants predominantly practising the insertive role in anal intercourse (AI), being circumcised was associated with a significantly reduced risk of HIV infection (HR 0.15, 95% CI 0.03-0.80, p=0.026). Circumcised HIM participants also had a lower risk of incident syphilis (HR 0.35, 95% CI 0.15-0.84, p=0.019), however circumcision status had no significant effect on the remainder of prevalent and incident STIs examined. Second, risk factors for pharyngeal gonorrhoea and chlamydia were investigated. The BD ProbeTec nucleic acid amplification test (NAAT) had a positive predictive value (PPV) for pharyngeal gonorrhoea diagnosis of only 30.4% (95% CI 25.2-36.1%) when compared to a previously validated NAAT targeting the gonococcal porA pseudogene. Pharyngeal gonorrhoea was common in HIM, mostly occurred without concurrent anogenital infection and may frequently spontaneously resolve. Infection was independently associated with younger age (p-trend=0.001), higher number of male partners (p-trend=0.002), contact with gonorrhoea (p<0.001) and insertive oro-anal sex with casual partners (p-trend=0.044). Pharyngeal chlamydia was less common but a high prevalence/incidence ratio suggested that infection may persist in the pharynx for long periods. Pharyngeal chlamydia was independently associated with receptive penile-oral sex with casual partners (p-trend=0.009). In conclusion, circumcision may have a role as an HIV prevention intervention among the subgroup of homosexual men who predominantly practise insertive rather than receptive AI. Regular screening of the pharynx including a validated supplemental NAAT for gonorrhoea diagnosis may prevent much transmission to anogenital sites, whereas chlamydia occurs too infrequently in the pharynx to recommend routine screening in homosexual men.

This thesis is devoted to general practice (GP) networks in Belgium, their development and their activities within the Belgian health system context. These networks are specific research tools for the repeated or continuous collection and analysis of data related to diseases and other health events observed in general practice, including interventions of general practitioners. The thesis focuses on three not-for-profit general practice research networks which are operational today: (1) the national Network of Sentinel General Practices (SGP), coordinated by the Federal research institute Sciensano; (2) the Flemish Intego network, coordinated by the Academic Center for General Practice of Catholic University Leuven; (3) the network of the Fédération des maisons médicales et des collectifs de santé francophones (FMM) with its Monitoring Chart (Tableau de bord), which collects data from Wallonia and the Brussels-Capital Region. The thesis is divided into a general introduction, three main parts and a final discussion with concluding remarks. The general introduction outlines the importance of data from general practice and the contribution of GP networks to research. Furthermore, it points out the importance of general practice for the control of sexually transmitted infections (STIs), a specific field of action. The first main part of the thesis investigates the research question of how the three GP research networks developed within the specific context of the Belgian health system. It is based on the interpretation of written sources such as project reports, annual network reports, research publications, parliamentary documents, relevant websites and the existing research literature. The context analysis included a comparison with the Netherlands since the latter have strong traditions with regard to the position of the general practitioner in the health system (gatekeeper to secondary care, whereas in Belgium the patient generally chooses his/her health provider, and a Global Medical File administered by the general practitioner is not mandatory in Belgium), to general practice research networks and computerisation. It could be shown (1) that Belgium has held a middle position in the European Union regarding GP computerisation; (2) that, contrary to the Netherlands, an operational national GP network based on data from electronic health records (EHRs) could not be established; and (3) that Belgian health system computerisation, which advanced substantially in the last decade, put the issue of health data collection and storage by a new digital service on the agenda. Subsequently, three sub-chapters focus on the development of the three GP networks from their foundation until today. They demonstrate that the SGP and Intego were founded as innovative tools originating from Flemish general practice research, whereas the Monitoring Chart originated from the dynamism of Integrated Primary Health Care Centres (IPHCCs, Maisons médicales) in French-speaking Belgium. Acting as health observatories was both part of the mission of the IPHCCs and the demand of the Regional governments. With time, the research designs of the three GP networks became more sophisticated. Furthermore, European cooperation of the SGP with other GP networks since the late 1980s stands out, since the vision to establish a European sentinel general practice network led to joint influenza surveillance as one of its lasting achievements. In continuation of the developments described above, the second main part of the thesis addresses the missions and the organisation of the three GP networks today as well as their respective strengths and limitations in comparative perspective. It is based on network publications and reports, relevant websites and informal information from the networks themselves. The comparison shows that there is little overlap between the activities of the three GP networks, given the different areas of investigation and the complementarity of supplementary information collected by the SGP versus routine data extraction from EHRs in the other two networks. Furthermore, Intego and the Monitoring Chart essentially cover different parts of the country. The prospective research design of the SGP allows formulating hypotheses and designing research questionnaires with precise definitions of diagnoses before the start of a new research topic in order to minimise inter-observer variability, whereas the diagnosis in the other two networks is the result of the general practitioner's clinical judgement. The Intego network disposes of a substantial number of routine parameters collected over more than two decades by now. With these data, the researchers can design retrospective cohort studies without recording or recall bias by the GP who does not know during his/her daily routine for which research questions his/her data may be used later. The Monitoring Chart stands out by its comparatively strong presence in the Brussels-Capital Region and its data from the less well-to-do part of the population. The third main part of the thesis focuses on STIs which provided a research opportunity, given that Belgian public health efforts to control them have increased in recent years and that the three GP networks engaged in research activities in this regard. The first sub-chapter addresses challenges for the surveillance and monitoring of STIs due to the nature of the pathogens, followed by a sub-chapter about characteristics of STI surveillance and monitoring in Belgium. Afterwards, a sub-chapter describes health policy efforts in order to establish the Belgian HIV Plan 2014-2019. The development of the HIV Plan was analysed by applying the policy streams model of John Kingdon. The analysis was based on published government statements, parliamentary documents, and websites of stakeholders, and showed that the Federal Ministry of Health initiative to achieve the HIV Plan was characterised by a coordinating role with a participatory approach towards the other Belgian governments and stakeholders. The 2013 protocol agreement of the Belgian governments committed them to principles, actions, and cooperation regarding HIV prevention, testing, treatment of persons living with HIV and care for their quality of life, but not to budgets, priorities or target figures. The implementation of the plan, highlighting aspects relating to general practice, is addressed in the subsequent sub-chapter. Two further sub-chapters are based on the analysis of retrospective cohort studies with Intego data from 2009 to 2013, based on EHR routine registration by over 90 general practitioners in Flanders. In the first sub-chapter, the frequencies of gonorrhoea and syphilis diagnoses were investigated. Case definitions were applied. Due to small case numbers obtained, cases were pooled and averaged over the observation period. Frequencies were compared with those calculated from mandatory notification. A total of 91 gonorrhoea and 23 syphilis cases were registered. The average Intego annual frequency of gonorrhoea cases obtained was 11.9 (95% Poisson confidence interval (CI) 9.6; 14.7) per 100,000 population, and for syphilis 3.0 (CI 1.9; 4.5), respectively, while mandatory notification was calculated at 14.0 (CI: 13.6, 14.4) and 7.0 (CI: 6.7, 7.3), respectively. In spite of limitations such as small numbers and different case definitions, the data suggests that the general practitioner was involved in the large majority of gonorrhoea cases, while the majority of new syphilis cases did not come to the knowledge of the general practitioner. The second sub-chapter deals with the prescription of antibiotics to treat gonorrhoea in general practice in Flanders 2009-2013. Belgian guidelines recommended ceftriaxone or alternatively spectinomycin from 2008 onwards and azithromycin combination therapy since 2012. The study investigated to which extent contemporary gonorrhoea treatment guidelines were followed. Ninety-one gonorrhoea cases with ten chlamydia and one genital trichomonas coinfections in 90 patients were registered between 2009 and 2013. The proportion of cases with ceftriaxone and/or spectinomycin prescriptions rose from 13% (two of 15 cases) in 2009 to 56% (nine of 16 cases) in 2013. Combination therapy of ceftriaxone and/or spectinomycin together with azithromycin rose from 0 of 15 cases (0%) in 2009 to 7 of 16 cases (44%) in 2013. Although numbers are small, the results suggest that gonorrhoea therapy guideline adherence improved between 2009 and 2013. Future opportunities, recommended in the final discussion, include (1) extending provider-led STI testing in Belgium, with a prominent role for general practitioners; (2) investigating barriers and facilitators for the achievement of the Global Medical File, notably if sensitive and potentially stigmatising issues such as STIs or mental health are involved; (3) making task delegation by the general practitioner towards other primary health care providers more attractive; (4) facilitating general practitioners' tasks by the introduction of support features into the EHR in order to improve registration and quality of care in general; (5) eliciting Regional government support in order to investigate the diagnostic profiles of the patient population of IPHCCs; and (6) establishing an extended network for the collection and analysis of "production data" (such as the number of contacts, interventions, referrals, prescriptions and diagnostic requests) from general practitioners and other primary health care providers, proceeding from the know-how and the experience of the three investigated GP networks.<br>Doctorat en Sciences de la santé Publique<br>info:eu-repo/semantics/nonPublished

Gonococci that have been phagocytized by human neutrophils are killed very effectively. While much research has focused on defining the microbicidal mechanisms of the neutrophil arsenal, substantially less is known regarding why phagocytized bacteria die. Gonococci were examined, at the molecular level, following exposure to human neutrophil granule extracts (GE) in an effort to discover the "lethal lesion", that injury to the bacterial cell which results in its death. The cytoplasm-based metabolism of GE-treated gonococci continues to function normally for at least 30 minutes, although these same cells have lost the ability to divide and are reproductively dead. GE-treated gonococci were found to utilize less oxygen than control cells, indicative of damage to the cytoplasmic membrane. Visual examination of GE-treated gonococci by light microscopy revealed that the cells undergo very minimal division once exposed to GE. GE-treated gonococci visualized by transmission electron microscopy had outer membranes which suffered time-related disorganization and disruption; the effects began immediately upon contact with GE. GE-treatment was also observed to cause aberrant structure and orientation of forming bacterial septa. Investigation of gonococcal peptidoglycan, the structural component of the bacterial membrane, yielded interesting results when the effects of GE were scrutinized. GE caused subnormal incorporation of peptidoglycan precursors, and also induced a twofold higher rate of release of peptidoglycan turnover fragments than was seen from control cells. After analysis of peptidoglycan fragments released into culture supernatants by thin-layer chromatography and high-pressure liquid chromatography, it was found that the small amount of high-molecular-weight fragments exhausted by control cells was not present with treated cells. Investigation of the cell-associated peptidoglycan, by the above methods, after exposure to GE revealed differences in the digestion products. There was a distinct reduction in the amount of penicillin bound by the penicillin-binding proteins of GE-treated cells. There was, however, no observed change in the electrophoretic mobility between the PBPs of control and treated cells.

Chlamydia Trachomatis and Neisseria Gonorrhoeae rank as the two most commonly reported sexually transmitted diseases (STDs) in the United States. Under limited budget, publicly funded clinics are not able to screen and treat the two diseases for all patients. They have to make a decision as to which group of population shall go through the procedure for screening and treating the two diseases. Therefore, we propose a cubic integer programming model on maximizing the number of units of cured diseases. At the same time, a two-step algorithm is established to solve the cubic integer program. We further develop a web-server, which immediately make recommendation on identifying population groups, screening assays and treatment regimens. Running on the empirical data provided by the Centers for Disease Control and Prevention, our program gives more accurate optimal results comparing to MS Excel solver within a very short time.

The incidence of Gonococcal infection in Grampian region was the highest in Scotland in 1992, 1993 and 1994.  Despite a considerable decrease in 1994, it remained more than twice that of the whole country. Grampian also differed from other regions in that 1A isolates were commoner, accounting for around 78%, 73% and 75% of isolates; the 1A-2 serovar was particularly common. The proportion of 1B isolates in Grampian (22%, 27% and 25%) was low compared with the other centres where 1B accounted for around 70% of all isolates (Young & Moyes, 1996). Seventy-six 1992, 1993 and 1994 clinical isolates of various serotypes were obtained from the Scottish <i>Neisseria gonorrhoeae</i> Reference Laboratory including 40 strains from Grampian and 36 strains from Lothian. Genomic DNA was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with low-frequency cleavage (LFC) endonucleases <i>Nhe</i> I and <i>Spe</i> I.  The restriction patterns generated were reproducible, stable and easy to read.  PFGE was compared to restriction endonuclease analysis (REA) with high-frequency cleavage (HFC) endonuclease <i>Hind</i> III, and 16S rDNA ribotyping with <i>Eco</i>R I. The comparison of phenotypic based typing methods and DNA-based typing techniques to study the 76 <i>N. gonorrhoeae</i> isolates in the present study indicate that the DNA-based methods are more discriminatory. However, the patterns obtained by <i>Nhe</i>I and <i>Spe</i> I PFGE analysis were easily interpreted, reliable and more discriminatory than those of <i>Hind</i> III REA. The finding of this study clearly demonstrate the potential of PFGE as a high discriminatory tool, as compared to MAb-based serotyping, auxotyping, S/A classification, 16S rDNA ribotyping and REA for studying the molecular epidemiology of gonorrhoea.

\(Neisseria gonorrhoeae\) is an obligate human pathogen that can use both oxygen and nitrite as electron acceptors, but its electron transfer chain has only been partially characterised. The gonococcus encodes one azurin, Laz, and eight c-type cytochromes. The aim of this project was to determine the functions of these redox proteins. Single mutants lacking cytochromes c2, c4 and c5 reduced oxygen at 126%, 84% and 80% of the parental rate, respectively. It was not possible to construct a double mutant defective in both cytochromes c4 and c5, unless an ectopic copy of cytochrome c5 was present, the expression of which was induced by IPTG. It was concluded that cytochromes c4 and c5 form a bifurcated electron transfer pathway between the cytochrome bc1 complex and the cytochrome cbb3 oxidase. Candidates for electron donors to the nitrite reductase, AniA, were Laz and cytochromes c2, c4 and c5. Mutants lacking various combinations of these redox proteins retained the ability to reduce nitrite, implicating the presence of further electron transfer pathways to AniA. Mutants defective in the third heme-binding domain of CcoP or the second domain of cytochrome c5 reduced nitrite at 52% and 39% of the parental rate, respectively. The double mutant still reduced nitrite, but at only 12% of the parental rate. It was concluded that the third heme group of CcoP and the second heme group of cytochrome c5 constitute the main electron transfer pathways to AniA, but a third pathway remains to be identified. Previous research failed to demonstrate the presence of cytochrome c2 on an SDS-PAGE gel stained for covalently bound heme. This was shown to be due to the low constitutive level of expression of this protein. Although a precise role for cytochrome c2 could not be determined, the data presented suggest that the protein could function either as an electron donor to AniA, an electron donor to ScoI, or as a regulator of electron flux to the terminal reductases. The combined data show that the plasticity of the gonococcal electron transfer chains allows this bacterium to respond rapidly to changes in terminal electron acceptor availability.

Thesis (PhD Med.(Microbiology))--University of Limpopo, 2010.<br>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.

This work represents the first study of the regulation of cell division genes in the Gram negative coccus Neisseria gonorrhoeae, and this regulation was studied under aerobic and anaerobic growth conditions using two different gonococcal strains: CH811 and FA1090. Three different clusters of cell division genes were investigated: the mur-fts cluster, the ftsEX cluster, and the min cluster corresponding to homologues at the 2 min, the 78 min, and the 26 min region of the Escherichia coli chromosome, respectively. Driven by initial DNA sequencing of the pivotal cell division gene ftsZ in our laboratory, further genomic analysis using the raw data from the N. gonorrhoeae FA1090 Genome Project was used to identify the genetic organization of the mur-fts cluster as well as the min cluster. (Abstract shortened by UMI.)

The carAB operon from E. coli hybridized with the gonococcal clones carrying carA or carB genes under conditions of high stringency (i.e. detecting approximately 80% or greater similarity) suggesting that the nucleotide sequence of the carbamoylphosphate synthetase genes is very similar in these two organisms. Under these conditions for hybridization, the gonococcal clones carrying argB or argF genes did not hybridize with plasmids containing the corresponding E. coli gene. Hybridizations performed under conditions of lower stringency indicate that the nucleotide sequence of the argB gene is less conserved than that of argF in E. coli and N. gonorrhoeae. Co-complementation experiments established gene linkage only between carA and carB and between proA and proB gonococcal genes. Clones complementing a gene defect in argE were also able to complement an argA mutation. This suggests that the enzyme ornithine acetyltransferase from N. gonorrhoeae (encoded by argJ) may be able to complement both argA and argE mutations in E. coli. The prevalence of specific arginine biosynthesis gene defects was studied for 319 arginine-requiring clinical isolates of Neisseria gonorrhoeae by using the ability of the strains to utilize intermediates of arginine biosynthesis. Nearly 99% of the strains were defective either in the conversion of acetylornithine to ornithine (174 strains) or in the conversion of ornithine to citrulline (141 strains). Based on a nutritional requirement for carbamoyl phosphate, only 11% of the uracil-requiring strains defective in the carbamylation of ornithine to yield citrulline were apparently defective in carAB. Three argininosuccinate-requiring strains (i.e. probably defective in argG) of auxotype PAU were identified. A high polymorphism was observed in hybridization patterns of restricted genomic DNA from N. gonorrhoeae strains having the same auxotype and serotype with a gonococcal CPSase gene-specific probe suggesting that this probe may provide a useful epidemiological marker for N. gonorrhoeae. Some of the arginine auxotrophs of N. gonorrhoeae defective in carAB, argJ, argF or argG were complemented by genetic transformation with DNA from recombinant bacteriophages carrying characterized gonococcal arginine biosynthesis genes. Gene defects in proA (5 strains) and in proB (6 strains) were identified by gonococcal transformation assays with recombinant bacteriophages or plasmids carrying proline biosynthesis genes from N. gonorrhoeae. None of the eleven proline-requiring strains tested appears to be defective in proC. Polymerase chain reaction (PCR) amplifications using oligonucleotides specific to conserved areas of the E. coli carAB operon yielded amplified copies of various portions of the N. gonorrhoeae CPSase genes. Amplifications using primers specific to the duplicated region of the E. coli carB gene suggest that the gonococcal carB gene contains a similar duplication. (Abstract shortened by UMI.)

Neisseria gonorrhoeae (NG) har successivt utvecklat resistens mot många antimikrobiella medel och betraktas som ett av de tre reella hoten bland antibiotikaresistenta bakterier. Ciprofloxacin är ett bredspektrum-antibiotikum tillhörande gruppen kinoloner som, förutom att behandla urinvägsinfektioner, används mot NG och infektioner i mage och tarm. Dock har det rapporterats att ca 30 % av NG-isolat som samlats in genom gonokock-isolatövervakningsprojekt (GISP) under 2017 var resistenta mot ciprofloxacin. På molekylnivå är resistens mot ciprofloxacin starkt associerad med en enda mutation i kodon 91 i gyras-genen (gyrA). Detta projekt har undersökt om det går att använda molekylärbiologiska metoder för att detektera NG-isolat med gyrA mutationen. Analysen gjordes med två olika PCR-system, ”7500 Fast Real-Time PCR System” från Applied Biosystems (ABI) och Panther Fusion från Hologic. Proberna som användes designades för påvisning av vildtyp gyrA (ciprofloxacin-känslig) och mutant gyrA (ciprofloxacin-resistent) hos NG. I projektet analyserades 50 NG-positiva prov (analyserade med screeningtest APTIMA COMBO2 från Hologic), från 43 patienter som provtagits under januari-februari 2020 i Region Skåne. Några patienter testades flera gånger vid olika tillfällen. NG-odling hade utförts parallellt från motsvarande tagna prov från patienterna. ABI-metoden påvisade genen hos 90 % (45/50) av NG-positiva prover (APTIMA COMBO2) medan endast 24 av de 49 proven (49 %) kunde odlas med traditionell metodik för att därefter resistensbestämmas. Av de 45 prov där gyras-genen kunde detekteras med ABI-metoden, uppvisade 28 (62 %) av proven en muterad gen och därmed en potentiell resistens för ciprofloxacin. Panther Fusion-metoden påvisade genen hos 80 % (40/50) av NG-positiva prover (APTIMA COMBO2), och såsom tidigare nämnts, kunde endast 24 av de 49 proven (49 %) odlas med traditionell metodik för att därefter resistensbestämmas. Av de 40 prov där gyras-genen kunde detekteras med Panther Fusion-metoden, uppvisade 26 av proven (65 %) en muterad gen och därmed en potentiell resistens för ciprofloxacin. En jämförelse mellan resultaten från PCR-metoderna och odlingarna visar att av de 24 odlingarna som kunde resistensbestämmas fick ABI-metoden resultat för 23 och Panther Fusion för 22. PCR-metodernas resultat överensstämde perfekt med resultaten från odling med samma 8 känsliga och 15 respektive 14 resistenta NG-isolat som odling. De båda PCR-metoderna och traditionell odling uppvisade jämförbara resultat. Av de 24 prov som kunde odlas och därmed resistensbestämmas, detekterades med ABI-metoden gyras-genen i 23 av dessa prov och i 22 av proven med Panther Fusion-metoden. Resistens mot ciprofloxacin uppvisades genom odling i 16 av de 24 odlingsbara prov, och av dessa 24 odlingsbara prov uppvisade ABI-metoden en muterad gen i 15 av proven och Panther Fusion-metoden en muterad gen i 14 av proven. Traditionell odling kunde bara genomföras på 24 av proven och PCR-metoderna identifierade signifikant fler prov innehållande vildtyp eller muterad gyras-gen, 45 respektive 40 prov. Projektet visade tydligt att PCR-metoderna kan identifiera fler prov än genom traditionell odling och kan därmed upptäcka fler prov med förväntad ciprofloxacin-resistens än vad som kan bestämmas genom traditionell odling.<br>Neisseria gonorrhoeae (NG) has been developing a resistance towards several different antibiotics and is viewed as one of the three real threats among resistant bacteria. Ciprofloxacin is a broad-spectrum-antibiotic belonging to the group quinolone antibiotics which, in addition to being used to treat urinal infections, is used to treat NG and infections in the stomach and intestines. However, it has been reported that 30 % of NG-isolates that have been gathered through the Gonococcal Isolate Surveillance Project (GISP) throughout 2017 were resistant to ciprofloxacin. On a molecular level, resistance to ciprofloxacin is strongly associated with a single mutation in kodon 91 in the gyras-gene (gyrA). This project sought to examine if it is possible to use methods from molecular biology to detect which NG that have the gyrA-mutation. The test was done using two different PCR-systems, ”7500 Fast Real-Time PCR System” from Applied Biosystems (ABI) and Panther Fusion from Hologic. The probes used were designed to show wild type gyrA (ciprofloxacin sensitive), and mutated gyrA (ciprofloxacin resistant) in NG. In this project 50 NG-positive samples (analysed with screentest APTIMA COMBO2 from Hologic), from 43 patients that had been tested during January-February 2020 in Region Skåne, were analysed. Some patients were tested several times, within the time period. NG-cultivation had been done in parallel from corresponding samples taken from the patients. The ABI-method showed the gene in 90 % (45/50) of NG-positive samples (APTIMA COMBO2) while only 24 of the 49 samples (49 %) could be cultivated by traditional methodology, and then tested for resistance. Of the 45 samples where the gyras-gene could be detected with the ABI-method, 28 samples (62 %) exhibited a mutated gene and thus a potential resistance to ciprofloxacin. The Panther fusion-method showed the gene in 80 % (40/50) of NG-positive samples (APTIMA COMBO2), and as mentioned earlier, only 24 of the 49 samples (49 %) could be cultivated by traditional methodology to then be tested for resistance. Of the 40 samples where the gyras-gene could be detected with the Panther Fusion-method, 26 samples (65 %) exhibited a mutated gene and thus a potential resistance to ciprofloxacin. The two PCR-methods and traditional cultivation exhibited comparable results. Of the 24 samples that could be cultivated and thus tested for resistance, the ABI-method detected the gyras-gene in 23 of these samples and the Panther Fusion-method detected the gene in 22 of the samples. Cultivation exhibited resistance to ciprofloxacin in 16 of the 24 samples that could be cultivated, and of these 24 cultivatable samples the ABI method exhibited a mutated gene in 15 of the samples and the Panther Fusion-method exhibited a mutated gene in 14 of the samples. Traditional cultivation could only be done on 24 of the samples and the PCR-methods could identify significantly more samples containing either wild type or mutated gyras-gene, 45 and 40 samples, respectively. The project clearly showed that more samples can be identified with the PCR-methods than through traditional cultivation, and thereby discover more samples with expected ciprofloxacin-resistance, than can be determined through traditional cultivation.

Neisseria gonorrhoeae, the etiologic agent of gonorrhea, utilizes TonB-dependent transporters to import essential nutrients such as iron. Study of TonB-dependent transporters is extremely important due to the fact that they make excellent vaccine targets. In order to learn more about the structure, function, expression, and regulation of selected TonB-dependent transporters, three goals were established for this study. The first goal was to examine the role of two highly conserved regions of TbpB in lipidation. One of the conserved regions of TbpB, the LSAC motif, was shown to be critical for lipidation. The second goal was to determine whether MisR/MisS regulates expression of TbpA and TbpB. MisR/MisS was shown to regulate the expression of TbpA and TbpB. The third goal was to assess the ability of recombinant TdfJ to bind hemin when expressed in E. coli. Recombinant TdfJ was shown to specifically bind hemin when expressed in E. coli.

To understand the rates and mechanisms of Neisseria gonorrhoeae opacity (opa) gene variation, the 11 opa genes were amplified independently so that an opa allelic profile could be defined for any isolate from the sequences at each locus. Initial examination of 14 unrelated gonococcal isolates showed that no opa alleles were shared. Analysis of closely related isolates showed these typically shared most opa alleles and so the mechanisms by which recent changes occurred at individual opa loci could be determined. The great majority of changes were due to recombination among existing alleles that either duplicated an opa allele present at another locus or resulted in a mosaic of existing opa alleles. Single nucleotide changes or the insertion/deletion of a single codon also occurred, but few of these events were assigned to mutation, the majority being assigned to localised recombination. Introduction of novel opa genes from co-infecting strains was rare and all but one occurred in the same sexual network. Changes at the eleventh opa locus (opa11) occurred much more rapidly than at other opa loci, almost always differing even between recent sexual contacts. Examination of the neighbouring pilE gene showed that changes at opa11 and pilE often occurred together, although this linkage may not be a causal one. The Opa protein sequences encoded by the opa genes were determined and the regions spanning the two hyper-variable regions (HVR1 and HVR2) were analysed. An almost equal number of Opa protein and opa nucleotide sequences were detected but there was a limited number of HVR combinations, indicating that a large proportion of differences are located elsewhere in the protein. Very few novel HVRs were generated by recombination, even at opa11 where the rate of variation was greatest. Most changes resulted in re-assortment of existing HVRs, most likely due to protein function restriction/benefits.

The aims of this study were (i) to investigate the value of newly developed monoclonal antibody coagglutination reagents for serotyping gonococci according to Protein I variation between strains; (ii) to determine if these or similar monoclonal antibodies could be used in an immunological method for the non -cultural detection of N. gonorrhoeae; and (iii) to produce monoclonal antibodies directed against gonococcal beta- lactamase and to examine their value in a non - cultural diagnostic test for penicillinase- producing gonococci. A total of 1201 gonococcal isolates, obtained from 967 patients attending a sexually transmitted diseases clinic in Edinburgh, were classified serologically using two independently developed panels, each of 14 monoclonal antibodies. The distribution of serogroups WI and WII /III was shown to be 51% and 49% respectively. Using a combination of the two panels of reagents, WI strains could be subdivided into 19 serovars and WII /III strains into 53 serovars: The individual panels recognised 7 (Ph) and 12 (GS) WI serovars and 22 (Ph) and 24 (GS) WII /III serovars. Temporal and geographical variations in serovar patterns were observed, illustrating the dynamic nature of the circulating gonococcal population. A statistically significant correlation between the WII /III serovar combination Bropyt /Back and homosexually acquired infection was found (P < 0.001). This serovar combination accounted for 57% of homosexually acquired infections compared with 3% and 0.8% of infections in heterosexual men and women. A statistically significant correlation between the WII /III serovar Bajk and concomitant rectal infection in women was found (0.05 > P > 0.02). This serovar accounted for 28% of women with genital and rectal infection compared with 17% of women in whom infection was restricted to the genital site. Statistically significant associations between serogroup WII /III and certain serovars and penicillin susceptibility were also observed. In addition to their value in correlating certain strains with particular anatomical sites and in predicting the antibiotic susceptibility of an isolate, these serovar studies proved valuable in tracing infected contacts. A previously developed dot -blot immunoassay using polyclonal antisera raised against whole cell gonococci demonstrated poor sensitivity (26 %) in detecting gonococcal antigen in female cervical specimens: The specificity was 81.7 %. Replacement of polyclonal antisera with a panel of anti -Protein I monoclonal antibodies failed to improve the system, even though coagglutination reagents prepared with these antibodies had detected 99.7% of gonococcal strains. An immunoperoxidase staining technique was developed for the detection of gonococci directly in patient smears. An alternative panel of monoclonal antibodies developed for detection of gonococcal antigen was employed. Coagglutination reagents prepared using these antibodies had previously detected 97% of gonococcal strains. The sensitivity and specificity of this test when applied to 30 male urethral smears was 75% and 100% respectively. However, serovar analysis suggested that the incorporation of additional monoclonal antibodies to the panel would improve the sensitivity of this system. None of the hybridomas obtained from the fusions of myeloma cells and spleen cells from mice immunised with purified gonococcal beta -lactamase enzyme secreted antibodies specific for gonococcal beta -lactamase. It was concluded from this study that (i) the resolution achieved using two panels of monoclonal antibody coagglutination reagents was better than that achieved with either panel alone and increases the value of serotyping for the study of gonococcal epidemiology; (ii) a large variety of antigenic types of gonococci are circulating in the population at any one time and this antigenic pattern does not remain static, therefore constant surveillance of gonococcal strains will be necessary to ensure that immunological detection techniques remain effective; and (iii) the immun peroxidase assay using monoclonal antibodies against PrI has potential for use as an 'on- the -spot' test for the diagnosis of gonococcal infection.

La résistance aux antibiotiques, évidente chez des souches telles que Neisseria gonorrhoeae, est une crise sanitaire mondiale. Il est vital de comprendre son mécanisme, en particulier la transformation naturelle par les Pili de type IV de ces bactéries. Bien qu'elle ait été découverte en 1928, la transformation naturelle n'a toujours pas été élucidée. La dépendance de Neisseria gonorrhoeae à l'égard des Pili de type IV en fait un modèle idéal pour l'étude de ce processus.Cette thèse se lance dans une exploration intensive des processus complexes de transformation de l'ADN au sein de Neisseria gonorrhoeae, répondant ainsi à l'urgence de ce problème critique. Le rôle central de cette étude approfondie est attribué aux pili de type IV, un acteur multifonctionnel et essentiel dans l'orchestration de la transformation de l'ADN.Cette recherche pose méticuleusement les bases, en introduisant des techniques de biologie moléculaire essentielles pour le génie génétique chez N. gonorrhoeae. Nous explorons le développement d'outils, en particulier l'optimisation des milieux et les outils de microscopie adaptés à l'étude de l'absorption et de la transformation de l'ADN. En outre, nos recherches ont mis en évidence une corrélation intrigante entre l'amidon et les acides gras, qui a un impact significatif sur la croissance des gonocoques. Pour comprendre la dynamique des pili de type IV pendant l'absorption de l'ADN, nous avons mis au point des outils et un flux de travail rationalisé capables de visualiser et de quantifier à la fois les pili et les molécules d'ADN. En outre, nous avons automatisé l'analyse des micropiliers d'hydrogel afin d'étudier les propriétés mécaniques des rétractions des pili. En outre, des adaptations des revêtements des micropiliers nous ont permis d'étudier les rétractions des pili en interaction avec l'ADN.Cette étude approfondie comprend également l'examen du comportement des mutants ΔPilV, ΔPilC, et ΔPilD, dévoilant ainsi de profondes connaissances sur les mécanismes de régulation des pili de type IV et leur impact sur la dynamique de l'absorption de l'ADN. Il est particulièrement intéressant de noter que les mutations ΔPilV induisent des altérations dans la translocation de PilE, ce qui entraîne l'émergence de pili plus courts mais efficaces. Cette découverte souligne la nature adaptative de N. gonorrhoeae dans la manipulation de la diversité des pili de type IV pour optimiser les processus d'absorption de l'ADN, une révélation qui revêt une importance considérable dans la lutte contre la résistance aux antibiotiques.En outre, nos études sur d'autres pilines mineures mettent en lumière les altérations du phénotype sans entraver le mécanisme de rétraction des pili. Nos études sur les mutants PilV et PilD mettent en évidence l'influence des modifications post-traductionnelles sur PilE, accentuant ainsi la composition hétérogène des pili de type IV et leur fonctionnalité robuste en tant que polymère.Nous incluons également une courte étude examinant l'interaction entre les espèces de Neisseria commensales et pathogènes dans le contexte de l'absorption de l'ADN, ce qui élargit le champ des implications, invitant à une enquête plus approfondie et élargissant les horizons de ce domaine captivant.Bien que cette expédition laisse certaines questions sans réponse, sa profondeur et son ampleur permettent de mieux comprendre les mécanismes complexes de transformation de l'ADN et le rôle dynamique joué par les pili de type IV dans la remarquable adaptabilité de Neisseria gonorrhoeae. Non seulement cette thèse apporte des solutions aux questions existantes, mais elle ouvre également de nouvelles voies pour la recherche future, suscitant la curiosité et la fascination pour l'élucidation des complexités fonctionnelles des structures pili et leurs implications profondes dans la transformation de l'ADN<br>Antibiotic resistance, evident in strains like Neisseria gonorrhoeae, is a global health crisis. Understanding its mechanism, particularly natural transformation through Type IV Pili in these bacteria, remains vital. Despite discovery in 1928, the specifics of natural transformation remain elusive. Neisseria gonorrhoeae's dependence on Type IV Pili makes it an ideal model for studying this process.This thesis embarks on an intensive exploration into the intricate processes of DNA transformation within Neisseria gonorrhoeae, responding to the urgency of this critical concern. Central to this comprehensive study is the pivotal role attributed to Type IV Pili, a multifunctional and essential player in orchestrating DNA transformation.This research meticulously lays the groundwork, introducing molecular biology techniques essential for genetic engineering within N. gonorrhoeae. We explores tool development, particularly in medium optimization and microscopy tools tailored to study DNA uptake and transformation. Moreover, our investigations uncovered an intriguing correlation between starch and fatty acid, significantly impacting gonococcal growth. To understand Type IV pili dynamics during DNA uptake, we engineered tools and a streamlined workflow capable of visualizing and quantifying both pili and DNA molecules. Additionally, we automated the analysis of hydrogel micropillars to delve into the mechanical properties of pili retractions. Furthermore, adaptations to the micropillar coatings enabled us to study pili retractions interacting with DNA.This in-depth investigation also involves scrutinizing the behaviour of ΔPilV, ΔPilC, and ΔPilD mutants, unveiling profound insights into the regulatory mechanisms of Type IV Pili and their consequential impact on the dynamics of DNA uptake. Particularly noteworthy is the revelation that ΔPilV mutations induce alterations in PilE translocation, resulting in the emergence of shorter yet efficient pili. This discovery underscores the adaptive nature of N. gonorrhoeae in manipulating the diversity of Type IV Pili to optimize DNA uptake processes, a revelation that holds immense significance in combating antibiotic resistance.Furthermore, our studies on other minor pilins sheds light on phenotype alterations without impeding the mechanics of pili retraction. Our studies on PilV and PilD mutants, highlight the influence of posttranslational modifications on PilE, thereby accentuating the heterogeneous composition of Type IV Pili and their robust functionality as a polymer.We also include a short study examining the interplay between commensal and pathogenic Neisseria species within the context of DNA uptake broadens the scope of implications, inviting further inquiry and expanding the horizons of this captivating field.While this expedition leaves certain questions unanswered, its depth and breadth offer extensive insights into the intricate mechanisms of DNA transformation and the dynamic role played by Type IV Pili in the remarkable adaptability of Neisseria gonorrhoeae. Not only does this thesis provide solutions to existing queries, but it also illuminates novel pathways for future research, sparking curiosity and fascination in unravelling the functional intricacies of pili structures and their profound implications in DNA transformation

The continuing evolution of antibiotic resistance strains of Neisseria gonorrhoeae coupled with the paucity of new antimicrobial agents makes the treatment of gonococcal infections challenging. A major cause of resistance is the expression of a multidrug efflux pump termed MtrCDE, which exports a wide range of antimicrobial agents. Efflux pumps are membrane-bound systems and consequently challenging to study and target with drugs. The transcriptional regulator (MtrR) of the efflux pump, however, is a soluble protein and therefore more amenable to study and drug target validation investigations. This thesis serves to investigate the hypothesis that substrates for the MtrCDE efflux pump are also ligands for the regulator MtrR. Isothermal titration calorimetry (ITC) was used to show that MtrR binds commercial antibiotics and antimicrobial peptides. -lactam antibiotics not only bind MtrR but are hydrolysed by the multidrug protein. Evidence for this novel enzymatic activity is provided by ITC, mass spectrometric and microbiological techniques. A series of peptides derived from LL-37 were synthesised and screened for binding to MtrR. A key region of LL-37 with a higher affinity to MtrR than the natural product was then identified. The peptide binding site in MtrR was elucidated via a photoactivated peptide binding study. Electrophoresis mobility shift assays indicated that the peptides do not induce derepression of the genes controlled by MtrR, although the peptide derivatives of LL-37 were shown to be substrates for the MtrCDE efflux pump.