Gonorrhea, caused by the human pathogen Neisseria gonorrhoeae, is a severe public health problem worldwide with more than 82 million new infections each year. N. gonorrhoeae is transmitted by sexual contact and primarily causes urogenital mucosal infections in men and women. Left untreated, this infection may cause severe complications, especially in females. Eye infections of the newborn can occur. Gonorrhea infections enhance HIV transmission. The highly prevalent antibiotic resistance and the emergence of new drug resistances render treatment of the infections increasingly difficult. Close monitoring of antimicrobial susceptibility of this pathogen is crucial, and enhanced knowledge of molecular mechanisms of gonococcal antimicrobial resistance is urgently needed. There are no vaccines available against N. gonorrhoeae. Control of gonorrhea relies on comprehensive strategies which can be better formulated by understanding, at molecular levels, how N. gonorrhoeae is transmitted in communities. My research aimed to illustrate the severe burden of antimicrobial resistance in N. gonorrhoeae temporally and geographically in China and to reveal the molecular mechanisms of antibiotic resistance particularly the development of reduced susceptibility to ceftriaxone in N. gonorrhoeae isolates. To determine specific strain distributions, N. gonorrhoeae isolates were characterized using molecular typing methods such as a modified porB-based typing scheme and the N. gonorrhoeae Multi-Antigen Typing (NG-MAST) method, compared to traditional epidemiological approaches. The ultimate goal was to provide information for better formulating disease control strategies for gonorrhea. In this research, male patients with gonorrhea and their sex partners were recruited in Shanghai (2005 and 2008) and in Urumchi (2007-2008), China. Epidemiological information pertaining to sexual contacts was collected. N. gonorrhoeae isolates were investigated for their antimicrobial susceptibility. Molecular mechanisms of antimicrobial resistance were explored by analysis of potential resistant determinants (gyrA, parC, porB, mtrR, ponA and penA). The molecular data were combined with bioinformatic analysis and traditional epidemiological data. High percentages of N. gonorrhoeae isolates (11% - 19% in Shanghai, 4.5% in Urumchi) exhibited reduced susceptibility to ceftriaxone (MICs = 0.125-0.25 mg/L), the first line drug recommended for the treatment of gonorrhea in China. The majority of isolates (>98%) were susceptible to spectinomycin, an alternative regimen for gonorrhea treatment; however, the proportion of isolates having intermediate levels of susceptibility increased from 1.9% in 2005 to 9.9% in 2008. The majority of isolates tested were resistant to penicillin (80% - 93%), tetracycline (56% - 65%) and ciprofloxacin (98% - 100%). Plasmid-mediated resistance in N. gonorrhoeae isolates were highly prevalent (51% - 79%) in Shanghai and Urumchi. Analysis of 60 clinical isolates revealed that reduced susceptibility to ceftriaxone is mediated by porB1b allele and is associated with specific mutations in penicillin binding protein 2 and in the DNA binding and dimerization domains of MtrR. Penicillin binding protein 1 is not involved in reduced susceptibility to ceftriaxone. Although mutation patterns in quinolone resistant determinant regions (QRDRs) varied, the majority of ciprofloxacin resistant isolates had double mutations in GyrA (S91F and D95G/A/N) and most isolates also carried a S87R/N mutation in ParC. The presence of mutations in the QRDR of ParC is correlated with elevated ciprofloxacin MICs. A modified porB-based molecular typing scheme was developed and involved ~82% of the DNA sequence of gonococcal porB. This typing method proved to have high discriminatory ability (index of discrimination = 0.93 0.96), and was cost effective and easy to perform as compared to the NG-MAST analysis. Using the modified porB-based typing method, N. gonorrhoeae isolates were reliably differentiated, and transmission clusters were identified. Molecular epidemiology using the porB-based method confirmed direct sexual connections and identified sexual networks otherwise unrevealed by the patient self-reporting or traditional case-tracing methods.

DNA mismatch repair ensures faithful transmission of genetic material from parents to progeny, which is required for the survival of the organism. The studies on E. coli MMR proteins have formed the basis for the study of the MMR system in eukaryotic organisms, because the functions of MMR proteins believed to be been conserved. In organisms that harbor MutH protein, it is known that MutH acts as a monomer which nicks the unmethylated daughter strand and is activated in a MutS-MutL- dependent manner. The cleavage specificity of MutH is very stringent. Till recently, it was not clear as to how MutH distinguishes hemimethylated DNA from fully or unmethylated DNA. The co-crystal structures of MutH-DNA complexes revealed that Y212, R184 and P185 were in close proximity to the methyl-adenine. Clustal-W sequence alignment of MutH with Sau3AI showed that Sau3AI has PCT residues instead of L183, R184, and P185. A triple mutant MutH-L183P-R184C-P185T was found to cleave both unmethylated and methylated DNA. The nicking endonuclease activity of the LRP→ PCT triple mutant was enhanced in the presence of Haemophilus influenzae MutL. The mutL gene of Neisseria gonorrhoeae was cloned and the gene product purified. It was shown that the homodimeric Neisseria gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of manganese or magnesium or calcium ions unlike human MutLα which shows endonuclease activity only in the presence of manganese. Further more the C-terminal domain of Neisseria gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460 to 658 also exhibits Mn2+ dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, it was shown that the latter protein exhibits highly reduced endonuclease activity. Surface plasmon resonance spectroscopy was used to determine the kinetics of DNA binding by NgoL. The DNA binding was carried out in absence of metal ions. Interaction studies with NgoL with ssDNA in SPR spectroscopy revealed a KD value of 4.7 × 10–8 M. While the human MutLα endonuclease activity was shown to be stimulated by ATP, ATP inhibits NgoL endonuclease activity. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, it was shown that the latter protein exhibits highly reduced endonuclease activity. NgoL ATPase activity was enhanced in the presence of DNA. The fact that NgoL ATPase activity is stimulated ~ 2.5-fold by dsDNA and ~ 2-fold by ssDNA is a further evidence for the interaction between NgoL and DNA. The results presented above show that NgoL harbors a nicking endonuclease activity which is present in the C-terminal domain. NgoL and NgoL-CTD are dimers in solution and DMHA(X)2E(X)4E motif present in the CTD is required for the nicking endonuclease activity. These results suggest that DNA mismatch repair mechanism in N. gonorrhoeae is different from that in E. coli. In the absence of MutH homolog, N. gonorrhoeae is able to repair the DNA by virtue of MutL nicking endonuclease activity.

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

The possibility of identifying single nucleotide polymorphisms (SNPs) that would be useful for rapid bacterial typing was investigated. Neisseria meningitidis was the organism chosen for modelling the approach since informative SNPs could be found amongst the sequence data available for multi-locus sequence typing (MLST) at http://www.mlst.net. The hypothesis tested was that a small number of SNPs located within the seven gene fragments sequenced for MLST provide information equivalent to MLST. Preliminary investigations revealed that a small number of SNPs could be utilised to highly discriminate sequence types (STs) of clinical interest. Laboratory procedures demonstrated that SNP fingerprinting of N. meningitidis isolates is achievable. Further tests showed that laboratory identification of a defining SNP in the genome of isolates was to be a practical method of obtaining relevant typing information. Identification of the most discriminating SNPs amongst the ever-increasing amount of MLST sequence data summoned the need for computer-based assistance. Two methods of SNP selection devised by the author of this thesis were translated into computer-based algorithms by contributing team members. Software for two computer programs was produced. The algorithms facilitate the optimal selection of SNPs useful for (1) distinguishing specific STs and (2) differentiating non-specific STs. Current input information can be obtained from the MLST database and consequently the programs can be applied to any bacterial species for which MLST data have been entered. The two algorithms for the selection of SNPs were designed to serve contrasting purposes. The first of these was to determine the ST identity of isolates from an outbreak of disease. In this case, isolates would be tested for their membership to any of the STs known to be associated with disease. It was shown that one SNP per ST could distinguish each of four hyperinvasive STs of N. meningitidis from between 92.5% and 97.5% of all other STs. With two SNPs per ST, between 96.7% and 99.0% discrimination is achieved. The SNPs were selected from MLST loci with the assistance of the first algorithm which scores SNPs according to the number of base mismatches in a sequence alignment between an allele of an ST of interest and alleles belonging to all other STs at a specified locus. The second purpose was to determine whether or not isolates from different sources belong to the same ST, regardless of their actual ST identity. It was shown that with seven SNPs, four sample STs of N. meningitidis could, on average, be discriminated from 97.1% of all other STs. The SNPs were selected with the aid of the second algorithm which scores SNPs at MLST loci for the relative frequency of each nucleotide base in a sequence alignment as a measure of the extent of their polymorphism. A third algorithm for selecting SNPs has been discussed. By altering the method of scoring SNPs, it is possible to overcome the limitations inherent in the two algorithms that were utilised for finding SNPs. In addition, the third approach caters for finding SNPs that distinguish members of a complex from non-members.

Background: Neisseria meningitidis is a gram-negative bacterium with the potential to cause invasive disease. Invasive meningococcal disease (IMD) can be fatal if delay to antibiotic therapy. There are six serogroups, which are capable of causing invasive disease in humans; A, B, C, W, X and Y. Since 2015, serogroup W and serogroup Y account for the majority of IMD cases reported in Sweden. Aim: To investigate the clinical presentations of IMD caused by Neisseria meningitidis serogroup W and Y. Method: Two databases, PubMed and Cochrane, were used to find articles that described the clinical picture of IMD. Articles with description of clinical features of the studied serogroups and with eight cases or more in every study were included. In addition, only original articles were included. Results: A total of 633 articles were found and 11 fulfilled all the inclusion criteria. Five out of seven articles found meningococcemia as the predominating presentation of serogroup W IMD. Two out of the four articles that studied serogroup Y IMD found meningitis at a higher number. Conclusion: The results of this systematic review suggest meningococcemia as a relatively common presentation of serogroup W IMD while meningitis and pneumonia might occur more frequently in serogroup Y IMD. However, these results should be interpreted carefully because the included articles were mostly retrospective studies and future prospective studies are needed to better identify clinical presentations of serogroup W and Y IMD.

As vacinas antimeningocócicas têm se demonstrado efetivas contra os sorogrupos A e C, no entanto ainda não existe vacina contra o sorogrupo B devido à similaridade entre a estrutura capsular do polissacáride B e o ácido polisiálico que faz parte do tecido cerebral humano, podendo levar à autoimunidade. O objetivo deste estudo foi investigar as propriedades adjuvantes dos toxóides Stx1 e Stx2 (STEC) de Escherichia coli, administrados em preparações antigênicas com vesículas de membrana externa nativa (NOMV) de Neisseria meningitidis B, comparando duas vias de imunização prime-boost ou intramuscular, em camundongos BALB/c com idade entre 6-8 semanas. A determinação dos níveis de anticorpos empregando a técnica de ELISA, mostrou elevadas concentrações de anticorpos IgG em soros de animais imunizados pela via intramuscular com Stx1+NOMV, mas não com NOMV, o que sugere que por esta via (intramuscular apenas) Stx1 possa ter atuado como adjuvante. No ensaio de Immunoblotting, soros de animais imunizados com Stx1+NOMV reconheceram maior número de antígenos de NOMV quando comparado ao grupo que recebeu Stx2+NOMV. O sistema prime-boost mostrou-se efetivo quando comparamos os níveis de anticorpos presentes no soro após a dose intramuscular (reforço), entretanto, não melhor do que quando utilizamos duas doses apenas pela via intramuscular. Este estudo poderá contribuir no desenvolvimento de tecnologias associadas a novas preparações antigênicas utilizando antígenos de membrana externa de N. meningitidis B , empregando toxóides como adjuvantes. / The meningococcal vaccines have been shown to be effective against serogroups A and C, however there is still no vaccine against serogroup B. The capsular polysaccharide from serogroup B meningococci polysialic acid moiety mimetic of many human glycoproteins including the neural cell adhesion molecules and may lead to autoimmunity. This study aimed to investigate the adjuvant properties of toxoids Stx1 and Stx2 (STEC) from Escherichia coli and native outer membrane vesicles (NOMV) of Neisseria meningitidis B, comparing two ways of immunization prime-boost or only intramuscular in BALB/c mice. The results showed high concentrations of IgG antibodies in sera of animals immunized intramuscularly with Stx1+NOMV, suggesting that in this way may have Stx1 acted as an adjuvant. In the Immunoblotting assay, sera from animals immunized with Stx1+NOMV recognized more antigens of NOMV when compared to the group that received Stx2+NOMV. The prime-boost was effective however, no better than only two doses intramuscularly. This study may contribute to the development of new technologies and strategies against N. meningitidis B employing toxoids as adjuvants.

Philosophiae Doctor - PhD (Biotechnology) / Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo;s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes. / South Africa

Les hommes ayant des relations sexuelles avec d'autres hommes (HARSAH) ont un risque accru d'infections sexuellement transmissibles (IST). Les données sur les IST chez les HARSAH sont rares au Bénin. Cette étude visait à évaluer la prévalence et les facteurs associés à Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) et Treponema pallidum (TP) chez 204 HARSAH VIH-négatifs de Cotonou au Bénin initiant la prophylaxie préexposition en prévention du VIH. Un questionnaire a été complété et un médecin a réalisé un examen physique des participants. Des échantillons de sang, anaux, pharyngés et urinaires ont été prélevés. Les prévalences des IST ont été présentées avec un intervalle de confiance à 95% et la régression de Poisson a permis d'identifier leurs déterminants. La prévalence était de 18,6%, 15,2%, 9,8% et 27,9% pour CT, NG tous sites confondus, NG anorectal et l'infection par NG ou CT (NG/CT) respectivement. Les IST étaient généralement asymptomatiques (84,2% pour NG/CT) et de localisation extra-génitale (61,4% pour NG/CT). Un seul cas de syphilis non confirmée a été observé. En analyse multivariée, le fait d'avoir moins de 25 ans, de ne pas être célibataire et d'avoir plus de quatre partenaires féminines étaient associés à CT et NG/CT alors qu'un faible revenu mensuel était associé à NG/CT seulement. Le jeune âge, le sexe anal réceptif et ne pas avoir de rapports sexuels payants étaient associés à NG. Seul, le sexe anal réceptif était associé à NG anorectal. Les infections à NG et à CT sont fréquentes chez les HARSAH VIH-négatifs de Cotonou au Bénin. Leur caractère asymptomatique et extra-génital rend la prise en charge syndromique inefficace et le dépistage aux sites anal et pharyngé nécessaire. La syphilis est rare chez ces HARSAH. Les interventions de contrôle et de prévention des IST devraient cibler particulièrement les jeunes HARSAH et leurs partenaires sexuelles féminines. / Men who have sex with men (MSM) are at increased risk of sexually transmitted infections (STIs). Data on STIs among MSM are scarce in Benin. This study aimed to assess the prevalence and factors associated with Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Treponema pallidum (TP) among 204 HIV-negative MSM in Cotonou, Benin initiating pre-exposure prophylaxis (PrEP) for HIV prevention. A questionnaire was completed, and participants underwent a physical examination by a physician. Blood, anal, pharyngeal, and urinary samples were collected. Prevalences are presented with a 95% confidence interval. Poisson regression was used to determine factors associated with STIs. The prevalence was 18.6%, 15.2%, 9.8% and 27.9% for CT, NG-all sites, anorectal NG, and infection by either NG or CT (NG/CT) respectively. STIs were generally asymptomatic (84.2% for NG/CT) and of extra-genital location (61.4% for NG/CT). Only one case of unconfirmed syphilis was observed. In multivariate analysis, younger age, not being single, having more than four female sexual partners were associated with CT and NG/CT while lower monthly income was associated with NG/CT alone. Younger age, receptive anal sex and paid sex were associated with NG. Only receptive anal sex was associated with anorectal NG. NG and CT infections are common among HIV-negative MSM in Cotonou, Benin. Their asymptomatic and extra-genital nature makes syndromic management ineffective and screening at anal and pharyngeal sites necessary. Syphilis is rare among these MSM. STI control and prevention interventions should target young MSM and their female sexual partners.

Neisseria meningitidis causes 500,000 cases of meningitis and septicaemia annually worldwide, with a mortality rate of approximately 10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opa proteins are major meningococcal outer membrane proteins, and a limited number of Opa variants have been associated with hyperinvasive serogroup B meningococci, suggesting their use as a potential novel vaccine. Immunisation of mice with recombinant Opa elicited high levels of meningococcal-specific serum bactericidal antibody (SBA), demonstrating proof in principle of this approach. Opa proteins mediate bacterial adherence to host cells and modulate human cellular immunity, and there are conflicting data regarding their effects on CD4⁺ T cells. opa genes from N. meningitidis strain H44/76 were cloned into the plasmid vector pBluescript, disrupted using antibiotic resistance cassettes and transformed into H44/76 to sequentially disrupt the four opa genes. This produced a unique panel of 15 isogenic Opa-deficient strains, including an Opa-negative strain, which enabled investigation of the immunomodulatory role of surface-expressed Opa proteins. There was no consistent effect of Opa expressed on the surface of OMVs and inactivated bacteria on CD4⁺ T cells, with significant heterogeneity of responses between individuals. The rate of Opa phase variation was between 10^-3 and 10^-4, and increased 180-fold following transformation of bacteria with unrelated DNA. These data support further investigation of Opa as a potential meningococcal vaccine component, and highlight the importance of host and bacterial factors in the development of OMV vaccines.

Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill &amp; Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications. / Patogena organismer smittas till värd organismen genom alla möjliga kontaktnätverk och skapar en mångfald olika sjukdomstillstånd. Dock är det fortfarande vanligt förekommande behandlingsbara infektiösa sjukdomar som orsakar den största hälsoförlusten, sett från ett globalt perspektiv. Bill och Melinda Gates Stiftelsen samarbetade med RAND kooperation för att forma “The Global Health Diagnostics Forum”. Deras mål var att etablera och analysera matematiska modeller för vilka effekter en ny diagnostisk metod utrustat för fältarbete skulle ha i utvecklingsländer. Resultaten var häpnadsveckande, med potentiellt miljoner av liv som skulle kunna räddas på en årlig basis. Den etablerade standarden för diagnostik av patogena bakterier har länge varit kultiveringsmedia baserad. Miljö specialiserade biologer har estimerat att mindre än 1 % av alla bakterie arter går att kultivera. Dock erbjuder genetiska analyser potentialen att kunna identifiera alla mikrober från alla de biologiska rikena. Nukleinsyrebaserade diagnostiska metoder har märkbart förbättrats över de senaste årtionden. Nya tekniker erbjuder utökad sensitivitet, selektivitet, sänkta kostnader och parallella analyser av patient prover. Dock är de flesta metoderna begränsade till standardiserade laboratoriemiljöer. För att konstruera en väl fungerande diagnostisk fältutrustning för användning i problem områden, behöver världsledande tekniker identifieras och kombineras. Fokuseringsområdet för denna doktorsavhandling har varit att utveckla och utföra nukleinsyrebaserade metoder för patogen diagnostik. Metoder och experimentella utförande applicerades på två distinkta system i) sökning av antibiotika resistens relaterade mutationer i den patogena bakterien Neisseria gonorrhoeae och ii) genotypning av det cancer orsakande Humana Papillomaviruset (HPV). Den första delen av studien inriktade sig mot utveckling av snabba, direkta och multiplexa Pyrosekvenserings baserade nukleinsyreanalyser. Med förbättrad provprepareringsmetodologi kunde vi detektera multipla HPV infektioner med högre sensitivitet än vad tidigare beskrivits med liknande metodologi. Den andra delen av studien fokuserades på multiplexa nukleinsyre amplifikationer med “Molecular Inversion Probe” tekniken med sista steg Pyrosekvenserings analys. “PathogenMip assay” erbjuder ett komplett detektionsprotokoll för alla kända patogena organismer. Vi introducerar även den nya “Connector Inversion Probe”, en “Padlock Probe” kapabel att genomföra kompletta gap fyllningar för multiplex nukleinsyre amplifiering. / QC 20100624