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Neisseria meningitidis is the most common cause of bacterial meningitis in the Western world and the second leading cause of mortality in 1-5 year-olds in the United Kingdom. A signature-tagged mutagenesis screen of approximately 3,000 insertional mutants of a serogroup B isolate of N. meningitidis, C311⁺, identified 73 genes required for pathogenesis in an infant rat model of meningococcal septicaemia. Homology-based searches indicate that two of the genes identified, NMB0342 and NMB0345, have homologues in other pathogenic bacteria and exist within a potential operon of seven genes (NMB0342-NMB0348). NBM0342 is a homologue of ispA (intracellular septation protein) of Shigella flexneri, required for effective intercellular spread and plaque formation in epithelial cells. NMB0345 is a homologue of cbf, a Campylobacter jejuni gene that encodes a 29 kDa protein which is a major antigenic peptide. PCR and Southern analyses of genes in the NMB0342-NMB0348 locus show that they are conserved across a wide range of pathogenic isolates and serogroups of N. meningitidis. However, these genes are present only in a subset of commensal strains. N. meningitidis mutants with transpoon insertions in NMB0342 and NMB0345 are highly attenuated when directly competed with the wild-type bacterium in the infant rat model of infection. Mutations have been introduced into other genes within the locus and the resulting mutants analyzed for their ability to cause systemic disease. Mutants with transpoon insertions in the NMB0343 and NMB0344 genes are significantly attenuated, while mutants with insertions in NMB0347 and NMB0348 are attenuated to a lesser degree. Complementation of the NMB0342 mutation by ectopic chromosomal integration of a wild-type copy of NMB0342 almost completely restores the virulence of the bacterium. In vitro cell-culture analyses and microscopic analysis of mutants in association with host cells demonstrate that a mutant with a transpoon insertion in NMB0345 is significantly reduced in its adherence to Chang epithelial cells compared to the wild-type bacterium. Whole blood and serum-sensitivity assays show that the NMB0342 and NMB0345 mutants are highly serum-sensitive compared to the wild-type bacterium. Preliminary experiments have demonstrated that immunization of mice with recombinant NMB0345 protein confers protection against challenge with the serogroup B isolate MC58. In vitro assays demonstrate that recombinant NMB0345 is an active peptidyl polyl cistrans isomerase (PPIase). Together, the results from these investigations support a role for genes of the NMB0342-NMB0348 locus in the pathogenesis of N. meningitidis infections. Further investigation is needed to assess the potential of NMB0345 as a vaccine candidate.

The population structure of Neisseria meningitidis was investigated by MLEE and REA. Using MLEE 107 strains were characterised studying 6 loci. Clones were defined as 5 loci were identical in the strains studied. Seventy two strains fell into twenty six multimember clones. The clones identified contained between 2 and 6 members. The remaining (n=35) strains were regarded as unique. The genetic diversity of the population was estimated as 0.700. This high degree of diversity seems to be because the majority of the strains were isolated from sporadic cases. Isolates within multimember clones contain isolates of outbreaks and sporadic cases. Clones were also identified in a collection of serogroup C. Neisseria meningtidis (n=34) strains by REA using endonuclease StuI. Eleven multimember clones were recognised containing between 2 and 15 members. Seven strains were regarded as unique. The largest multimember clone (n=15) contained 6 rifampicin resistant meningococci and also strains sensitive to rifampicin. This finding seems to be in the favour of this hypothesis that the resistant phenotype arose once and spread through the United Kingdom. Similarly matrices between 11 serogroup C and 5 serogroup B meningococcal strains were determined. The extensive Dice similarity coefficient between strains of serogroup C and B and also close genetic distance between these two serogroups, on the one hand, and very low Dice similarity coefficient (>50%) and distant genetic relations between serogroups C meningococcal isolates, on the other, demonstrated serogrouping cannot be regarded as a reflection of overall genetic similarity; although its practicality in epidemics and its convenience renders it useful as a first step in hierarchical typing system.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree Master of Medicine in Paediatrics (MMed) Johannesburg 2013 / Introduction Invasive pneumococcal disease, including meningitis, caused by Streptococcus pneumoniae is a major cause of morbidity and mortality world-wide. The introduction of pneumococcal polysaccharide-protein conjugate vaccine (PCV) in the United States has resulted in a reduction in incidence of pneumococcal meningitis. PCV was introduced into the South African expanded programme on immunization (EPI) in 2009. Objective We evaluated the temporal association which the introduction of PCV into the South African EPI had on the incidence of pneumococcal meningitis in children. Methods The study was undertaken in Soweto. All children admitted to Chris Hani Baragwanath Academic Hospital (CHBAH) <14 years of age with meningitis from January 2006 to November 2011 were identified through an electronic database and their microbiological records reviewed to identify the causative bacteria. The results were time framed into two groups: prior to introduction of PCV 1 January 2006 to 31 March 2009 (pre-vaccine era) and post PCV-introduction 1 April 2009 to 30 November 2011 (post-vaccine era). Results 783 patients were admitted with suspected meningitis during the study period, of these 243 (31.0%) met the criteria for bacterial meningitis. The incidence of pneumococcal meningitis was decreasing in the CHBAH in-patient paediatric population by 4.7% per annum prior to the introduction of the vaccine in April 2009. The decline in incidence after PCV introduction accelerated to 18% per annum post-vaccine introduction (P=0.391). In the population most at risk for pneumococcal meningitis, children <1 year of age, the annual reduction in incidence of pneumococcal meningitis accelerated from 1.1% in the pre-vaccine era to 43.4% following PCV introduction (P= 0.011). Conclusions The introduction of PCV resulted in a decline in the incidence of pneumococcal meningitis in all age groups. This decline was most dramatic in the <1 year age group.