In the African “meningitis belt,” outbreaks of meningococcal meningitis occur in cycles, representing a model for the role of host-pathogen interactions in epidemic processes. The periodicity of the epidemics is not well understood, nor is it currently possible to predict them. In our longitudinal colonization and disease surveys, we have observed waves of clonal replacement with the same serogroup, suggesting that immunity to noncapsular antigens plays a significant role in natural herd immunity. Here, through comparative genomic analysis of 100 meningococcal isolates, we provide a high-resolution view of the evolutionary changes that occurred during clonal replacement of a hypervirulent meningococcal clone (ST-7) by a descendant clone (ST-2859). We show that the majority of genetic changes are due to homologous recombination of laterally acquired DNA, with more than 20% of these events involving acquisition of DNA from other species. Signals of adaptation to evade herd immunity were indicated by genomic hot spots of recombination. Most striking is the high frequency of changes involving the pgl locus, which determines the glycosylation patterns of major protein antigens. High-frequency changes were also observed for genes involved in the regulation of pilus expression and the synthesis of Maf3 adhesins, highlighting the importance of these surface features in host-pathogen interaction and immune evasion.

A GMMA vaccine produced from a recombinant African N. meningitidis W strain with deleted capsule locus, lpxL1, gna33 and overexpressed fHbp v.1 has potential as an affordable vaccine with broad coverage against strains from all main serogroups currently causing meningococcal meningitis in sub-Saharan Africa.

Genomic studies on pathogenic and environmental mycobacteria are of growing interest for understanding of their evolution, distribution, adaptation, and host-pathogen interaction. Since most mycobacteria are slow growers, material from in vitro cultures is usually scarce. The robust mycobacterial cell wall hinders both experimental cell lysis and efficient DNA extraction. Here, we compare elements of several DNA preparation protocols and describe a method that is economical and practical and reliably yields large amounts-usually 10-fold increased compared to earlier protocols-of highly pure genomic DNA for sophisticated downstream applications. This method was optimized for cultures of a variety of pathogenic and environmental mycobacterial species and proven to be suitable for direct mycobacterial DNA extraction from infected insect specimens.Mycobacterial diseases are a major health concern for humans (i.e., Mycobacterium tuberculosis, M. leprae, M. ulcerans, M. avium, and M. paratuberculosis) (4, 13, 18, 29, 30) (13,20). Efficient methods for DNA preparation are required both for the identification and genotyping of such pathogens and for population genomics, which is developing into an important tool to study bacterial evolution, virulence, and epidemiology.Extraction of mycobacterial genomic DNA is especially demanding since (i) many mycobacterial species are among the most extreme slow growers, accounting for small amounts of starting material, and (ii) a robust and waxy cell wall renders mycobacteria difficult to lyse. Published protocols for mycobacterial DNA preparations and commercially available extraction kits are mainly designed for the isolation of small amounts of genomic material suitable for conventional PCR application (2,7,9,11,14,15,23,24,27,28,33), such as for testing of potentially contaminated milk (6,8,17). However, such DNA quantities and qualities are usually not sufficient for more sophisticated molecular analyses.M. ulcerans, the causative agent of the devastating human skin disease Buruli ulcer, is one of the slowest growers among mycobacterial species, and the development of molecular tools is crucial for studying its transmission and microepidemiology. The objective of this study was to develop an optimized extraction protocol for DNA of both high quantity and quality from scarce material of in vitro-cultivated M. ulcerans disease isolates. We compared elements of several protocols and developed a DNA preparation method that is optimized in each individual step and thus ready to use for virtually all mycobacterial species to yield a maximum of pure genetic material. In addition, we applied the established method to cultures of a variety of pathogenic and environmental mycobacterial species and tested it by isolating DNA from insects experimentally infected with M. ulcerans. MATERIALS AND METHODSMycobacterial strains and preparation of mycobacterial cell suspensions. The strains used for this investigation and their origins are as follows: M. ulcerans Agy99, Malaysia 1615, and Japa...

Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.