The genetic diversity and population structure of

The genetic diversity and population structure of the species have been elucidated by the use of two related methods, multilocus enzyme electrophoresis (MLEE) starting in the early 1980s and multilocus sequencing typing (MLST) at the end of the 1990s (Caugant et al., 1986a; Maiden et al., 1998). Both methods assess genetic variation among isolates by indexing their whole genome through a small subset of representative housekeeping genes. With the availability of high-throughput Sanger DNA sequencing, MLEE was replaced by MLST, which presents the additional advantage of being fully portable through an Internet database (www.pubmlst.org/neisseria). Both methods produce equivalent data and the basic features of meningococcal populations first elucidated by MLEE were confirmed by MLST. Of the thousands of genotypes, distinguished by MLEE and MLST most are rarely, if ever, associated with disease, in saha histone to the handful of clonal complexes responsible for epidemics or even pandemics (Yazdankhah et al., 2004). Many serogroup B outbreaks since the 1970s have been caused by N. meningitidis isolates belonging to the ST-32 clonal complex, previously designated electrophoretic type (ET)-5 complex. ET-5 was first identified from a case of serogroup B meningococcal disease in Norway in 1969 (Caugant et al., 1987). In the succeeding years, a hyper-endemic wave of serogroup B meningococcal disease started in Norway with an incidence reaching 8.7 per 100,000 population in 1983, subsequently decreasing over the years to less than 1.0 per 100,000 population in 2000. Similar or closely related clones expressing the same or different major antigenic outer membrane proteins were responsible for high incidence of serogroup B disease in several other European countries in the 1980s and 1990s, as well as outbreaks and epidemics in Latin America, including Cuba, Chile, Brazil, and Argentina (Bygraves et al., 1999; Caugant et al., 1986b; Cruz et al., 1990; Sacchi et al., 1992; Wedege et al., 1995). The ST-32/ET-5 complex has also caused a prolonged outbreak in the Pacific-Northwest of United States in 1993 to 2007 (Diermayer et al., 1999). A long-lasting outbreak in Normandy, France, in the past decade has also been caused by the ST-32 complex (Rouaud et al., 2006). While serogroup B disease is rare in Asia and Africa, the few available serogroup B isolates were also linked to the same clonal complex. Thus, the ST-32 complex caused disease globally over a 40-year period (Caugant et al., 1987). Complete “finished” genomes for two ST-32 complex isolates, MC58 and H44/76, have been published, the former originating from the United Kingdom in the 1980s and the latter from a case in Norway in 1976 (Tettelin et al., 2000; Piet, 2011). Both isolates have been extensively used in serogroup B vaccine research with MC58 pivotal to the design of the 4CMenB vaccine (BexSero®) through a technique known as reverse vaccinology and H44/76 used in the design of several outer membrane vesicle-based vaccines (Serruto et al., 2012; van der Ley and Poolman, 1992). Serogroup B vaccine research has focussed on surface-expressed proteins which, while being immunogenic, might also be strongly under selection pressure. It is, therefore, essential to elucidate how these vaccine antigens might change over time and during worldwide spread of a hyper-invasive clone. WGS provides a new means to elucidate genomic variation within a clonal complex of N. meningitidis and this paper presents a gene-by-gene description of WGS data from a global selection of isolates belonging to the ST-32 clonal complex. A pipeline for the population annotation of WGS has been developed (Fig. 1) combining the use of: i) the Bacterial Isolate Genome Sequence platform (BIGSdb) hosted on the www.pubmlst.org/neisseria database which currently enables the curation of over 2000 Neisseria genes and, ii) the prokaryotic annotation tool, prokka for novel gene discovery (Seemann, 2014). Through comparison with reference genomes, the Lineage 5 core genome (Lineage 5 cgMLST) was defined and compared between isolates revealing three distinct clusters of isolates grouping by PorA type within which small localised clusters were also visible. Novel gene discovery identified the Lineage 5 pan genome (Lineage 5 pgMLST) and included type IV secretion systems (T4SS), haptoglobin–haemoglobin receptors associated with iron acquisition, as well as a gonococcal conjugative plasmid.