La vanguardia y la “otra vanguardia”

Typhoid fever is a human systemic infection that is caused by Salmonella enterica serovar Typhi (S. Typhi). This human-restricted and highly adapted pathogen is transmitted via the oral-faecal route. S. Typhi is responsible for 21.7 million infections and results in approximately 217,000 deaths worldwide annually (Crump & Mintz, 2010). The disease primarily causes acute systemic infection with life-threatening complications, and the recovering patient may develop into a chronic carrier state (Gonzalez-escobedo et al., 2011). The risk of developing gallbladder diseases, including carcinoma, is also higher among typhoid carriers (Crawford et al., 2010; Gonzalez-escobedo et al., 2011; Shukla et al., 2000).

Typhoid is endemic, with periodic outbreaks and sporadic cases occur in developing countries, particularly in Southeast Asia, South-central Asia, Latin America and Southern Africa where sanitary conditions are poor (Crump & Mintz, 2010). Among the 13 states of Malaysia, Kelantan has a significantly higher incidence of typhoid fever (MOH, 2006). A large typhoid outbreak occurred in Kelantan state, which resulted in 735 cases of infection and two deaths in a short period of three months (from April to June of 2005) (MOH, 2006). The representative clonal strain of this outbreak, S. Typhi BL196 was isolated from the blood samples of a severe typhoid case in Kelantan during the year 2005 notorious outbreak. In the year 2007, an S. Typhi strain CR0044 was isolated from a stool sample of an asymptomatic carrier in

Kelantan, Malaysia. This strain was highly similar to the outbreak strain BL196 by pulsed-field gel electrophoresis (PFGE) (Appendix B).

Human carriers are the main reservoirs of S. Typhi transmission, but the genetic basis, and the underlying mechanisms, in particular, are unclear (Holt et al., 2008). It has been suggested that a carrier strain will likely lack gene acquisition capabilities and have little fitness advantages compared with those strains causing symptomatic infections because the human reservoir is small and physiologically isolated (Holt et al., 2008; Vaishnavi et al., 2005). Although typhoid fever is endemic in many countries, including Malaysia, little is known about the mechanism of survival and persistence of S. Typhi in the host. Therefore, it is of great interest to know as to what extent these closely related strains differed or shared in its genomic contents despite being isolated from these two distinct epidemiological settings. It was hypothesized that the genome sequences of the underlying strains would provide more insights to enhance understanding of endemicity or persistence of typhoid in Kelantan. On the contrary, an S. Typhi strain, ST0208 was isolated from the stool sample of a typhoid fever patient admitted to University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia, in 2008. The strain was characterized by pulsed-field gel electrophoresis (PFGE), repetitive extragenic palindromic (REP)-PCR, and antimicrobial susceptibility profiling (Tiong et al., 2010). PFGE analyses showed that this strain is more distantly related to the outbreak and carrier strains, but the epidemiological link is unknown. The genome sequence of this sporadically associated strain will provide insights into possible genetic events that would confer a fitness advantage.

Recent whole-genome sequencing of S. Typhi has demonstrated that the pathogen shows limited genetic variation with little evidence of purifying selection, antigenic variation or recombination between isolates (Achtman, 2008; Holt et al.,

severity in different regions (Parry et al., 2002). Previous PFGE studies have also demonstrated genome size variations and distinct PFGE patterns in relation to fatal and non-fatal typhoid cases (Thong, et al., 1996; Thong et al., 1997). Although the health conditions of the host cannot be completely ruled out, various reports have suggested that the gain and loss of genes through mutations and gene transfers that have occurred independently in different lineages have markedly contributed to the varying pathogenic potentials (Fraser-liggett, 2005; Holt et al., 2008). However, these important factors are poorly understood because there is limited genomic information for S. Typhi, mainly involving strains that are associated with diverse epidemiological settings. Its genomic heterogeneity is likely due to the adaptation of the pathogen to the host and its exposure to mobile elements, such as bacteriophages (Dobrindt & Hacker, 2001). Organisms having common core genomes could differ in their dispensable (strain-specific) genes, reflecting their unique physiological and virulence properties (Ahmed, Dobrindt, Mayne, Wright, & Cartmel, 2008; Wren, 2000). Although not all genetic variations are essential for adaptation, some dispensable genes are believed to be responsible for conferring fitness advantages to the pathogen to thrive in its host. Horizontal gene transfer is also thought to be the predominant force in bacterial evolution, which contributes to novel gene acquisition. The acquired genes provide new characteristics, which either aid in host adaptation and persistence or enhance virulence capabilities (Dobrindt & Hacker, 2001; Lawrence, Ochman, & Ragan, 2002; Ahmed et al., 2008; Wren, 2000). A previous study on the pan-genome of Salmonella enterica revealed that the pan-genome (total known genomic content) of all strains will continue to increase as new genomes are sequenced (Medini, Donati, Tettelin, Masignani, & Rappuoli, 2005). With the availability of robust next-generation sequencing technologies, high-quality whole genome sequences can be generated and analysed, which will be especially useful for capturing fine variations among highly conserved S.

Typhi strains. Multiple whole-genome sequence comparisons of closely related strains will not only lead to the better understanding of their relationships but also provide novel insights into the functional roles of strain-specific genes.

In this study, detailed and comprehensive comparative functional analyses were performed on three sequenced genomes of S. Typhi strains that were isolated from typhoid patients during a massive outbreak in 2005, a sporadic case in 2008 and an asymptomatic carrier from Malaysia, where typhoid is endemic. These Malaysian S. Typhi strains were compared with previously published S. Typhi genomes with the following aims: 1) to determine and describe the genomes signatures and conserved and unique regions of the strains that were being studied; 2) to elucidate the phylogeny and genetic relatedness of these Malaysian strains compared with 17 other published strains using phylogenomic analysis; 3) to compare those strains that have been associated with various epidemiological settings (outbreak, carrier, and sporadic cases), and particularly the regions of plasticity that may contribute to the varying pathogenic potentials; 4) to identify potential novel pathogenic factors that are harboured by the analysed strains; 5) to provide insight into the possible differential functionalities of the genes, focusing mainly on virulence- and persistence-related genes based on non- synonymous SNPs of closely related strains and particularly on carrier strains to gain insight into the persistence of the carrier state; and 6) to understand how the potential nsSNPs affect protein structures and functions. The data that are generated will be useful for the profiling of strains, marker development and the increased understanding of outbreak, sporadic and less studied asymptomatic typhoid carriage infection.