Serovar Typhi is a young, genetically monomorphic bacterial pathogen that has not been in existence long enough to generate extensive sequence polymorphisms (15
). Here, we have investigated the population structure of serovar Typhi associated with Indonesia by using SNP typing and flagellin-based PCR analysis. In the collections we identified nine haplotypes composed of two dominant and seven less common haplotypes. Our data show that in a relatively small area (~10.5 km2
) there are several different haplotypes of serovar Typhi circulating within the local population, suggesting the existence of independent transmission clusters. These data are in agreement with other studies, which report multiple strain types circulating within a specific location (28
). Furthermore, we identified bacteria of the same haplotype isolated over a 30-year period, providing further support for a vital role of persistent carriage and/or prolonged dissemination of serovar Typhi in the environment.
Typhoid fever remains a significant public health problem in many parts of Southeast Asia. Dissemination of serovar Typhi is of particular importance considering the wave of multiple drug-resistant strains that is currently spreading across Asia and the movement of people associated with current rapid economic growth. There is a pressing need for more effective epidemiological scrutiny of this organism, enabling better understanding of the nature of spread, which will ultimately facilitate control policies. Molecular methods appear to be the most robust, but such methods require standardization for the communication and analysis of the resulting data. The SNP-based approach is reproducible, results are applicable in any setting, and the data can be integrated into datasets produced in multiple laboratories using a variety of SNP detection technologies. This is in contrast to other schemes that detect strain differences but produce variable results in different laboratory settings. In addition, such schemes typically provide little or no information on phylogeny or phenotype and are unable to identify clonal expansion in a region.
The high-throughput system utilized here has been used to perform MLST on a number of Neisseria meningitidis
reference strains (12
). We have demonstrated the effectiveness of the same platform for detecting haplotypes in a genetically monomorphic gram-negative bacterial pathogen. Further flexibility can be anticipated when additional sensitivity is incorporated after more SNP variation is discovered.
Although the system has benefits, high-throughput SNP typing is currently not suitable for routine use in laboratories without specific infrastructure or financial support to run such assays. However, SNP detection technology has been driven in recent years by the need for massive throughput (currently >1 million SNPs for human genotyping studies). Microbial genotyping is much less demanding, with studies such as this providing epidemiologically informative data by assaying fewer than 100 SNP loci. With current high-throughput SNP detection technology, running costs for bacterial genotyping assays should be feasible for clinical laboratories, although setup costs may be prohibitive in some settings. The development of novel medium-throughput SNP detection technologies will be an important step toward the adoption of SNP-based microbial genotyping in clinical laboratories worldwide.
A practical solution for short-term development would be to house SNP typing facilities in regional reference laboratories linked via the internet through a global database. Simple “in the field” assays can also be established to detect a subset of discriminatory SNPs using a targeted approach. For example, MLST is performed routinely in many laboratories, while PCR analysis of nine gene fragments would have detected all of the haplotypes circulating in the present study. The resulting data would provide information about population structure in a particular region. Indeed, we are currently developing such a method based on PCR that can distinguish many of the major serovar Typhi haplotypes, which could potentially be universally adopted in clinical and research laboratories alike. However, it is important to note that the validity of this kind of simplified genotyping scheme depends on prior knowledge of the underlying phylogeny provided by comprehensive SNP typing studies.
It was originally hypothesized that z66+
serovar Typhi isolates from Indonesia were the precursor to global serovar Typhi, and it was assumed that the possession of a second phase of the flagella antigen was an ancestral state which has subsequently been lost (19
). Conversely, an “out of Africa” hypothesis was proposed as the global source of Typhi (25
), which would predict that the z66 gene was acquired later, possibly by horizontal gene transfer. We now know that z66 is present in only a single haplotype, and that the z66 flagellin gene [fljB
(z66)] and fliC
(z66)] are located on a plasmid, indicating a relatively recent origin (1
). The present study demonstrates that the acquisition of the pBSSB1 linear plasmid permitting the expression of z66 antigen most likely occurred only once and does not readily transfer to other genotypes. Furthermore, we suggest that pBSSB1 was horizontally transferred into an serovar Typhi strain carrying the d fliC
allele, and over time the gene became truncated in some strains to form the j flagellin epitope. Expression of the z66 antigen is the default in serovar Typhi stains harboring the fljB
(z66) locus, and fliC
is effectively silenced (2
). The truncated j allele can be found in three haplotypes, i.e., H50, H59, and H85, suggesting either that deletion has occurred spontaneously on several independent occasions or that homologous recombination has taken place between different haplotypes. However, it seems that strains harboring fljB
(z66) preferentially possess the j fliC
In conclusion, our results demonstrate the adaptation of a global serovar Typhi genotyping scheme for a single country and ultimately a localized typhoid endemic region. The methodology offers a high level of sensitivity, thus allowing interrogation of phenotypes or pinpointing the locality of specific strains. The technique can in principle be used to define serovar Typhi circulating globally and is also potentially applicable to other bacterial pathogens. Our data prove that a number of distinct haplotypes of serovar Typhi can circulate in relatively small geographical area. However, somewhat paradoxically, we found that H58 strains that are currently circulating in other parts of South Asia and are associated with multiple drug resistance were not detected in Indonesia. Furthermore, strains harboring the z66 antigen are associated with the Indonesian archipelago, there is no evidence for the spread of these organisms to other countries where typhoid is endemic. It is likely that understanding the “Indonesian exception” would aid our understanding of the global epidemiology of typhoid fever. By combining haplotyping with phenotyping we were able to gain considerable insight into the population structure of serovar Typhi circulating in this region. Surveillance of strains using these methods combined with assessment of social and medical practices over a prolonged period will add vital information on how serovar Typhi is evolving and spreading in the human population.