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Many of the developing countries of the Southeast Asian region are significantly affected by endemic typhoid fever, possibly as a result of marginal living standards. It is an important public health problem in countries such as Papua New Guinea, which is geographically close to some of the foci of endemicity in Asia. The severity of the disease varies in different regions, and this may be attributable to genetic diversity among the native strains. Genome sequence data on strains from different countries are needed to clearly understand their genetic makeup and virulence potential. We describe the genomes of two Salmonella Typhi isolates from patients with fatal and nonfatal cases of typhoid fever in Papua New Guinea. We discuss in brief the underlying sequencing methodology, assembly, genome statistics, and important features of the two draft genomes, which form an essential step in our functional molecular infection epidemiology program centering on typhoid fever. The comparative genomics of these and other isolates would enable us to identify genetic rearrangements and mechanisms responsible for endemicity and the differential severity of pathogenic salmonellae in Papua New Guinea and elsewhere.
Typhoid fever is a major pestilence in the developing world (20), and its prevalence is significant (1,000 cases per 100,000 individuals per year) in Papua New Guinea, which is geographically close to Southeast Asia (15). DNA profiling, which was used previously (6, 14, 20, 21, 22), could not fully explore genome diversity, as Salmonella enterica serovar Typhi isolates from Papua New Guinea showed limited heterogeneity, perhaps because of recent clonal expansion from a single endemic/ancestral strain (the disease was rarely seen before 1985) (17, 20). Minimal selection pressure and confinement to a specific geographical region might explain this limited genetic diversity (21, 22) despite horizontal gene transfer (13).
We hypothesized that the genome sequences of Salmonella Typhi isolates from patients with typhoid fever due a fatal strain (UJ308A) or a nonfatal strain (UJ816A) would provide significant insights into the association among disease phenotypes and strain characteristics. Two such strains were isolated from blood samples from patients and were found to be sensitive to common antibiotics. Strain UJ308A (phage type VS1) was obtained from a patient who died of typhoid, while UJ816A (phage type DI) was from a patient who recovered.
The 73-bp paired-end sequence data (insert size, 300 bp) were determined with an Illumina Genome Analyzer (GA2x, pipeline version l.6). About 95× and 105× coverage was achieved for strains UJ308A and UJ816A, respectively, comprising 1.9 and 2.0 Gb of data, respectively. De novo assembly was done as described previously (1, 2, 4, 8, 19); initial assembly generated 416 and 335 contigs for UJ308A and UJ816A, respectively, using Velvet (23) with a hash length of 39. The scaffolds were generated from contigs by using SSPACE (5) and further assembled and curated to give a consensus draft. The following statistics were gleaned upon analysis at RAST (3). The sizes of the chromosomes for UJ308A and UJ816A were approximately 4,724,875 and 4,736,723 bp, respectively, with a G+C contents of 51.89 and 51.94%, respectively. The coding percentage for both strains was ~86.8%; UJ308A and UJ816A contained approximately 4,720 and 4,710 protein coding sequences with average lengths of 869 and 873 bp, respectively. The data were further validated by Glimmer (7) and EasyGene (12). RNAmmer (11) revealed that the genome of UJ308A has 78 tRNA and 21 rRNA genes and the genome of UJ816A contains 77 tRNA and 22 rRNA genes. All of the major virulence markers encoded by pathogenicity islands and the genes relevant to the assembly of a type III secretion system (16) were identified in both the strains. The Vi antigen (10, 18), which plays major role in immune evasion, was present in both strains, as in Salmonella Typhi CT18 (16). The homologues of Campylobacter toxin cdtB and Bordetella pertussis toxin (9) were also present.
In view of this, further efforts are needed to determine the true extent of strain diversity in terms of (i) gene gains and losses over an evolutionary time scale, (ii) geographic gene flow, (iii) core versus accessory genome dynamics, (iv) virulence acquisition and attenuation, and (v) the preponderance of highly virulent versus “docile” strains across the regions of Asia where typhoid fever is endemic.
The GenBank accession numbers for the genomes reported here are AJTD00000000 (UJ308A) and AJTE00000000 (UJ816A).
We thankfully acknowledge support received from the University of Malaya High Impact Research Grant (UM.C/625/1HIR/MOHE/02 [A000002-5000 1]) Molecular Genetics. This genome program was completed under the wider umbrella of the Indo-German International Research Training Group, Internationales Graduiertenkolleg (GRK1673), Functional Molecular Infection Epidemiology, an initiative of the German Research Foundation (DFG) and the University of Hyderabad (India), of which N.A. is a speaker. N.A. is an Adjunct Professor of Molecular Biosciences at the University of Malaya, Kuala Lumpur, Malaysia, and an Adjunct Professor of Chemical Biology at the Institute of Life Sciences, Hyderabad, India. We are also grateful to M/s Genotypic Technology Pvt. Ltd., Bengaluru, India, for their untiring efforts with Illumina sequencing. We acknowledge the Bioinformatics Facility (BIF) at the Department of Biotechnology, University of Hyderabad, for the use of its computational infrastructure. We are thankful to Akash Ranjan for enabling access to the high-speed computing infrastructure at CDFD, Hyderabad.
All of the members of the Ahmed and Thong labs are gratefully acknowledged for their help and support.