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Single nucleotide polymorphism typing of global Salmonella enterica serovar Typhi isolates by use of a hairpin primer real-time PCR assay.

ABSTRACT: Salmonella enterica serovar Typhi is highly homogeneous. Single nucleotide polymorphisms (SNPs) have been shown to be valuable markers for molecular typing of S. enterica serovar Typhi. Here, we used a hairpin primer real-time PCR assay for SNP typing of S. enterica serovar Typhi isolates. Forty-two SNPs were selected from a comparison of 19 published S. enterica serovar Typhi genomes and sequences from other studies. The SNPs were used to type 71 global S. enterica serovar Typhi isolates and differentiated these S. enterica serovar Typhi isolates and the 19 genome sequenced strains into 25 SNP profiles. Phylogenetic analysis revealed that these SNP profiles were grouped into six major clusters. These clusters can be identified by using five SNPs, while the full differentiation of the 25 SNP profiles requires a minimum of 24 SNPs. This real-time PCR-based SNP typing method will be useful for global epidemiological analysis.

SUBMITTER: Octavia S

PROVIDER: S-EPMC2953126 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Single nucleotide polymorphism typing of global Salmonella enterica serovar Typhi isolates by use of a hairpin primer real-time PCR assay.

Octavia Sophie S Lan Ruiting R

Journal of clinical microbiology 20100728 10

Salmonella enterica serovar Typhi is highly homogeneous. Single nucleotide polymorphisms (SNPs) have been shown to be valuable markers for molecular typing of S. enterica serovar Typhi. Here, we used a hairpin primer real-time PCR assay for SNP typing of S. enterica serovar Typhi isolates. Forty-two SNPs were selected from a comparison of 19 published S. enterica serovar Typhi genomes and sequences from other studies. The SNPs were used to type 71 global S. enterica serovar Typhi isolates and di ...[more]

PMID: 20668124

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Project description:BackgroundTyphoid fever is an acute systemic infection of humans caused by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). In chronic carriers, the bacteria survive the harsh environment of the gallbladder by producing biofilm. The phenotype of S. Typhi biofilm cells is significantly different from the free-swimming planktonic cells, and studies have shown that they are associated with antibiotic resistance, immune system evasion, and bacterial persistence. However, the mechanism of this transition and the events leading to biofilm formation are unknown. High throughput sequencing was performed to identify the genes involved in biofilm formation and to postulate the mechanism of action.ResultsPlanktonic S. Typhi cells were cultured using standard nutrient broth whereas biofilm cells were cultured in a stressful environment using high shearing-force and bile to mimic the gallbladder. Sequencing libraries were prepared from S. Typhi planktonic cells and mature biofilm cells using the Illumina HiSeq 2500 platform, and the transcriptome data obtained were processed using Cufflinks bioinformatics suite of programs to investigate differential gene expression between the two phenotypes. A total of 35 up-regulated and 29 down-regulated genes were identified. The identities of the differentially expressed genes were confirmed using NCBI BLAST and their functions were analyzed. The results showed that the genes associated with metabolic processes and biofilm regulations were down-regulated while those associated with the membrane matrix and antibiotic resistance were highly up-regulated.ConclusionsIt is proposed that the biofilm phenotype of S. Typhi allows the bacteria to increase production of the membrane matrix in order to serve as a physical shield and to adhere to surfaces, and enter an energy conservation state in response to the stressful environment. Conversely, the planktonic phenotype allows the bacteria to produce flagella and increase metabolic activity to enable the bacteria to migrate and form new colonies of infection. This data provide a basis for further studies to uncover the mechanism of biofilm formation in S. Typhi and to discover novel genes or pathways associated with the development of the typhoid carrier state.

Project description:The pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever in humans, is mainly attributed to the acquisition of horizontally acquired DNA elements. Salmonella pathogenicity islands (SPIs) are indubitably the most important form of horizontally acquired DNA with respect to pathogenesis of this bacterium. The insertion or deletion of any of these transferrable SPIs may have impact on the virulence potential of S. Typhi. In this study, the virulence potential and genetic relatedness of 35 S. Typhi isolates, collected from 2004 to 2013 was determined by identification of SPI and non-SPI virulence factors through a combination of techniques including virulotyping, Whole Genome Sequencing (WGS), and Variable Number of Tandem Repeats (VNTR) profiling. In order to determine the virulence potential of local S. Typhi isolates, 56 virulence related genes were studied by PCR. These genes are located in the core as well as accessory genome (SPIs and plasmid). Major variations among studied virulence determinants were found in case of SPI-7 and SPI-10 associated genes. On the basis of presence of virulence related genes, the studied S. Typhi isolates from Pakistan were clustered into two virulotypes Vi-positive and Vi-negative. Interestingly, SPI-7 and SPI-10 were collectively absent or present in Vi-negative and Vi-positive strains, respectively. Two Vi-negative and 11 Vi-positive S. Typhi strains were also analyzed by whole genome sequencing (WGS) and their results supported the PCR results. Genetic diversity was tested by VNTR-based molecular typing. All 35 isolates were clustered into five groups. Overall, all Vi-negative isolates were placed in a single group (T5) whereas Vi-positive isolates were grouped into four types. Vi-negative and Vi-positive isolates were mutually exclusive. This is the first report on the comparative distribution of SPI and non-SPI related virulence genes in Vi-negative and Vi-positive S. Typhi isolates with an important finding that SPI-10 is absent in all Vi-negative isolates.

Dataset Information

Single nucleotide polymorphism typing of global Salmonella enterica serovar Typhi isolates by use of a hairpin primer real-time PCR assay.

Publications

Single nucleotide polymorphism typing of global Salmonella enterica serovar Typhi isolates by use of a hairpin primer real-time PCR assay.

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