Retrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar Typhi outbreak in Zambia during 2010 to 2012. Ninety-four isolates were susceptibility tested by MIC determinations. Whole genome sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the MLST, haplotype, plasmid replicon, antimicrobial resistance genes, and the genetic relatedness by Single Nucleotide Polymorphism (SNP) analysis and genomic deletions. The outbreak affected 2,040 patients with a fatality rate of 0.5%. Most isolates (83.0%) were multi-drug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the haplotype; H58B. Most isolates contained a chromosomally translocated region containing seven antimicrobial resistance genes; catA1, blaTEM-1, dfrA7, sul1, sul 2, strA, and strB, fragments of incQ1plasmid replicon, class 1 integron, and the mer operon. The genomic analysis revealed an overall 415 SNPs difference and 35 deletions among 33 of the isolates whole genome sequenced. In comparison with other genomes of H58, the Zambian isolates separated from genomes from Central Africa and India with 34 and 52 SNPs, respectively. The phylogenetic analysis indicates that 32 isolates of the 33 sequenced belonged to a tight clonal group, distinct from other H58 genomes included in the study. The small numbers of SNPs identified within this group are consistent with short-term transmission that can be expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for the outbreak during which occasional other S. Typhi lineages including sensitive ones continued to co-circulate. The common view is that the emerging global S. Typhi haplotype; H58B, containing the MDR incHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-Saharan Africa; we found that a new variant of the haplotype harbouring a chromosomally translocated region containing the MDR islands of incHI1 plasmid emerged in Zambia. This could chance the perception of the term "classical MDR typhoid" currently being solely associated with the incHI1 plasmid. It might be more common than anticipated that S. Typhi haplotype; H58B harbour either the incHI1 plasmid and /or a chromosomally translocated MDR region.
Whole genome sequencing (WGS) shows great potential for real-time monitoring and identification of infectious disease outbreaks. However, rapid and reliable comparison of data generated in multiple laboratories and using multiple technologies is essential. So far studies have focused on using one technology because each technology has a systematic bias making integration of data generated from different platforms difficult. We developed two different procedures for identifying variable sites and inferring phylogenies in WGS data across multiple platforms. The methods were evaluated on three bacterial data sets and sequenced on three different platforms (Illumina, 454, Ion Torrent). We show that the methods are able to overcome the systematic biases caused by the sequencers and infer the expected phylogenies. It is concluded that the cause of the success of these new procedures is due to a validation of all informative sites that are included in the analysis. The procedures are available as web tools.
Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly 'real-time' monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS) shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE revealing that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.
Multidrug-resistant Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02-03/002 (?st313-td) followed by complementation (02-03/002-C). ?st313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n?=?82) and 100% of S. Dublin strains (n?=?50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n?=?82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313.
The comparison of the publicly available genomes of the virulent Salmonella serovar Typhimurium strains SL1344, 14028s, D23580 to that of the virulence attenuated isolate LT2 revealed the absence of a full sequence of bacteriophage ST64B in the latter. Four selected ST64B regions of unknown function (sb7-sb11, sb46, sb49-sb50, and sb54) were mapped by PCR in two strains collections; 1) 310 isolates of S. Typhimurium from human blood or stool samples and from food, animal and environmental reservoirs, and 2) 90 isolates belonging to other serovars. The region sb49-sb50 was found to be unique to S. Typhimurium and was strongly associated to strains isolated from blood samples (100% and 28.4% of the blood and non-blood isolates, respectively). The region was cloned into LT2 and knocked out in SL1344 and these strains were compared to wild type isogenic strains in in vitro assays used to predict virulence association. No difference in invasion of the Int407 human cell line was observed between the wild type and mutated strains, but the isolate carrying the whole ST64B prophage was found to have a slightly better survival in blood. The study showed a high prevalence and a strong association between the prophage ST64B and isolates of S. Typhimurium collected from blood, and may indicate that such strains constitute a selected subpopulation within this serovar. Further studies are indicated to determine whether the slight increase in blood survival observed in the strain carrying ST64B genes is of paramount for systemic infections.
White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSV-WSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ?28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein-protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture.
Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria.
Many species of marine shrimp in the Family Penaeidae, viz. Penaeus (Litopenaeus) vannamei, Penaeus monodon, Penaeus (Fenneropenaeus) chinensis, and Penaeus (Marsupenaeus) japonicus, are animals of economic importance in the aquaculture industry. Yet information about their DNA and protein sequences is lacking. In order to predict their collective proteome, we combined over 270,000 available EST and cDNA sequences from the 4 shrimp species with all protein sequences of Drosophila melanogaster and Caenorhabditis elegans. EST data from 4 other crustaceans, the crab Carcinus maenas, the lobster Homarus americanus (Decapoda), the water flea Daphnia pulex, and the brine shrimp Artemia franciscana were also used.
The advances and decreasing economical cost of whole genome sequencing (WGS), will soon make this technology available for routine infectious disease epidemiology. In epidemiological studies, outbreak isolates have very little diversity and require extensive genomic analysis to differentiate and classify isolates. One of the successfully and broadly used methods is analysis of single nucletide polymorphisms (SNPs). Currently, there are different tools and methods to identify SNPs including various options and cut-off values. Furthermore, all current methods require bioinformatic skills. Thus, we lack a standard and simple automatic tool to determine SNPs and construct phylogenetic tree from WGS data.
Two cases of extremely drug-resistant Salmonella enterica serovar Senftenberg isolated from patients in Zambia were investigated by utilizing MIC determinations and whole-genome sequencing. The isolates were resistant to, and harbored genes toward, nine drug classes, including fluoroquinolones and extended-spectrum cephalosporins, contained two plasmid replicons, and differed by 93 single-nucleotide polymorphisms.
More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with ?(70) (including phoP, slyA, and invF) from which we identified the -10 and -35 motifs of ?(70)-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.
Technological advances in high throughput genome sequencing are making whole genome sequencing (WGS) available as a routine tool for bacterial typing. Standardized procedures for identification of relevant genes and of variation are needed to enable comparison between studies and over time. The core genes--the genes that are conserved in all (or most) members of a genus or species--are potentially good candidates for investigating genomic variation in phylogeny and epidemiology.
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