Towards the development of a DNA-sequence based approach to serotyping of Salmonella enterica

BACKGROUND: The fliC and fljB genes in Salmonella code for the phase 1 (H1) and phase 2 (H2) flagellin respectively, the rfb cluster encodes the majority of enzymes for polysaccharide (O) antigen biosynthesis, together they determine the antigenic profile by which Salmonella are identified. Sequencing and characterisation of fliC was performed in the development of a molecular serotyping technique. RESULTS: FliC sequencing of 106 strains revealed two groups; the g-complex included those exhibiting "g" or "m,t" antigenic factors, and the non-g strains which formed a second more diverse group. Variation in fliC was characterised and sero-specific motifs identified. Furthermore, it was possible to identify differences in certain H antigens that are not detected by traditional serotyping. A rapid short sequencing assay was developed to target serotype-specific sequence motifs in fliC. The assay was evaluated for identification of H1 antigens with a panel of 55 strains. CONCLUSION: FliC sequences were obtained for more than 100 strains comprising 29 different H1 alleles. Unique pyrosequencing profiles corresponding to the H1 component of the serotype were generated reproducibly for the 23 alleles represented in the evaluation panel. Short read sequence assays can now be used to identify fliC alleles in approximately 97% of the 50 medically most important Salmonella in England and Wales. Capability for high throughput testing and automation give these assays considerable advantages over traditional methods

A multi-country outbreak of Salmonella newport gastroenteritis in Europe associated with watermelon from Brazil, confirmed by whole genome sequencing:October 2011 to January 2012

In November 2011, the presence of Salmonella Newport in a ready-to-eat watermelon slice was confirmed as part of a local food survey in England. In late December 2011, cases of S. Newport were reported in England, Wales, Northern Ireland, Scotland, Ireland and Germany. During the outbreak, 63 confirmed cases of S. Newport were reported across all six countries with isolates indistinguishable by pulsed-field gel electrophoresis from the watermelon isolate. A subset of outbreak isolates were whole-genome sequenced and were identical to, or one single nucleotide polymorphism different from the watermelon isolate. In total, 46 confirmed cases were interviewed of which 27 reported watermelon consumption. Further investigations confirmed the outbreak was linked to the consumption of watermelon imported from Brazil. Although numerous Salmonella outbreaks associated with melons have been reported in the United States and elsewhere, this is the first of its kind in Europe. Expansion of the melon import market from Brazil represents a potential threat for future outbreaks. Whole genome sequencing is rapidly becoming more accessible and can provide a compelling level of evidence of linkage between human cases and sources of infection, to support public health interventions in global food markets

A multi-country outbreak of Salmonella Newport gastroenteritis in Europe associated with watermelon from Brazil, confirmed by whole genome sequencing: October 2011 to January 2012

In November 2011, the presence of Salmonella Newport in a ready-to-eat watermelon slice was confirmed as part of a local food survey in England. In late December 2011, cases of S. Newport were reported in England, Wales, Northern Ireland, Scotland, Ireland and Germany. During the outbreak, 63 confirmed cases of S. Newport were reported across all six countries with isolates indistinguishable by pulsed-field gel electrophoresis from the watermelon isolate. A subset of outbreak isolates were whole-genome sequenced and were identical to, or one single nucleotide polymorphism different from the watermelon isolate. In total, 46 confirmed cases were interviewed of which 27 reported watermelon consumption. Further investigations confirmed the outbreak was linked to the consumption of watermelon imported from Brazil. Although numerous Salmonella outbreaks associated with melons have been reported in the United States and elsewhere, this is the first of its kind in Europe. Expansion of the melon import market from Brazil represents a potential threat for future outbreaks. Whole genome sequencing is rapidly becoming more accessible and can provide a compelling level of evidence of linkage between human cases and sources of infection, to support public health interventions in global food markets

What's in a Name? Species-Wide Whole-Genome Sequencing Resolves Invasive and Noninvasive Lineages of Salmonella enterica Serotype Paratyphi B

For 100 years, it has been obvious that Salmonella enterica strains sharing the serotype with the formula 1,4,[ 5], 12: b:1,2-now known as ParatyphiB-can cause diseases ranging from serious systemic infections to self-limiting gastroenteritis. Despite considerable predicted diversity between strains carrying the common Paratyphi B serotype, there remain few methods that subdivide the group into groups that are congruent with their disease phenotypes. Paratyphi B therefore represents one of the canonical examples in Salmonella where serotyping combined with classical microbiological tests fails to provide clinically informative information. Here, we use genomics to provide the first high-resolution view of this serotype, placing it into a wider genomic context of the Salmonella enterica species. These analyses reveal why it has been impossible to subdivide this serotype based upon phenotypic and limited molecular approaches. By examining the genomic data in detail, we are able to identify common features that correlate with strains of clinical importance. The results presented here provide new diagnostic targets, as well as posing important new questions about the basis for the invasive disease phenotype observed in a subset of strains. IMPORTANCE Salmonella enterica strains carrying the serotype Paratyphi B have long been known to possess Jekyll and Hyde characteristics; some cause gastroenteritis, while others cause serious invasive disease. Understanding what makes up the population of strains carrying this serotype, as well as the source of their invasive disease, is a 100-year-old puzzle that we address here using genomics. Our analysis provides the first high-resolution view of this serotype, placing strains carrying serotype Paratyphi B into the wider genomic context of the Salmonella enterica species. This work reveals a history of disease dating back to the middle ages, caused by a group of distinct lineages with various abilities to cause invasive disease. By quantifying the key genomic differences between the invasive and noninvasive populations, we are able to identify key virulence-related targets that can form the basis of simple, rapid, point-of-care tests.Peer reviewe

Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella

Background: Foodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 patients in our hospital. This outbreak was linked to community, national and European-wide cases. Hospital patients with Salmonella are at high risk, and require a rapid response. We initially investigated this outbreak by whole-genome sequencing using a novel rapid protocol on the Illumina MiSeq; we then integrated these data with whole-genome data from surveillance sequencing, thereby placing the outbreak in a national context. Additionally, we investigated the potential of a newly released sequencing technology, the MinION from Oxford Nanopore Technologies, in the management of a hospital outbreak of Salmonella. Results: We demonstrate that rapid MiSeq sequencing can reduce the time to answer compared to the standard sequencing protocol with no impact on the results. We show, for the first time, that the MinION can acquire clinically relevant information in real time and within minutes of a DNA library being loaded. MinION sequencing permits confident assignment to species level within 20 min. Using a novel streaming phylogenetic placement method samples can be assigned to a serotype in 40 min and determined to be part of the outbreak in less than 2 h. Conclusions: Both approaches yielded reliable and actionable clinical information on the Salmonella outbreak in less than half a day. The rapid availability of such information may facilitate more informed epidemiological investigations and influence infection control practices

Studies on the activation of azo-dyes into direct-acting genotoxic agents by enterococcus faecalis.

The ability of the intestinal bacterium Enterococcus faecalis to reduce a range of azo dyes was studied and the optimal pH and temperature for azoreductase activity were determined. The rate of dye reduction was highly variable between dyes. No obvious correlation existed between chemical structure of dye and reduction rate. Both glucose and lactate acted as electron donors for dye reduction. lodoacetate, HQNO and PCMB inhibited azo reduction whereas sodium azide did not, thus demonstrating that azoreduction involves a flavin-based electron transport system. The genotoxic and mutagenic activity of the dyes was investigated prior to and following reduction by Ent. faecalis using a variety of assays. Again there was considerable variation among the dyes tested with regard to the production of genotoxic and mutagenic agents. Although the majority of dyes showed little or no mutagenicity in the Salmonella/mutagenicity assay (Ames test), the M13 mutagenesis assay and the SOS Chromotest many were active using the Fluctuation assay. These dyes were also highly genotoxic using a differential killing assay with repair-proficient and repair-deficient E. coli strains. Although it was thought that reductive cleavage of the azo-compound generates metabolites that are able to damage DNA, activation was not always dependent on dye reduction by Ent. faecalis. While mutagenicity was not observed in the M13 mutagenesis assay the rate of bacteriophage transfection was lowered after exposure to the dyes under certain conditions, which may again be a reflection of DNA damage. The precise nature and significance of this damage in terms of toxicity and/or carcinogenicity remains to be determined. The electrochemical characteristics of the azo dyes were studied in aqueous buffer systems. Upon electrolytic reduction of the dyes a single irreversible reduction process was observed when using repeat scan cyclic voltammetry (CV). The reduction potentials of the first forward wave could be roughly correlated with the bacterial metabolism of the dyes as measured spectrophotometrically. Although a reverse wave for the azo-reduction process was absent, an associated oxidation wave was frequently observed in the return sweep of the CV due to the formation of a new redox-active species. No data has been obtained to suggest that the damaging compound produced upon reductive cleavage of the azo bond or the mechanism of genotoxicity is related to the new redox-active complex

Salmonella enterica subspecies II infections in England and Wales--the use of multilocus sequence typing to assist serovar identification

Identifying rare Salmonella serotypes by conventional serotyping can be a problem for diagnostic or reference microbiology laboratories. We report two cases of the seldom encountered serovar Salmonella Dubrovnik, which is known as Salmonella subspecies II 41 : z : 1,5, in an elderly man and a young child. Multilocus sequence typing, a technique that is being used more frequently in our laboratory, was used to assist serovar identification as serotyping proved to be inconclusive. To our knowledge, these cases were not linked geographically and there were no known associations between them although they occurred within a very short time of each other

Characterization of new Salmonella serovars by whole-genome sequencing and traditional typing techniques

International audienceSerotyping forms the basis of all national and international surveillance networks for Salmonella. Public health microbiology is currently being transformed by high-throughput DNA sequencing, which opens the door to serovar determination using this powerful technique. Twenty-nine Salmonella isolates referred to the Public Health England between 1994 and 2004 for serovar identification were selected for this study, and they all presented with novel antigenic formulae. Results from a combination of traditional phenotypic and molecular assays were compared. Twenty-two isolates (76 %) were subsequently independently confirmed as new types; of these, 18 (82 %) were grouped as Salmonellaenterica subspecies I, and four (18 %) were S. enterica subspecies II. In general, it is shown that there is concordance between the DNA sequence type and traditional phenotypic serotype, but it would be necessary to analyse a larger data set to confirm this. Traditional multilocus sequence typing (MLST) by Sanger sequencing also correlates to insilico whole-genome sequencing MLST. This permits the continuation of traditional serovar nomenclature alongside sequence type methods and enhances the ability to infer true phylogenetic relationships between isolates

Emergence and Characterization of Salmonella enterica Serovar Typhimurium Phage Type DT191a

The emergence of a previously undefined phage type of Salmonella enterica serovar Typhimurium, designated DT191a, occurred in England and Wales in July 2008. The new strain exhibits a number of distinctive phenotypic and genotypic features. This report provides the tools necessary to track S. Typhimurium DT191a globally