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

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 Serovar Enteritidis, England and Wales, 1945–2011

In England and Wales, the emergence of Salmonella enterica serovar Enteritidis resulted in the largest and most persistent epidemic of foodborne infection attributable to a single subtype of any pathogen since systematic national microbiological surveillance was established. We reviewed 67 years of surveillance data to examine the features, underlying causes, and overall effects of S. enterica ser. Enteritidis. The epidemic was associated with the consumption of contaminated chicken meat and eggs, and a decline in the number of infections began after the adoption of vaccination and other measures in production and distribution of chicken meat and eggs. We estimate that >525,000 persons became ill during the course of the epidemic, which caused a total of 6,750,000 days of illness, 27,000 hospitalizations, and 2,000 deaths. Measures undertaken to control the epidemic have resulted in a major reduction in foodborne disease in England and Wales

Identification of Salmonella for public health surveillance using whole genome sequencing

In April 2015, Public Health England implemented whole genome sequencing (WGS) as a routine typing tool for public health surveillance of Salmonella, adopting a multilocus sequence typing (MLST) approach as a replacement for traditional serotyping. The WGS derived sequence type (ST) was compared to the phenotypic serotype for 6,887 isolates of S. enterica subspecies I, and of these, 6,616 (96%) were concordant. Of the 4% (n = 271) of isolates of subspecies I exhibiting a mismatch, 119 were due to a process error in the laboratory, 26 were likely caused by the serotype designation in the MLST database being incorrect and 126 occurred when two different serovars belonged to the same ST. The population structure of S. enterica subspecies II–IV differs markedly from that of subspecies I and, based on current data, defining the serovar from the clonal complex may be less appropriate for the classification of this group. Novel sequence types that were not present in the MLST database were identified in 8.6% of the total number of samples tested (including S. enterica subspecies I–IV and S. bongori) and these 654 isolates belonged to 326 novel STs. For S. enterica subspecies I, WGS MLST derived serotyping is a high throughput, accurate, robust, reliable typing method, well suited to routine public health surveillance. The combined output of ST and serovar supports the maintenance of traditional serovar nomenclature while providing additional insight on the true phylogenetic relationship between isolates

Packed with Salmonella--investigation of an international outbreak of Salmonella Senftenberg infection linked to contamination of prepacked basil in 2007

Salmonella Senftenberg is uncommon in the United Kingdom. In January-June 2007, the Health Protection Agency reported on 55 primary human cases of Salmonella Senftenberg in England and Wales. In May 2007, fresh basil sold in the United Kingdom was found to be contaminated with Salmonella Senftenberg. We launched an investigation to elucidate the cause of this outbreak. Isolates were examined using plasmid profiling and pulsed-field gel electrophoresis, and the outbreak strain (SSFTXB.0014) was identified. We enquired via Enter-net whether other countries had isolated the outbreak strain, analyzed samples of fresh herbs from U.K. retailers, and interviewed patients on food history. Thirty-two patient-cases were referred to this outbreak in England and Wales. Onsets of illness occurred between 5 March and 6 June 2007. Fifty-six percent of patient-cases were females and 90% adults (>20 years old); three were admitted to hospital as a result of Salmonella infection. Scotland, Denmark, the Netherlands, and the United States reported on 19 cases of Salmonella Senftenberg infection presenting with the outbreak strain since January 2007. Eight samples of prepacked fresh basil imported from Israel tested positive with the same strain. A minority of patients could recall the consumption of basil before illness, and some reported consumption of products where basil was a likely ingredient. Environmental investigations in Israel did not identify the contamination source. Microbiological evidence suggested an association between contamination of fresh basil and the cases of Salmonella Senftenberg infection, leading to withdrawal of basil from all potentially affected batches from the U.K. market