Whole Genome Sequence Analysis of Salmonella Enteritidis PT4 Outbreaks from a National Reference Laboratory’s Viewpoint

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Introduction: In April and May 2014, two suspected egg-related outbreaks of Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) were investigated by the Belgian National Reference Laboratory of Foodborne Outbreaks. Both the suspected food and human isolates being available, and this for both outbreaks, made these the ideal case study for a retrospective whole genome sequencing (WGS) analysis with the goal to investigate the feasibility of this technology for outbreak investigation by a National Reference Laboratory or National Reference Centre without thorough bioinformatics expertise.

Methods: The two outbreaks were originally investigated epidemiologically with a standard questionnaire and with serotyping, phage typing, multiple-locus variable-number of tandem repeats analysis (MLVA) and antimicrobial susceptibility testing as classical microbiological methods. Retrospectively, WGS of six outbreak isolates was done on an Illumina HiSeq. Analysis of the WGS data was performed with currently available, user-friendly software and tools, namely CLC Genomics Workbench, the tools available on the server of the Center for Genomic Epidemiology and BLAST Ring Image Generator (BRIG).

Results: To all collected human and food outbreak isolates, classical microbiological investigation assigned phage type PT4 (variant phage type PT4a for one human isolate) and MLVA profile 3-10-5-4-1, both of which are common for human isolates in Belgium. The WGS analysis confirmed the link between food and human isolates for each of the outbreaks and clearly discriminated between the two outbreaks occurring in a same time period, thereby suggesting a non-common source of contamination. Also, an additional plasmid carrying an antibiotic resistance gene was discovered in the human isolate with the variant phage type PT4a.

Discussion: For the two investigated outbreaks occurring at geographically separated locations, the gold standard classical microbiological subtyping methods were not sufficiently discriminative to distinguish between or assign a common origin of contamination for the two outbreaks, while WGS analysis could do so. This case study demonstrated the added value of WGS for outbreak investigations by confirming and/or discriminating food and human isolates between and within outbreaks. It also proved the feasibility of WGS as complementary or even future replacing (sub)typing method for the average routine laboratory.

Whole Genome Sequencing for the Retrospective Investigation of an Outbreak of Salmonella Typhimurium DT 8

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Background:
Salmonella enterica serovar Typhimurium DT8 is uncommon within the European Union. An increase in this phage type was reported in the summer of 2013 in the States of Jersey.

Methods:
A total of 21 human cases with this phage type were microbiologically confirmed. Salmonella isolates from mayonnaise made using raw eggs were also confirmed as being Salmonella Typhimurium DT8. The epidemiological investigations strongly supported a link between mayonnaise consumption and illness. Whole genome sequencing (WGS) was used to retrospectively investigate this outbreak with a view to assess the similarity between the suspect food and the human isolates and to characterise a known point source outbreak to assist in development of algorithms for outbreak detection.

Results:
Sequence data showed that the outbreak associated isolates, including the food isolates, formed a tightly clustered monophyletic group, with a maximum pairwise distance of 3 single nucleotide polymorphisms.

Conclusions:
WGS data is useful in confirming the causative agent of outbreaks where food and clinical isolates are available. This dataset, comprising a known outbreak, will be useful in the development of automatic algorithms for outbreak detection.