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

Abstract

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.

Keywords: Salmonella; Whole genome sequence; disease outbreak; infectious disease.

BRIG analysis pSLA5

De novo assemblies of reads of the outbreak isolates that did not map to S. Enteritidis P125109 are shown as concentric rings with plasmid pSLA5 as reference on the inner black circle. Absence of colour in a ring indicates absence of the region. Isolates of the outbreak in Flanders are represented by a blue colour, those of the Walloon outbreak by a purple colour.

BRIG analysis pSD107

The de novo assembly of S. Enteritidis P125109 unmapped reads of outbreak isolate S14BD01672 is shown as a concentric ring with plasmid pSD107 as reference on the inner black circle. Absence of colour in the ring indicates absence of the region.

CSI Phylogeny analysis

Radial phylogenetic tree of the 6 outbreak isolates with S. Enteritidis P125109 as reference. Isolates of the outbreak in Flanders are represented by a blue colour, those of the Walloon outbreak by a purple colour.