Staphylococcal Enterotoxin Gene Cluster: Prediction of Enterotoxin (SEG and SEI) Production and of the Source of Food Poisoning on the Basis of v Saβ Typing

Abstract

Currently, only 5 (SEA to SEE) out of 27 known staphylococcal enterotoxins can be analyzed using commercially available kits. Six genes (seg, sei, sem, sen, seo, and seu), encoding putative and undetectable enterotoxins, are located on the enterotoxin gene cluster (egc), which is part of the Staphylococcus aureus genomic island vSaβ. These enterotoxins have been described as likely being involved in staphylococcal food-poisoning outbreaks. The aim of the present study was to determine if whole-genome data can be used for the prediction of staphylococcal egc enterotoxin production, particularly enterotoxin G (SEG) and enterotoxin I (SEI). For this purpose, whole-genome sequences of 75 Staphylococcus aureus strains from different origins (food-poisoning outbreaks, human, and animal) were investigated by applying bioinformatics methods (phylogenetic analysis using the core genome and different alignments). SEG and SEI expression was tested in vitro using a sandwich enzyme-linked immunosorbent assay method. Strains could be allocated to 14 different vSaβ types, each type being associated with a single clonal complex (CC). In addition, the vSaβ type and CC were associated with the origin of the strain (human or cattle derived). The amount of SEG and SEI produced also correlated with the vSaβ type and the CC of a strain. The present results show promising indications that the in vitro production of SEG and SEI can be predicted based on the vSaβ type or CC of a strain. IMPORTANCE Besides having infectious properties in human and animals, S. aureus can produce different enterotoxins in food. The enterotoxins can cause vomiting and diarrhea, often involving many people. Most of these outbreaks remain undiscovered, as detection methods for enterotoxins are only available for a few enterotoxins but not for the more recently discovered enterotoxins G (SEG) and I (SEI). In this study, we show promising results that in vitro production of SEG and SEI can be predicted based on the whole-genome sequencing data of a strain. In addition, these data could be used to find the source (human or cattle derived) of an outbreak strain, which is the key for a better understanding of the role SEG and SEI play in foodborne outbreaks caused by S. aureus.

Keywords: Staphylococcus aureus; egc; enterotoxin.

FIG 1

Representation of the newly defined S. aureus genomic island vSaβ types XVI to XXII. The virulence-associated genes, and other hypothetical genes located on vSaβ, are also presented. For each vSaβ type, one reference strain is shown. Arrows show the orientation of open reading frames. FIG numbers are hp genes that were assigned to a FIG number by the RAST (Rapid Annotations using Subsystem Technology) pipeline. ent1 and ent2 of vSaβ type XVIII are genes that were already described by Collery and Smyth (78). *, truncated or fragmented gene.

FIG 2

Maximum likelihood phylogenetic tree based on the core genome (nucleotidic sequences) showing the evolutionary relationship among 75 isolates of Staphylococcus aureus (all strains positive for the enterotoxin gene cluster) recovered from human, animal, environment, and food samples (left). At the right, for each strain its clonal complex (CC), origin of the strain, source of the strain, and involvement in staphylococcal food poisoning outbreak (SFPO) is given. Bootstrap values of >80 are shown. Production of enterotoxin G (SEG) and I (SEI) for the 32 analyzed strains is also given (last two columns). These are shown as L for low enterotoxin production and H for high enterotoxin production. *, statistical outliers; **, unknown CC.

FIG 3

Amount (nanograms per milliliter) of staphylococcal enterotoxins G and I (A and B, respectively), measured with enzyme-linked immunosorbent assay. Each point is the average measurement from three biological replicates, and the corresponding bars represent standard deviations. Strains were incubated in brain heart infusion (BHI) for 24 h at 37°C. The limit of detection of the corresponding enterotoxin is presented by red dashed line (LOD SEG, 0.001 ng/ml; LOD SEI, 0.037 ng/ml).