Production of staphylococcal enterotoxins in food. Team: Justyna Schubert, Magdalena Gonet, Jarosław Bystroń, Jacek Bania.
Production of enterotoxins by Staphylococcus aureus was identified as a factor of Staphylococcal Food Poisoning (SFP). Dairy products have been associated with SFP. Staphylococcal enterotoxin H (SEH), a gastrointestinal toxin, was first described in 1995. SEH was already implicated in SFP outbreaks associated with reconstituted and raw milk. All data on SEH expression is based on experiments conducted in microbiological broths. Very limited information on its production in food is available.
Milk was reported by many teams as unfavorable environment for enterotoxin production. However, this statement was based on limited number of S. aureus strains producing SEC. We tested a number of S. aureus strains carrying genes encoding SEH as well as SEH together with SEA or SEC. We found that the main feature allowing S. aureus to secrete large amounts of SEH seems to be in part related to the ability to decrease pH of the environment by this microorganism. Microbial broth (brain heart infusion - BHI) tested by us allowed each S. aureus strain to decrease pH during growth which was related to high level of SEH expression and moderate differences in level of secreted SEH between strains. Milk seems not to offer the same opportunity for a significant part of S. aureus strains tested in our laboratory. Some other S. aureus strains growing in milk were not restricted in SEH and SEA expression, unlike SEC which remained down-regulated in milk. Moreover, the level of SEC seems to be down-regulated in milk also in S. aureus producing high levels of SEH in milk. Therefore low food safety risk related to strains producing SEC in milk as suggested previously may not pertain to certain SEA and SEH-producing strains. Occurrence of staphylococci carrying sea and seh genes may pose an increased risk for enterotoxin production in milk-related products (Schubert et al. IJFM, 235, 36-45, 2016).
We demonstrated recently that in a number of S. aureus strains crying sed and ser genes production of SED and SER was significantly decreased in milk when compared to meat juice, supporting previous reports. However, certain strains secreted high amounts of SED and SER irrespective of environment. No clear relation of enterotoxin protein level and expression of known regulators was established. Our results indicate that enterotoxin production in meat is likely to pose higher risk for food safety than enterotoxin production in milk, suggesting area of routine food control should be expanded. Nevertheless, food safety risk related to enterotoxin production in milk should still be considered important (Schubert et al. FPD, 2016 – in press).
Enterotoxins of coagulase-negative staphylococci. Team: Sylwia Krakowiak, Ewa Wałecka-Zacharska, Jacek Bania.
According to the EU legislation foodstuffs are being examined for staphylococcal enterotoxins only if the presence of coagulase-positive staphylococci, like S. aureus is confirmed. The fact that also coagulase-negative staphylococci might possess enterotoxin genes was revealed not long ago, but current status of knowledge remains to scarce to provide insights into their significance for public health.
In the genome sequence of Staphylococcus epidermidis strain, designated 4S, isolated from ready-to-eat food we identified 21,426-bp region flanked by direct-repeats, encompassing sec and sel genes, corresponding to the previously described composite staphylococcal pathogenicity island (SePI) in S. epidermidis. We provided the first evidence that S. epidermidis occurring in food bears an element encoding an orthologue to Staphylococcus aureus SEC, and that SECepi can be produced in microbial broth, milk and meat juices. Regarding that only enterotoxins produced by S. aureus are officially tracked in food in EU, the ability to produce enterotoxin by S. epidermidis pose real risk for food safety (Podkowik et al. IJFM, 229, 52-59, 2016).
Our data indicate that among enterotoxigenic coagulase-negative staphylococci appear strains with stable enterotoxin genes (like in S. epidermidis 4S), similarly as in all known S. aureus strains, and strains that are heterogeneous in term of enterotoxin genes content. This latter seem to constitute mixed assemblage of bacterial cells where some are positive and some are negative for enterotoxin genes. We think that instability of enterotoxin genes perceived in some coagulase-negative staphylococci might contribute to overall enterotoxicity evolution of the genus Staphylococcus. According to our hypothesis enterotoxins of coagulase-negative staphylococci may impact staphylococcal pathogenicity in a twofold manner. Firstly, acting directly as virulence factors of coagulase-negative staphylococci; secondly, they may constitute the gene pool to supply pathogenic staphylococcal species with virulence factors.
In order to explain the significance of enterotoxins of coagulase negative staphylococci, a number of questions remain to be answered. They include:
- Evaluation of heterogeneity of coagulase-negative strains, and assessment of laboratory conditions to stabilize heterogenic cultures,
- Evaluation of possible intra- and interspecies transfer of mobile genetic elements harbouring enterotoxin genes.
- Characterization of mobile genetic elements harboring enterotoxin genes, their insertion sites in the genomes of coagulase-negative staphylococci, and identification of motives associated with enterotoxin gene transfer. Resolving of abovementioned issues allow to assess real safety hazards for public health posed by enterotoxins of coagulase negative staphylococci (ongoing NCN project 2015/19/B/NZ7/00447).
