Elevated enterotoxin A expression and formation in Staphylococcus aureus and its association with prophage induction.

Staphylococcus aureus strains producing the bacteriophage-encoded staphylococcal enterotoxin A (SEA) were divided into two groups, high- and low-SEA-producing strains, based on the amount of SEA produced. After growth under favorable conditions in batch cultures, 10 of the 21 strains tested produced more than 1,000 ng/ml SEA, and 9 strains produced less than 10 ng/ml SEA; two enterotoxigenic strains, MRSA252 and Newman, produced intermediate levels of SEA (around 450 ng/ml). The differences in the production of SEA were found to be associated with the expression level of sea and whether the strains hosted the sea(1) or sea(2) version. Furthermore, differences in nucleotide sequence in the Siphoviridae phage region showed two clonal lineages of the high-SEA-producing strains. One of these lines was correlated with the capacity for a massive increase in SEA levels by prophage induction as demonstrated using mitomycin C (MC). This was also confirmed by the occurrence of additional sea expression, presumed to be initiated by a latent phage promoter located upstream of the endogenous sea promoter. Remarkably, the SEA level was increased up to 10-fold in some strains due to prophage induction. The low-SEA-producing group and the high-SEA-producing subgroup lacking phage-activated sea transcription showed no increase in SEA formation after the addition of MC. This study demonstrates that sea expression in enterotoxigenic strains is correlated with the clonal lineage of sea-carrying phages. The high-SEA-producing group, in particular the prophage-inducible sea(1) group, may be more relevant to staphylococcal food poisoning than the low-SEA-producing group, harboring mainly sea(2).

The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment.

The recent finding that the formation of staphylococcal enterotoxins in food is very different from that in cultures of pure Staphylococcus aureus sheds new light on, and brings into question, traditional microbial risk assessment methods based on planktonic liquid cultures. In fact, most bacteria in food appear to be associated with surfaces or tissues in various ways, and interaction with other bacteria through molecular signaling is prevalent. Nowadays it is well established that there are significant differences in the behavior of bacteria in the planktonic state and immobilized bacteria found in multicellular communities. Thus, in order to improve the production of high-quality, microbiologically safe food for human consumption, in situ data on enterotoxin formation in food environments are required to complement existing knowledge on the growth and survivability of S. aureus. This review focuses on enterotoxigenic S. aureus and describes recent findings related to enterotoxin formation in food environments, and ways in which risk assessment can take into account virulence behavior. An improved understanding of how environmental factors affect the expression of enterotoxins in foods will enable us to formulate new strategies for improved food safety.

Extended staphylococcal enterotoxin D expression in ham products.

Staphylococcal enterotoxin D (SED) is one of the most frequently recovered enterotoxins in staphylococcal food poisoning (SFP) outbreaks. The expression and production of SED were investigated in three ham products, i.e. boiled ham, smoked ham and dry-cured Serrano ham incubated at room temperature for seven days. Staphylococcus aureus was also, as a reference, grown in cultivation broth during optimal growth conditions for seven days. In boiled and smoked ham, continuous sed expression was observed throughout the incubation period with a second increase in sed expression found after five days of incubation. In smoked ham, nine times less SED per colony-forming unit of S. aureus was detected than in boiled ham. In boiled ham, the SED levels unpredictably decreased after three days of incubation. In the Serrano ham, SED was detected after five days of incubation although S. aureus growth was poor and sed expression was too low to determine. After five days of incubation, all three products contained enough SED to cause SFP. These results show that the specific production levels of SED vary in the different ham products, and that toxin production was in part uncoupled from bacterial growth.

Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus.

BACKGROUND: The effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA) expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea. RESULTS: The sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea-containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study. CONCLUSIONS: Our findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the sea-encoding prophage, linking SEA production to the lifecycle of the phage.

Prolonged expression and production of Staphylococcus aureus enterotoxin A in processed pork meat.

The bacteriophage-encoded staphylococcal enterotoxin A (SEA) is the toxin most frequently reported to be involved in staphylococcal food poisoning. In this study, sea expression and SEA formation were studied in four processed pork products: boiled ham, hot-smoked ham, Serrano ham (dry-cured Spanish ham) and black pepper salami. The products were selected because of their differences in intrinsic factors. As a reference, Staphylococcus aureus was cultivated under favorable planktonic growth conditions. Expression was mainly linked to bacterial growth for both meat products and broth cultures. In liquid broth, however, the relative level of sea mRNA peaked in the late exponential phase and then rapidly declined, while in the meat products allowing immediate growth, i.e. boiled and smoked ham, active sea expression occurred throughout the incubation period of seven days. Lower levels of sea mRNA and SEA were found in smoked ham compared to boiled ham, although viable counts of S. aureus on the two products were similar. Furthermore, the SEA concentration in the boiled ham reached a maximum after three days of incubation and then unpredictably decreased. In the Serrano ham, no increase in cell number was observed until day seven, and sea expression and extracellular SEA could only be detected on days five and seven. Finally, the black pepper salami with low pH and competing microbiota proved to be a difficult environment for the survival of S. aureus. The molecular mechanism behind the behaviour of S. aureus SEA expression is discussed in connection to the life-cycle of the SEA-encoding bacteriophage and the microbial communities in these pork products.