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ABSTRACTStaphylococcus aureusstrains 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 ofseaand whether the strains hosted thesea1orsea2version. Furthermore, differences in nucleotide sequence in theSiphoviridaephage 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 additionalseaexpression, presumed to be initiated by a latent phage promoter located upstream of the endogenousseapromoter. 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-activatedseatranscription showed no increase in SEA formation after the addition of MC. This study demonstrates thatseaexpression in enterotoxigenic strains is correlated with the clonal lineage ofsea-carrying phages. The high-SEA-producing group, in particular the prophage-induciblesea1group, may be more relevant to staphylococcal food poisoning than the low-SEA-producing group, harboring mainlysea2.

BackgroundThe 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.ResultsThe 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.ConclusionsOur 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.

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