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

Abstract

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).

Fig 1

Growth, SEA production, and relative sea expression of S. aureus Mu50. The values were obtained from 10 independent batch cultivations grown in duplicate (duplicates A [filled symbols and bars] and B [open symbols and bars]). Circles denote the average growth, expressed as ln OD₆₂₀, and squares denote the average extracellular SEA levels of duplicates A and B. Bars show the average relative sea expression (RE).

Fig 2

Comparison of relative expression, PFU, and growth (OD₆₂₀) of S. aureus Sa17 (a) and PFU and growth of Sa51 (b) in noninduced and induced cultures. The relative expression of short and long sea transcripts, together with PFU, was analyzed at 3 h, 4 h, and 6 h of cultivation. Filled symbols denote the results for noninduced (without MC) cultures, and open symbols denote the results for induced (with MC) cultures. Solid lines show the relative sea expression levels, with circles representing the short transcript and triangles the long transcript. Dashed lines show PFU, and dotted lines show growth. No relative sea expression and long transcript levels were detectable in Sa51.

Fig 3

Assumed gene map of sea promoter regions in 21 S. aureus strains. P1 is the endogenous sea gene promoter, and P2 represents the latent promoter activated by phage induction in some sea₁ strains. The black arrow indicates the sea gene. Nucleotide sequence analysis of the sea promoter region was performed on the sequenced (whole genome) S. aureus strains MRSA252 (GenBank accession no. BX571856), MSSA476 (GenBank accession no. BX571857), Mu3 (GenBank accession no. AP009324), Mu50 (GenBank accession no. BA000017), MW2 (GenBank accession no. BA000033), and Newman (GenBank accession no. AP009315).