| Summary: | Biofilm formation and slow growth by <i>Staphylococcus aureus</i> in platelet concentrates (PCs) cause missed detection of this bacterium during routine PC screening with automated culture systems. This heightens the chances of false-negative screening transfusions and pre-disposes transfusion patients to an elevated risk of sepsis due to secretion of staphylococcal enterotoxins (SEs) in PCs. A hybrid approach of comparative RNAseq analyses and CRISPR mutagenesis of SE genes was employed to investigate the effect of SEs in <i>S. aureus</i> growth and biofilm formation in PCs. RNAseq data showed no differential expression for key biofilm genes, whereas SE genes were upregulated (>0.5- to 3.6-fold change) in PCs compared to trypticase soy broth (TSB). Remarkably, growth and biofilm formation assays revealed increased growth for the <i>S. aureus</i> SE mutants, while their ability to form biofilms was significantly impaired (−6.8- to −2.4-fold change) in comparison to the wild type strain, in both PCs and TSB. Through the well-established superantigen mechanism of SEs, we propose three roles for SEs during biofilm development in PCs: (1) provide a scaffold for biofilm matrix, (2) mediate cell-to-cell aggregation, and (3) guarantee biofilm survival. Furthermore, SE contribution to both growth and biofilm development seems to be centrally regulated by <i>agr</i> via quorum sensing and by <i>saeSR</i> and <i>sigB</i>. This study reveals new roles for SEs, which enforce their relevance in ensuring PC safety for transfusion patients. It further deciphers the underlying reasons for failed <i>S. aureus</i> detection in PCs during screening with automated culture systems.
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