(457) Staphylococcus aureus expresses Enterotoxin Gene Cluster toxins in cystic fibrosis infections

Background: Staphylococcus aureus is one of the most prevalent pathogens in cystic fibrosis (CF). Clinical isolates of S. aureus collected from patients with CF often encode Enterotoxin Gene Cluster (EGC) toxins. Patients infected with egc+ isolates have lower lung function, but it is not known whether EGC toxins are expressed in vivo. Our goals were to measure EGC toxins in CF infections, determine whether infected patients produce antibodies versus EGC toxins, and assess inflammatory responses to these toxins in human airway epithelial cultures.

Methods: With IRB approval and following written consent, we enrolled patients with CF and control subjects in an observational study of S. aureus infections. We collected oropharyngeal swabs and/or sputum to measure EGC toxins and serum to assess IgG responses to EGC. We collected S. aureus proteins by rinsing oropharyngeal swabs in saline. We compared subjects with CF who had egc+ S. aureus confirmed by whole genome sequencing versus control subjects who were negative for S. aureus by oropharyngeal culture. With sample blinding, we measured EGC toxins from the oropharynx by quantitative dot immunoblot, using antibodies raised against purified toxin. Samples were compared to a standard curve with purified toxin. We measured the IgG response to EGC toxins by enzyme-linked immunosorbent assay (ELISA), using purified toxins adsorbed to the ELISA plate. The ELISA standard was mixed-donor IVIG versus Toxic Shock Syndrome Toxin (TSST), defined as 1:640. We measured cytokine responses to purified EGC toxin SEl-I after adding the toxin to polarized human airway epithelial cultures.

Results: We probed 42 oropharyngeal swabs collected from 36 patients (28 CF EGC+, 8 Control) by immunoblot. We found that the EGC toxins of SEG, SEl-I and SEl-O were all detectable above the control baseline (p < 0.05, Wilcoxon rank sum) in our CF samples. ELISAs were conducted on 26 samples from 26 patients (22 CF EGC+, 4 Control). We found that IgG response to EGC toxins was detectable in both groups, but higher in the CF population for SEl-O and SEl-U (p < 0.05, Wilcoxon rank sum). The IgG response trended higher for SEl-I, SEl-M, and SEl-N in patients with CF. Purified SEl-I stimulated the secretion of IL-8 by human airway epithelial cultures as measured by a multiplex cytokine panel.

Conclusions: EGC toxins were detected in oropharyngeal swabs from patients with CF who have confirmed S. aureus infections. These patients also have detectable antibody responses to some of these toxins. These data suggest that EGC toxins are expressed during chronic respiratory infection or colonization. It is not known whether antibody responses to these toxins are predictive of eventual eradication of S. aureus. Because SEl-I induced IL-8 expression, production of EGC toxins could play a role in disease pathogenesis by stimulating neutrophilic inflammation.

Acknowledgements: Supported in part by CFF Research Grant FISCHE21GO

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