April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Effectiveness Of Novel Chemical Inhibitors Of Staphylococcus aureus Alpha-toxin In Vitro And In Vivo
Author Affiliations & Notes
  • Anastasia C. Weeks
    Microbiology, University of Mississippi Medical Center, Jackson, Mississippi
  • Charles L. Balzli
    Microbiology, University of Mississippi Medical Center, Jackson, Mississippi
  • Richard J. O'Callaghan
    Microbiology, University of Mississippi Medical Center, Jackson, Mississippi
  • Footnotes
    Commercial Relationships  Anastasia C. Weeks, None; Charles L. Balzli, 12/773,556 (P); Richard J. O'Callaghan, 12/773,556 (P)
  • Footnotes
    Support  NIH Grant EY010974
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 5811. doi:
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      Anastasia C. Weeks, Charles L. Balzli, Richard J. O'Callaghan; Effectiveness Of Novel Chemical Inhibitors Of Staphylococcus aureus Alpha-toxin In Vitro And In Vivo. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5811.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Alpha-toxin is a key mediator of corneal damage in S. aureus keratitis. Cyclodextrin complexes containing cholesterol have been shown to significantly reduce alpha-toxin activity in vitro and in a keratitis model. This study seeks a potent inhibitory effect in toxin-mediated corneal pathology by various sterols complexed with methyl- or hydroxypropyl- forms of beta-cyclodextrin.

Methods: : Eleven sterols were allowed to complex with either cyclodextrin via equilibration in an ethanolic solution followed by lyophilization and reconstitution in sterile phosphate buffered saline. For quantitative determinations of alpha-toxin inhibition in vitro, the hemolytic activity of alpha-toxin was determined in the presence of two-fold dilutions of each complex using rabbit erythrocytes (107/well).The ability of each sterol complex to inhibit alpha-toxin in vivo was tested by incubating the complex (1% w/v) with 12 hemolytic units of alpha-toxin for 1 hr prior to intrastromal injection (10 µL) of rabbit corneas (n ≥ 5). Topical drops of the inhibitors (1%) were applied to rabbit eyes at 60, 45, 30, and 15 minutes before toxin injection. Epithelial erosions were measured (mm2) at 2, 4, 6, and 8 hours after injection. Methyl-cyclodextrin complexed with cholesterol served as a positive control of inhibition for both the in vitro and in vivo assays.

Results: : Of the 22 complexes tested in vitro, three (methyl-cyclodextrin:lanosterol, hydroxypropyl-cyclodextrin:desmosterol, hydroxypropyl-cyclodextrin:squalene; 0.1% solution) showed greater than 32-fold reduction of toxin activity. Of these three, lanosterol was similar to the methyl-cyclodextrin:cholesterol complex, demonstrating a ≥8000 fold reduction in lytic activity. However, in vivo, hydroxypropyl-cyclodextrin:demosterol (P ≤ 0.004), methyl-cyclodextrin:lanosterol (P ≤ 0.022), hydroxypropyl-cyclodextrin:squalene (P ≤ 0.021) had significant reductions in the size of the toxin-induced epithelial erosion.

Conclusions: : The methyl-cyclodextrin:cholesterol complex produces the most potent inhibition of S. aureus alpha-toxin in vitro and in vivo. The cyclodextrin complex with lanosterol had potent in vitro inhibitory activity and three sterol complexes tested produced significant in vivo inhibition of alpha-toxin mediated corneal damage.

Keywords: keratitis • Staphylococcus • microbial pathogenesis: experimental studies 

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