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Judy M. Moreau, Dalia O. Girgis, Emma B. H. Hume, Joseph J. Dajcs, Megan S. Austin, Richard J. O’Callaghan; Phospholipase A2 in Rabbit Tears: A Host Defense against Staphylococcus aureus. Invest. Ophthalmol. Vis. Sci. 2001;42(10):2347-2354.
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purpose. This study analyzed rabbit tears for anti-staphylococcal activity, the
role of phospholipase A2 (PLA2) in this reaction, and the
ability of enzyme inhibitors to promote bacterial survival.
methods. Contact lenses with Staphylococcus aureus were applied
to scarified rabbit eyes. The colony-forming units (CFU) per cornea or
lens were determined and pathology was scored by slit-lamp examination
(SLE). The bactericidal activity was measured by incubating bacteria
with rabbit tears or PLA2 at 33° or 37°C. Radiolabeled S.
aureus was incubated with PLA2 or tears to quantify the release
of a membrane component that was identified by thin-layer
chromatography. Inhibitors of these reactions were also analyzed.
results. Application of Staphylococcus, on contact lenses, to
rabbit corneas resulted in bacterial killing and limited inflammation.
Incubation of tears and bacteria (1:1; v/v) in tryptic soy broth at
33°C decreased CFU approximately 4 logs. Tears (≥30 μl) or PLA2
(≥30 U) incubated with bacteria in phosphate-buffered saline
were bactericidal. PLA2 (≥0.2 U) or tears (≥2 μl) cleaved bacterial
membranes, liberating arachidonic acid. Spermidine or tetracaine
inhibited cleavage of bacterial membranes by tears or PLA2 and
spermidine promoted bacterial survival and growth in tears. Tears (60μ
l) killed >99% of the bacterial inoculum, whereas bacteria
incubated in tears plus spermidine approximately doubled in number.
conclusions. PLA2 in rabbit tears kills Staphylococcus by hydrolyzing
bacterial membranes to release arachidonic acid. Spermidine and
tetracaine inhibited PLA2 activity and spermidine protected Staphylococcus from PLA2 in rabbit
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