Phospholipase A(2) in rabbit tears: a host defense against Staphylococcus aureus.

PURPOSE: This study analyzed rabbit tears for anti-staphylococcal activity, the role of phospholipase A(2) (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 degrees or 37 degrees 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 degrees C decreased CFU approximately 4 logs. Tears (> or =30 microl) or PLA2 (> or =30 U) incubated with bacteria in phosphate-buffered saline were bactericidal. PLA2 (> or =0.2 U) or tears (> or =2 microl) 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 (60 microl) 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 tears.

The effectiveness of tobramycin and Ocuflox in a prophylaxis model of Staphylococcus keratitis.

PURPOSE: To determine the effectiveness of prophylactic antibiotic treatment prior to intra-corneal infection with Staphylococcus aureus. METHODS: One topical drop of Tobrex (0.3% tobramycin), tobramycin (0.3%) in the Tobrex vehicle with 0.05% dodecyl maltoside (DDM)/4.0% hydroxypropylmethycellulose (HPMC), Ocuflox (0.3% ofloxacin) or DDM/HPMC vehicle were applied to rabbit eyes at one or five hours prior to injection of bacteria. Approximately 500 colony-forming units (CFU) of S. aureus strain 8325-4 were injected into the corneal stroma. Rabbits were sacrificed five hours after infection and corneal homogenates were cultured to determine the number of colony forming units (CFU) per cornea. RESULTS: Rabbits treated at five hours prior to infection with tobramycin-DDM/HPMC reduced the bacterial load by approximately 2.4 log CFU/cornea as compared to the untreated control (3.47 +/- 0.98 vs. 5.71 +/- 0.14 log CFU/cornea, respectively; P = 0.0010); however, Ocuflox, Tobrex, or DDM/HPMC vehicle did not significantly reduce the log CFU (P >or= 0.4837). Rabbits treated at 1 hour prior to infection with Ocuflox or tobramycin-DDM/HPMC had significantly reduced CFU/cornea (1.31 +/- 0.86 and 0.48 +/- 0.31 log CFU/cornea, respectively) as compared to the untreated group (5.71 +/- 0.14 log CFU/cornea; P or= 0.2312). CONCLUSIONS: This pre-treatment model of Staphylococcus keratitis quantitatively measured the prophylactic effectiveness of topical antibiotic formulations. An important finding was that a tobramycin-DDM/HPMC formulation was highly effective as a prophylactic medication.