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M. E. Sanders, E. W. Norcross, Q. C. Moore, III, M. E. Marquart; A Comparison of Pneumolysin Activity and Concentration in vitro and in vivo in a Rabbit Endophthalmitis Model. Invest. Ophthalmol. Vis. Sci. 2008;49(13):495.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study was to determine whether the in vitro activity and concentration of Streptococcus pneumoniae pneumolysin correlated to the pathogenesis of S. pneumoniae endophthalmitis.
Five S. pneumoniae clinical endophthalmitis strains were grown in media to similar optical densities (OD), and extracellular milieu was separated from intracellular content. Intracellular and extracellular samples were tested for pneumolysin activity by hemolysis of rabbit red blood cells. Pneumolysin concentration was determined using a sandwich ELISA. Strains with low (< 50%) and high (> 80%) extracellular hemolysis relative to a 100% lysis control were compared in a rabbit endophthalmitis model. Rabbit vitreous was injected with 102 colony-forming units (CFU) of 2 different groups of low hemolytic activity strains (n = 18) or 3 different groups of high hemolytic activity strains (n = 24). Severity of endophthalmitis infection was graded by two observers using slit lamp examination (SLE) at 24 hours post-infection. Eight parameters were each assigned a grade ranging from 0 (normal) to 4 (most severe), resulting in a total score of 0-32. Vitreous was removed from rabbits. Serial dilutions of the vitreous were plated on blood agar for bacterial CFU quantitation, and vitreal pneumolysin was quantitated.
Two clinical strains had low hemolytic activity and 3 strains had high hemolytic activity. Pneumolysin concentration and percent hemolysis in vitro correlated in 60% (intracellular) and 80% (extracellular) of the strains. Pneumolysin concentration in the vitreous following infection with S. pneumoniae was comparable to the in vitro concentration in 60% of the intracellular samples and 40% of the extracellular samples. For all 5 strains, extracellular hemolysis correlated to pathogenesis in vivo. Strains 1, 2, and 3 had high hemolytic activity and average SLE scores of 25.21 ± 2.1493, 14.45 ± 3.3575, and 16.65 ± 3.6413 respectively. Strains 4 and 5 had low hemolytic activity and average SLE scores of 8.33 ± 2.7586 and 5.3958 ± 2.1310 respectively. The SLE scores of strains 1 and 4, 2 and 5, and 3 and 5 were significantly different (P = 0.0007, 0.0459, and 0.0236 respectively), however, CFU recovered from the vitreous were not significantly different (P ≥ 0.6612).
Pneumolysin hemolytic activity in vitro directly correlates to the pathogenesis of S. pneumoniae endophthalmitis. Pneumolysin concentration is not a reliable indicator of pneumolysin activity. Other factors could be contributing to the pathogenesis of S. pneumoniae endophthalmitis.
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