Flow cytometry to monitor phagocytosis and oxidative burst of anaerobic periodontopathogenic bacteria by human polymorphonuclear leukocytes

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Abstract

The reduced susceptibility to phagocytosis found among some periodontopathogenic anaerobes may account for the differences between invasive and non-invasive strains. We applied flow cytometry as a powerful tool to analyze and quantify phagocytosis using standardized cultures of oral anaerobes (Porphyromonas gingivalis, Prevotella intermedia, P. nigrescens, Capnocytophaga gingivalis, C. ochracea, C. Sputigena, Fusobacterium nucleatum and Peptostreptococcus micros) and heparinized whole blood. Bacteria were labeled by a fluorescein-methylester and their esterase activity, resulting in green fluorescence. Ingested bacteria could be detected easily and quantified by a shift towards green fluorescence in the PMNL population involved and a concomitant decrease in the bacterial population. Furthermore, the oxidative burst of PMNLs was detected in parallel assays using the dye DHR123 which becomes fluorescent upon oxidation during the oxidative burst process. We found a great diversity in phagocytosis susceptibility determined by estimating the portion of phagocytosing PMNLs, ranging from 10.6% (strain W83) to > 99.4% (e.g. ATCC 33277T) in P. gingivalis and from 15.9% (strain MH5) to > 95% (ATCC 33563T) in P. nigrescens. In contrast, almost all P. intermedia strains as well as the representatives of the other anaerobic, putative periodontopathic species tested showed no or only moderate resistance in the phagocytosis assay. Comparison of clinical data of patients and the extent of phagocytosis resistance of the corresponding P. gingivalis strains suggests that this virulence factor may contribute to the clinical outcome.

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