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

The reduced susceptibility to phagocytosis found among some periodontopatho genic anaerobes may account for the differences between invasive and non-in vasive strains. We applied Row cytometry as a powerful tool to analyze and quantify phagocytosis using standardized cultures of oral anaerobes (Porphy romonas gingivalis, Prevotella intermedia, P. nigrescens, Capnocytophaga gi ngivalis, C. ochracea, C. sputigena, Fusobacterium nucleatum and Peptostrep tococcus micros) and heparinized whole blood. Bacteria were labeled by a fl uorescein-methylester and their esterase activity, resulting in green fluor escence. Ingested bacteria could be detected easily and quantified by a shi ft towards green fluorescence in the PMNL population involved and a concomi tant decrease in the bacterial population. Furthermore, the oxidative burst of PMNLs was detected in parallel assays using the dye DHR123 which become s fluorescent upon oxidation during the oxidative burst process. We found a great diversity in phagocytosis susceptibility determined by estimating th e portion of phagocytosing PMNLs, ranging from 10.6% (strain W83) to > 99.4 % (e.g. ATCC 33277(T)) in P. gingivalis and from 15.9% (strain MH5) to > 95 % (ATCC 33563(T)) in P. nigrescens. In contrast, almost all P. intermedia s trains as well as the representatives of the other anaerobic, putative peri odontopathic species tested showed no or only moderate resistance in the ph agocytosis assay. Comparison of clinical data of patients and the extent of phagocytosis resistance of the corresponding P. gingivalis strains suggest s that this virulence factor may contribute to the clinical outcome.