The influence of lysostaphin on phagocytosis, intracellular bactericidal activity, and chemotaxis of human polymorphonuclear cells.

Lysostaphin, a microbicidal enzyme that lyses Staphylococcus aureus, was introduced to study phagocytosis and ICBA (Tan et al.3) on the presumption that it does not penetrate into the phagocytic cells. It was recently suggested, however, that LS enters the cells and kills ingested bacteria. By using two methods to study phagocytosis and bactericidal activity, the old one based on disruption of PMNs and plating technique and a new one that does not require disruption, we found that LS did not influence phagocytosis or phagocytic index but altered intracellular kill of Staphylococcus. LS eliminated almost completely extracellular bacteria, but centrifugation and washing of PMN at the end of phagocytic assay were almost equally efficient. Since the method of disruption of PMN and plating of bacteria cannot distinguish penetration of LS to the cells from its adherence to the outer wall of PMN, we employed a new, recently described acridine orange/crystal violet method, which can measure simultaneously phagocytosis and ICBA and eliminates completely extracellular microorganisms. This method has shown that in the presence of LS, a significantly higher proportion of staphylococci were killed intracellularly--91% +/- 2.7 vs. 74% +/- 2.9 (p less than 0.001), i.e., that LS either penetrated to the cells or enhanced ICBA. It was also found that trypsin, which was used as an inhibitor of LS, was unable to abolish bactericidal activity of LS. It is suggested that LS should not be used for assessment of ICBA but may be employed for studies of phagocytosis over short incubation periods. A new method based on acridine orange/crystal violet staining was found to be useful for investigation of phagocytosis and ICBA of human PMNs.