Elimination of extracellular bacteria by antibiotics in quantitative assays of bacterial ingestion and killing by phagocytes.

In vitro assays to quantify the bactericidal capacity of phagocytes require the removal of extracellular bacteria from the reaction mixture before intracellular bacteria are released from the phagocytic cells and counted. This may be achieved by using an antibiotic, such as gentamicin, which has a rapid bactericidal action coupled with limited ability to cross cytoplasmic membranes. In this study, we investigated the susceptibility of Escherichia coli and Yersinia enterocolitica to killing by mouse peritoneal macrophages. Extracellular bacteria in the reaction mixture were eliminated with gentamicin, whereafter the antibiotic was enzymatically inactivated using gentamicin acetyl transferase (GAT). The method has few washing stages, thus reducing the likelihood of inadvertent removal of macrophages or bacteria from the reaction mixture. The assay disclosed clear differences in the ability of E. coli and Y. enterocolitica to survive phagocytosis by macrophages.