Effect of bacterial invasion of macrophages on the outcome of assays to assess bacterium–macrophage interactions

Abstract

In vitro assays to quantify killing of bacteria by macrophages provide useful insights into host-pathogen relations. In the present study, we used strains of Yersinia enterocolitica and Escherichia coli which varied in their ability to invade mammalian cells to evaluate these assays. The results showed that 30 min and 24 h after incubation with murine bone marrow-derived macrophages, strains of Y. enterocolitica and E. coli which expressed invasin (an outer membrane protein which allows bacteria to penetrate mammalian cells) achieved significantly greater numbers in macrophages than otherwise isogenic bacteria which lacked this protein (P<0.01). When the 24-h data were corrected for the number of bacteria ingested by macrophages initially, the differences between invasin-positive and -negative bacteria were no longer evident (P>0.2). This study has shown (1) that invasin-mediated penetration of macrophages by bacteria is not associated with enhanced intracellular survival, and (2) that invasion of macrophages by bacteria may influence the interpretation of assays for bactericidal capacity unless allowance is made for the number of bacteria ingested during the early phase of the assay.

Introduction

The interaction of bacteria with macrophages plays a central role in the pathogenesis of many infectious diseases. Assays of these interactions in vitro can provide valuable insights into the ways in which bacteria overcome immune defenses and persist within host tissues. In a typical assay, macrophages are incubated with bacteria for a sufficient length of time to allow the phagocytes to ingest them. Extracellular bacteria are then removed by washing and by the addition of bactericidal antibiotics, such as gentamicin, which penetrate mammalian cells poorly (Homes et al., 1966; Tabrizi and Robins-Browne, 1993). After removal of the gentamicin, the cells are incubated with fresh medium. At particular time points thereafter, macrophages are lysed so that bacteria which persisted within them can be released and enumerated.

Results of these assays are usually expressed as the number of bacteria recovered from the macrophages, either as an absolute value or as the percentage of the original bacterial inoculum (for examples, see Jones et al., 1997; Skerrett and Martin, 1996; Tabrizi and Robins-Browne, 1993; Yamamoto et al., 1996). As such, these assays fail to take account of the number of bacteria ingested by the macrophages initially. Although this may not necessarily influence interpretation of the assay, these data could assume importance when a comparison is made between two or more bacterial strains with differing ability to enter macrophages, either by passive ingestion or active penetration. In this study, we used invasive and non-invasive strains of Yersinia enterocolitica and Escherichia coli to determine if active penetration of macrophages by bacteria can influence the interpretation of assays of macrophage–bacterium interactions.