Microbial Pathogenesis 1992; 12: 159-l 64 Quantitative assessment of the ability of Escherichia co/i to invade cultured animal cells Roy M. Robins-Browne’ and Vicki Bennett-Wood’ ‘Department of Microbiology and Infectious Diseases, Royal Children’s Hospital, and 2Department of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia (Received September 6, 1991; accepted in revised form October 18, 1991) Robins-Browne, R. M. (Dept of Microbiology and Infectious Diseases, Royal Children’s Hospital, Parkville, Victoria 3052, Australia) and V. Bennett-Wood. Quantitative assessment of the ability of Escherichia coli to invade cultured animal cells. Microbial Pathogenesis 1992; 12: 159-I 64. Assays to quantify bacterial invasion of epithelial cells generally fail to take account of the ability of the bacteria to adhere to the cells prior to invasion. We have developed a modified invasion assay to allow for this factor. We then used the assay to investigate diarrhoeagenic strains of Escherichia coli with differing ability to adhere to and invade HEp-2 epithelial cells. The results showed that enteroinvasive strains of E. coli were the most invasive variety, followed in order by enteropathogenic E. coli and enterotoxigenic E. coli. These findings correspond to what is known of the ability of the bacteria to invade the intestinal tract in vivo. The results also indicated that adhesins of diarrhoeagenic E. coli play no direct role in invasion, although they may facilitate invasion indirectly by promoting initial contact between bacteria and animal cells. Key words: invasion; adhesion; cell culture; E. co/i. Introduction The ability to penetrate epithelial ceils is an essential virulence determinant of a large variety of pathogenic bacteria. This property may be investigated in vitro using mammalian cell culture.‘-’ In a typical assay, bacteria are incubated with a susceptible cell culture and allowed from l-3 h to adhere and invade. Non-adherent bacteria are removed by washing, after which an antibiotic, commonly gentamicin, is added to kill any remaining extracellular bacteria.‘-’ The principle underlying this step of the assay is that aminoglycoside antibiotics penetrate mammalian cells poorly, achieving intracellular concentrations below those which interfere with bacterial viability.’ Once the extracellular bacteria have been killed, the antibiotic is removed and the cell culture is lysed to release intracellular bacteria, which then are quantified by determination of the viable count. Results generally are expressed as the percentage of the initial inoculum recovered from the cell culture. 3-7 For a typical invasive organism this may range from approximately 0.5 to 25%. Although adhesion is an essential prerequisite of invasion, typical invasion assays fail to take account of the ability of bacteria to adhere to the cells initially. Thus the assay could conceivably give misleading results if two bacterial strains with a similar 0882-401 O/92/0201 59+06 $03.00/O @ 1992 Academic Press Limited