HeLa cell infection by Yersinia enterocolitica: evidence for lack of intracellular multiplication and development of a new procedure for quantitative expression of infectivity

The in vitro invasive properties of bacteria have frequently been studied by the use of HeLa cell cultures in chamber slides, using microscopic examination to enumerate intracellular bacteria. When this system was used to examine invasive properties of Yersinia enterocolitica, it resulted in rapid internalization of high numbers of bacteria during the infection phase which prevented subsequent discrimination of intracellular multiplication. A modified procedure was developed which standardized the ratio of bacteria to HeLa cells (i.e., multiplicity), the time for the infection phase, and the addition of specific antiserum with gentamicin for restricting bacterial uptake during the intracellular growth phase. Studies with this modified chamber slide system found that strains of human isolates of Y. enterocolitica (serotypes O:3, O:8, O:5,27, and O:6,30) exhibited different degrees of cell infection but did not multiply intracellularly. A second test system was developed that used roller tubes and viable cell counts for the enumeration of intracellular bacteria. This roller tube system confirmed that internalized bacteria did not multiply inside HeLa cells over a 24-h period. The roller tube system with viable cell counts for enumeration is a simplified technique for quantitative comparison of in vitro infectivity of HeLa cells by Y. enterocolitica.