Virus-Like Plaque Formation in Human Cell Culture by Pseudomonas aeruginosa ∗

Abstract

Summary

A chance contamination in the preparation of HeLa cultures in tubes with an organism identified as P. aeruginosa resulted in the formation of plaques within 24 hr which were indistinguishable from viral plaques. At 48 hr, the medium in such tubes became turbid, revealing the presence of bacteria. Filtration of the contaminated medium through a Selas filter or the addition of neomycin eliminated both plaques and turbidity. Stock strains of P. aeruginosa, P. fluorescens, and Alcaligenes fecalis formed similar plaques on HeLa and fibroblastic-like cell line cultured in antibiotic-free medium, whereas strains of E. coli and S. aureus did not. All human epitheloid and fibroblastic-like cell lines tested developed plaques with P. aeruginosa. A linear relationship was observed between bacterial concentration and plaque counts or the time required for plaques to appear. Bacterial plaques were not formed when newborn calf serum (NBCS) was omitted from the medium. At low concentrations of NBCS (1 and 2%), plaques were detected as early as 8 hr, whereas at high concentrations (5-20%), plaques were not detectable until sometime between 13 and 23 hr after infection. Addition of neomycin at the time of infection was more effective in inhibiting plaque formation than addition of the antibiotic 1 hr before or after infection. Growth curve studies indicated that the number of bacteria in the fluid phase (medium plus those released from the cell surface by trypsinization) exceeded the number in the cell phase (glass-homogenized HeLa) for 11 hr. Then the reverse occurred, for at 15 hr there was 1 log greater number in the cells than in the combined fluid. By 24 hr, cell disintegration due to plaques was sufficiently advanced to cause a release of organisms from the cells into the fluid.