Fig. 2.

Effect of E. coli infection on 5′-nucleotidase activity in the supernatant medium. a Nonpathogenic E. coli strains (HB101 and HS) or EPEC strains E2348/69 and JCP88 were subcultured in DMEM medium for 2 h, then used to infect T84 cells at a multiplicity of infection of 100:1 in phosphate-free DMEM. After 35 min to allow adherence, the medium was changed to nucleotidase buffer and aliquots were collected at various times after the medium change, filtered through a 0.45-μm filter to remove bacterial cells, then assayed for 5′-nucleotidase activity. Since the activity was measured in the cell-free sterile filtrates the activity is expressed as nmol/min per well. bE. coli suspensions were subjected to sterile filtration without treatment (light gray bars) or following treatment with 50 μg/ml polymyxin B, a lytic antibiotic (dark gray bars). Then the nucleotidase activity of a 100-μl aliquot was measured. The 100 μl volume was chosen because this was the volume of inoculum needed to achieve an MOI of 100:1 for the slowest growing strain (E2348/69) in a typical 48-well plate of T84 cells