Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection

Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing 10(9) CFU/ml of E. coli O157:H7 for 24 +/- 1 h at 4 degreesC. Rinsed samples were treated with 200 mg/liter (200 ppm) chlorine for 5 min at 22 degreesC. Viability of E. coli O157:H7 cells was evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E. coli O157:H7. Quantitative microscopic observations of viability were made at intact leaf surface, stomata, and damaged tissue. Most E. coli O157:H7 cells (68.3 +/- 16.2%) that had penetrated 30 to 40 mum from the damaged tissue surface remained viable after chlorine treatment. Cells on the surface survived least (25.2 +/- 15.8% survival), while cells that penetrated 0 to 10 mum from the damaged tissue surface or entered stomata showed intermediate survival (50.8 +/- 13.5 and 45.6 +/- 9.7% survival, respectively). Viability was associated with the depth at which E. coli O157:H7 cells were in the stomata. Although cells on the leaf surface were mostly inactivated, some viable cells were observed in cracks of cuticle and on the trichome. These results demonstrate the importance of lettuce leaf structures in the protection of E. coli O157:H7 cells from chlorine inactivation.