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 s canning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by i mmersing 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 p pm) chlorine for 5 min at 22 degreesC. Viability of E. coli O157:H7 cells w as evaluated by CSLM observation of samples stained with Sytox green (dead cell stain) and Alexa 594 conjugated antibody against E. coli O157:H7. Quan titative microscopic observations of viability were made at intact leaf sur face, 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 remain ed 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 da maged tissue surface or entered stomata showed intermediate survival (50.8 +/- 13.5 and 45.6 +/- 9.7% survival, respectively). Viability was associate d with the depth at which E. coli O157:H7 cells were in the stomata. Althou gh cells on the leaf surface were mostly inactivated, some viable cells wer e observed in cracks of cuticle and on the trichome. These results demonstr ate the importance of lettuce leaf structures in the protection of E. coli O157:H7 cells from chlorine inactivation.