Effects of mechanical injuries on leafy greens in relation to proliferation of Escherichia coli O157:H7 Danyelle Osorio 1, Daniel Labuz 1, Sofia Windstam 1, Beatrix Alsanius 2 1 Department of Biological Sciences, SUNY Oswego, Oswego, NY, USA 2 Department of Horticulture, Swedish University of Agricultural Sciences, Alnarp, Sweden Introduction: Recently, there has been a rising concern over the ability of enteric bacteria, such as Escherichia coli O157:H7 (E.coli), to survive on the surface of produce because of the increased global concern with food safety (Aruscavage et al, 2006). Enteric pathogens only appear on the phyllosphere (plant leaf surface) after plants have been contaminated by their environment (Aruscavage et al, 2006, Franz and van Bruggen, 2008) and their persistence is still under evaluation. In that time, Aruscavage et al. (2008) and Brandl (2008), both found an increase in growth of E.coli when plant leaves are mechanically damaged. The purpose of this experiment was to assess under which sugar conditions promoted the most growth of E.coli, intact vs. mechanically damaged, on the leaves of spinach, arugula, and red chard through the use of cell counts. Methods: Results: Conclusions: Romaine: In the end, there were two replicate assays that both had a significant increase of the growth of E.coli on both intact and bruised leaves over a 24 hour period. The second replicate assay appeared to have a greater growth of bacteria, but this may have been due to the size of the leaves being greater in the second assay. The mature romaine leaves also had significant increases over 24 hours over all treatments. Red Chard: There were three replicated assays done, where in the second assay, in both treatments, there was a significant increase over 24 hours and in the third there was no significant increase of growth on the intact leaves, but there was on the bruised leaves. There was less growth on the third assay compared to the second, which also may have been because of the size of the leaves that were used. References: Aruscavage, D., Lee, K., Miller, S., and Lejeune, J.T. (2006) Interaction affecting the proliferation and control of human pathogens on edible plants. Journal of Food Science. 71, Aruscavage, D., Miller, S. A., Lewis Ivey, M. L., Lee, K., and LeJeune, J.. (2008) Survival and dissemination of Escherichia coli O157:H7 on physically and biologically damaged lettuce plants. Journal of Food Protection. 71, Brandl, M.T. (2008) Plant lesions promote the rapid multiplication of Escherichia coliO157:H7 on postharvest lettuce. Applied and Environmental Microbiology. 74, Franz, E., and van Bruggen, A. H.C.(2008) Ecology of E.coliO157:H7 and Salmonella enterica in the primary vegetable production chain. Critical Reviews in Microbiology. 34, Images taken by Osorio, D. Harvested baby leaves (Red Chard (Beta vulgaris var. vulgaris) and Romaine (Lactuca sativa var. longifolia cv. Ovired) were submerged in solutions of 10 5 cfu × ml -1 E.coli or 0.085% NaCl and left intact or mechanically injured using tweezers. Leaves were incubated at 28˚C for 2,4, and 24 hrs at which point leaves were transferred to 40 ml of 0.1 M Tris in 50 ml tubes. Leaves were sonicated at 20˚C for 7 minutes, then vortexed for 20 s before serially diluting suspensions and plating dilutions in duplicate onto Luria Bertani agar amended with 100 µg × ml -1 ampicillin and 0.2% arabinose. Plates were incubated at 37˚C overnight before enumerating colonies. Table 3. E. coli O157:H7 on minimally processed mature romaine leaves after incubation at 28˚C for up to 24 hrs post inoculation (hpi). Each data point is the mean of three replicates and the number in parenthesis is the standard error (S.E.). Different letters following the mean and S.E. denote that treatments were significantly different at the level of p=0.05. a Control leaves were submerged in 0.085% NaCl b Nd = Not detected Table 2. E. coli O157:H7 on bruised baby romaine leaves after incubation at 28˚C for up to 24 hrs post inoculation (hpi). Each data point is the mean of three replicates and the number in parenthesis is the standard error (S.E.). Different letters following the mean and S.E. denote that treatments were significantly different at the level of p=0.05. Table 1. E. coli O157:H7 on bruised baby red chard leaves after incubation at 28˚C for up to 24 hrs post inoculation (hpi). Each data point is the mean of three replicates and the number in parenthesis is the standard error (S.E.). Different letters following the mean and S.E. denote that treatments were significantly different at the level of p=0.05. a Control leaves were submerged in 0.085% NaCl b Nd = Not detected Image 1. Daniel Labuz harvesting the first leaves of Red Chard plants. Image 2. 0hr time point plates of E.coli collected from bruised spinach leaves. The sample was plated through the use of a spiral plater. E. coli O157:H7 (log cfu × g -1 leaf) InoculationTime (hpi)IntactBruised Control a 0Nd b Nd Nd E. coli05.58 (0.05)ab5.42 (0.05)a (0.04)a5.00 (0.09)a (0.09)ab5.31 (0.17)ab (0.21)b6.40 (0.18)b E. coli O157:H7 (log cfu × g -1 leaf) Time (hpi)IntactBruisedCutShredded (0.03)a4.65 (0.05)a5.47 (0.24)ab5.09 (0.06)a (0.03)a4.76 (0.09)ab4.92 (0.04)a4.97 (0.05)a (0.09)ab5.00 (0.01)b5.40 (0.16)a5.48 (0.08)b (0.08)b5.53 (0.21)a5.71 (0.12)b6.37 (0.12)c E. coli O157:H7 (log cfu × g -1 leaf) InoculationTime (hpi)IntactBruised Control a 0Nd b Nd (0.23)4.78 (0.29) E. coli05.34 (0.05)a5.39(0.13)a (0.07)ab5.24(0.09)a (0.11)ab5.19(0.19)a (0.32)b7.11(0.14)b