Daniel Labuz1, Danyelle Osorio1, Beatrix Alsanius2, and Sofia Windstam1

1Department of Biological Sciences, SUNY Oswego, Oswego, NY, USA 2Department of Horticulture, Swedish University of Agricultural Sciences, Alnarp, Sweden

• Safety of produce has been put in the spotlight in recent times. Specifically, E. coli O157:H7 has been the main culprit of many food related pathogenic outbreaks in the United States (Doyle & Erickson 2008). E. coli O157:H7 (referred to as E. coli from now on) is of particular concern because it produces shiga-toxins which can induce HUS (Hemolytic Uremic Syndrome) and cause death in severe cases (Kaper et al. 2004). The dynamics of the phyllosphere are very important for E. coli to survive. Not known as an epiphytic bacterium, E. coli has been documented and found to live on parsley for up to 177 days when grown in contaminated manure (Islam et al. 2004). Moreover, Oliveira et al. (2012) found that E. coli persisted on lettuce leaves for up to five weeks during the fall when inoculated using a sprinkler irrigation system. Thus, it is important to study the mechanisms that assist E. coli surviving in this novel environment.

• When E. coli arrives to the phyllosphere it has difficulty surviving due to a lack of nutrients and moisture. Most importantly, nutrients are spread across the leaf surface and are concentrated around trichomes, veins, stomata, and other natural depressions (Aruscavage et al. 2006; Leveau and Lindow 2001). It is no surprise that most bacterial aggregates are found in these areas due to the abundance of nutrients (Brandl and Mandrell 2002). Additionally, bacteria will compete for the same nutrients in the phyllosphere making it even more difficult to obtain these sparse nutrients for immigrating bacteria like E. coli (Aruscavage et al. 2008). Furthermore, different plant species have different amount of sugars on the leaf surface, and bacteria will exhaust the amount of sugars at different rates depending on the plant species (Mercier and Lindow 2000). Overall, E. coli has to compete for these scattered nutrients in this intermediate habitat.

• Mechanical injury to a plant can support growth of larger E. coli populations which is presumed to be because of the increase in nutrients (Aruscavage et al. 2008). The reasoning for the increase in E. coli growth is not clearly understood. Brandl (2006) comments that nutrient leakage from damaged plant tissue give enteric pathogens a broad spectrum of available nutrients that they may not be accessible from healthy plant tissue. The actual connections between sugar availability and E. coli growth have not been simultaneously observed. This study will examine the effects of injured plant tissue on E. coli growth.

Preparation of E. coli Culture and PlantsE. coli O157:H7 was maintained on Luria-Bertani agar (LBA) at 37C with 100 µg × ml-1 ampicillin. For plant inoculations, bacteria were grown in LB broth and 100 µg × ml-1 ampicillin at 200 rpm and 37˚C for 16-18 hrs . Cells were harvested by centrifugation and washed with 0.85% NaCl twice before resuspending in 0.085% NaCl and adjusting the cell density to 105 CFU × ml-1.Trays filled with K-jord soil were prepared for planting of spinach (Spinacia oleracea) (Figure 2) and baby arugula (Eruca sativa). Plants were fertilized with 5 g per liter Superba brown weekly (Figure 1). Plants were carefully harvested after 3-4 weeks of initial planting.

Growth of E. coli O157:H7 on Spinach and ArugulaControls of all treatments and plants had no significant growth of E. coli (p < 0.05). Spinach plants had no significant differences (p < 0.05) on intact leaves. The 24-hour time point was significantly higher (p < 0.05) than 0-hour on bruised spinach. Baby arugula had no significant differences (p < 0.05) on intact leaves. The bruised arugula at 24-hour was significantly higher in growth (p > 0.05) than 0-hour. In both plants 24-hour was significantly higher than 0-hour bruised leaves. This follows results found by Brandl (2008) and Aruscavage et al. (2008) on romaine lettuce. In each study there was a significant growth of E. coli found on mechanically injured leaves, while the intact leaves had minimal or decreased growth over the same time period. Our data suggests that injured leaves promotes a better environment for E. coli to grow and survive in due to increased nutrient leakage (Aruscavage et al. 2008). Leaf Inoculation, Plating, and Plate Counts

At first, leaves were weighed, dipped in solution of E. coli or NaCl for 3 seconds and then injured (or left intact for controls) by use of forceps twice at the tip and at the central part of the leaf. Leaves were placed in a bag and incubate at 28C for 0, 2, 4, or 24 hr(s). Leaves were sonicated for seven minutes in 40 mL 0.1 M Tris HCL solution, vortexed, diluted to 10-1 and 10-3 for E. coli plates (0, 2, 4 hr(s)) and 10-2 and 10-4 for 24 hrs. Controls were diluted to 10-1. Using a spiral plater all treatments were plated with duplicate LB plates were incubated at 37C for 16-18 hrs. Plates were checked for E. coli counts (gfp under UV light) as well as total count of microorganisms present.

Mechanically damaged baby greens and the effect on Escherichia coli O157-H7 growth

Aruscavage, D., Lee, K., Miller, S. and LeJeune, J. T. (2006) Interactions affecting the proliferation and control of human pathogens on edible plants. Journal of Food Science. 8: 89-99Aruscavage, D., Miller, S. A., Lewis Ivey, M. L., Lee, K. and Lejeune, J. T. (2008) Survival and dissemination of Escherichia coli O157:H7 on physically and biologically damaged lettuce plants. Journal of Food Protection. 12: 2384-2388.Brandl, M. T. & Mandrell, R. E. (2002) Fitness of Salmonella enterica serovar Thompson in the cilantro phyllosphere. Applied and Environmental Microbiology. 68: 3614-3621. Brandl, M. T. (2006) Fitness of human enteric pathogens on plants and implications for food safety. Annual Review of Phytopathology. 44: 367-392.Brandl, M. T. (2008). Plant lesions promote the rapid multiplication of Escherichia coli O157:H7 on postharvest lettuce. Applied and Environmental Microbiology. 74: 5285-5289. Doyle, M. P. & Erickson, M. C. (2008) Summer meeting 2007 – the problems with fresh produce: an overview. Journal of Applied Microbiology. 105: 317-330.Islam, M., Doyle, M. P., Phatak, S. C., Millner, P. and Jiang, X. (2004) Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water. Journal of Food Protection. 67: 1365-1370.Kaper, J. B., Nataro, J. P. and Mobley, H. L. (2004) Pathogenic Escherichia coli Nature. 2: 125-140.Leveau, J. H. J. & Lindow, S. (2001) Appetite of an epiphyte: Quantitative monitoring of bacterial sugar consumption in the phyllosphere. Proceedings of the National Academy of Science. 98: 3446-3453.Mercier, J. & Lindow, S. E. (2000) Role of leaf surface sugars in colonization of plants by bacterial epiphytes. Applied and Environmental Microbiology. 66: 369-374.Oliveira, M., Vinas, I., Usall, J., Anguera, M. and Abadias, M. (2012) Presence and survival of Escherichia coli O157:H7 on lettuce leaves and in soil treated with contaminated compost and irrigation water. International Journal of Food Microbiology. 156: 133-140.

INTRODUCTION

RESULTS AND DISCUSSION

MATERIAL AND METHODS

REFERENCES

Figure 2. Mature (15-day old) baby spinach leaves grown in a tray filled with K-jord soil.

Figure 1. 15-day old (left) and 8-day old (right) baby greens (Clockwise: Spinach, romaine, arugula, and red chard) being fertilized with Superba brown.

E. coli O157:H7 (log cfu × g-1 leaf)Plant Inoculation Time Intact BruisedSpinach E. coli 0 5.13 (0.09)a 5.43 (0.23)a

2 5.32 (0.05)a 5.32 (0.05)ab4 5.30 (0.09)a 5.15 (0.04)ab24 6.22 (0.37)a 6.16 (0.20)b

Arugula E. coli 0 5.24 (0.05)a 5.09 (0.12)a2 5.22 (0.06)a 5.18 (0.09)a4 5.12 (0.10)a 4.96 (0.09)a24 5.32 (0.18)a 6.18 (0.02)b

Growth of E. coli O157:H7 on Mechanically Damaged RomaineIntact romaine leaves had no significant differences (p < 0.05) among time periods. Bruised leaves were significantly higher (p < 0.05) at 4-hour and 24-hour time points compared to 0-hour and 2-hour. Cut and shredded romaine leaves showed E. coli growth that were significantly higher (p < 0.05) at 24-hour compared to all other time points, and 4-hour had significantly higher (p < 0.05) growth than 2-hour. (Table 1). Brandl (2008) found similar results on bruised, cut, and shredded lettuce after 4 hours, while there was growth it was between a 4-11 fold difference. Our data shows a much larger increase in E. coli growth after 24 hours. In both our study and Brandl (2008) the shredded damaged romaine lettuce had the highest increase in growth of E. coli; most likely due to the high amount of nutrients being released.

Table 1. Growth of E. coli on damaged romaine. Standard errors are shown in parentheses and significantly different points are noted by a, b or c

Table 2. Growth of E. coli on baby spinach and arugula. Standard errors are shown in parentheses and significantly different points are noted by a, b or c

RESULTS AND DISCUSSION cont.