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Survey of Mid-Atlantic Farms for Analysis of Foodborne

Pathogens in Tomatoes, Irrigation Water and Soil Samples Pagadala S.1, Pham M.1, Coffie J.N.1, Khan N.1, Martinez L.M.1, Micallef S.A.1

Research Team: Wang F.1, Oni R.1, Buchanan R.1, Pahl D.1, Wallis A.1, Walsh C.S.1, Kline W.2, Marine S.3, Everts K.3

1University of Maryland, College Park; 2 Rutgers New Jersey Agricultural Experiment Station; 3Lower Eastern Shore Research and Education Center, University of Maryland

Lu, L., Hume, M. E., Sternes, K. L., & Pillai, S. D. (n.d.). Genetic diversity of Escherichia coli isolates in irrigation water. In Genetic diversity of Escherichia coli isolates in irrigation water. Elsevier. (Reprinted from Water Research, 38,

2004)

• No Salmonella or STEC were isolated from any of the produce, water or soil samples.

• Generic E. coli was detected in 15.18% of water samples. • Generic E. coli was isolated from 5.07% of tomatoes. • Generic E.coli was isolated from 10.20% of soil samples. • Spinach and lettuce samples are currently being analyzed until the end of

October 2012.

Irrigation water, soil and compost have the potential to harbor microorganisms such as E. coli, Salmonella, Listeria and others that can contaminate agricultural crops and soils. Understanding where, how, and to what extent food contamination occurs is critical in order to improve management techniques, prevent risks, and set fair regulations regarding food safety (Lu et al., 2004). The purpose of this experiment was to understand the current bacterial safety status of tomatoes, irrigation water, and soil collected from small farms in Maryland, New Jersey and Delaware. Four hundred and twenty four samples were tested over a 12 week period for indicator species and possible contamination by the enteric pathogens Salmonella and Shiga toxin producing-E. coli (STEC). Currently, there is no national consensus for good agricultural practices (GAP) or good handling practices (GHP) for domestic and imported produce. The information gathered from this experiment will be considered in a larger objective to provide microbiological data for the development of science-based metrics of food safety for leafy greens and tomatoes.

INTRODUCTION

Sample collection Twenty four farms in Western MD, Eastern Shore MD, DE and NJ were sampled during the 2012 tomato growing season. Tomatoes, water and soil/compost sample were collected. Samples were collected using gloves at all times. Upon collection, samples were kept in ice-packed coolers until processed.

Tomatoes: At each farm, 4 samples of 4 tomatoes each were taken from 2 random sites and 2 targeted sites for possible contamination (Figure 1).

Irrigation water: One liter each of source and end of line irrigation water samples were collected into sterile plastic bags (Figure 2).

Soil: A 100 g sample of field soil was collected into sterile plastic bags using a sterile scoop.

Sample processing

Tomatoes: Each sample was weighed, and buffered peptone water (BPW) was added to make a 1:1 dilution. The sample bag was rubbed by hand for 2 minutes and left to stand for 1 hour. The rinsate was serially diluted in 0.1% peptone water to make dilutions of 10-1 – 10-4. The dilutions were plated onto aerobic plate count (APC) and E.coli/total coliform (TC) petrifilms (3M). Total coliforms were counted after 24 h and E.coli after 48 h incubation at 35°C. APC petrifilms were analyzed for aerobic bacteria after 48 h incubation at 35°C (Figure 3).

Irrigation water: Samples were serially diluted in 0.1% peptone water to create dilutions of 100-10-1 or 100-10-3, depending on turbidity. Using a membrane filtration system, 100 ml and 250 ml of each sample and 10 ml of each dilution were filtered through a sterile 0.45µm membrane filter. The 250 ml sample filter was placed into a falcon tube containing 40 ml of BPW. The tube was vortexed, and then incubated at 37oC for 24 h for Salmonella and STEC analysis. The 100 ml and 10 ml sample filters were placed onto MI agar petri dishes and incubated at 37oC for 24 h (Figure 4). The plates were then analyzed for TC, APC, and fluorescence analysis was used to verify presence or absence of generic E. coli.

Soil: Ten grams of each soil sample was vortexed in 90 ml of BPW, and then diluted in 0.1% peptone water to make dilutions of 10-1-10-4. The dilutions were plated and analyzed the same way tomatoes were. The 10 g soil/BPW mixtures were incubated at 37oC for 24 h.

After 24 h incubation, all BPW suspensions were taken to another lab to test for Salmonella and STEC by PCR.

MATERIALS AND METHODS RESULTS

CONCLUSIONS

REFERENCES

Figure 4. (Left) Water testing by membrane filtration.

(Bottom right) Incubated MI plates of different dilutions for the

presence or absence of E.coli.

(Top right) Viewing the MI plates under UV light.

Figure 3. (Right) Tomato samples being washed for 2 minutes in a BPW

solution.

(Left) Petrifilms for various dilutions of a sample for quantification of

E.coil , coliforms and aerobic organisms.

Total Samples Analyzed (Total 432)

State Compost Pond sediment Soil Tomato End of

Line Source

MD 0 5 7 87 4 17

ES 2 1 12 86 16 18

NJ 4 3 23 60 23 24

DE 0 0 4 26 4 6

Figure 1. Target tomatoes with possible

contamination

Figure 2. Irrigation source used on one of

the farms.

ACKNOWLEDGEMENTS

We thank Sanghyun Han for his guidance, training and lab support. Funding Source: This Research was supported by USDA NIFA SCRI Award No. 2011-51181-30767

Bacterial quantification in soil samples

Bacterial quantification in tomato samples

Bacterial quantification in water samples

Frequencies

E. coli Salmonella stx1

(STEC) stx2

(STEC)

Tomato 5.07% 0.00% 0.34% 0.34%

water (source and end of line) 15.18% 0.00% 0.00% 0.77%

soil (compost, sediment, and soil) 10.20% 0.00% 0.00% 0.00%