Comparison of Bacteria on Surfaces of Iron Rich and Iron Poor Vegetables

1. Comparison of Bacteria on Surfaces of Iron Rich and Iron Poor Vegetables Jasmine Whitaker, Lakiyah Campbell, Scott Harrison, Wiley Hitchcock, Joseph Whittaker, Mary Smith and Jian HanBiology Department, College of Arts and Sciences, North Carolina Agricultural and Technical State University Results Conclusion Abstract Background cont. • Table 2. Iron-poor and iron-rich vegetables from which Gram negative and Gram-positive pathogens have been isolated and reported. Iron-poor: <0.5 mg per cup. Iron-rich: ≥ 0.5 mg per cup. Adapted from Buck et al 2003 and USDA 2004. Iron is required by cellular organisms for energetic function and other critical pathways. We investigated whether bacterial growth and variety is influenced by the relative presence of iron in different grocery store vegetables. In this study, the iron-rich vegetables were mustard green, the iron-poor vegetables were cucumber, and the iron-moderate vegetables were broccoli and rutabaga. Bacterial densities on the surface of these vegetables were measured by colony counting, and bacteria from these colonies were examined further by Gram staining. We found mustard green to have the most bacteria. Bacteria from the iron-rich and iron-moderate vegetables were both Gram positive and negative, while bacteria on cucumber were predominantly Gram negative. The abundance and variety of bacterial strains on vegetables therefore appears to be affected by the availability of iron, and this finding serves to motivate further investigation of bacteria across a wider range of plants based on nucleic acid sequence analysis, direct microscopic counting, and quantification of iron availability. • The iron-rich vegetable, mustard green, contained the most bacteria per measured sample (7,774). This was almost 300% more than that found for the iron-moderate vegetable rutabaga. There may be additional factors other than iron influencing abundance of bacteria on vegetables. • Bacteria on iron-rich vegetables showed more variety. They contained both Gram positive and Gram negative bacteria with various arrangements and morphologies. Gram-negative bacteria predominated in the iron-poor vegetable, cucumber, consistent with our analysis of reports of food-borne pathogens where Gram-negative bacteria were more frequently found on iron-poor vegetables. • Future studies will include broader sampling of different vegetables, nucleic acid sequence analysis of bacteria, direct microscopic counting, and quantification of iron availability on vegetable surfaces and tissues. 1A 1B Background References • Iron is an important mineral essential for the human body in order to perform functions such as oxygen delivery, energy production, and DNA synthesis. Adequate iron levels in the body ensure healthy organ and muscle function, and provide energy required to perform daily activities. • Iron can be obtained from various dietary resources. Table 1 shows the iron contents for serving amounts of different vegetables (1 cup = 240 mL; USDA 2004). • Table 1. Iron content in some vegetables • U.S. Department of Agriculture, Agricultural Research Service. 2004. USDA National Nutrient Database for Standard Reference, Release 17. Nutrient Data Laboratory Home Page, http://www.nal.usda.gov/fnic/foodcomp • Holt JG, Bergey DH, and Krieg NR. (1994) Bergey's Manual of Determinative Bacteriology. Lippincott Williams & Wilkins. ISBN# 0683006037. • Ryan K, Ray CG, Ahmad N, Drew WL, and Plorde J. (2004) Sherris Medical Microbiology (4thed). McGraw Hill. pp232-233. • Buck JW, Walcott RR, and Beuchat LR. (2003) Recent Trends in Microbiological Safety of Fruits and Vegetables. Plant Health Progress PMN. • Skaar EP. (2010) The battle for iron between bacterial pathogens and their vertebrate hosts. PLoSPathog 6(8): e1000949. Figure 1 Comparison of bacteria colonies for different vegetables. 1A: bacterial colonies on a nutrient agar plate. 1B: Mean number of bacterial colonies from sampling different types of vegetables in all stores. Each error bar represents the standard error of the mean. 2A 2B Hypothesis The hypothesis of this study is that the amount of iron in vegetables will influence the types of bacteria on those vegetables. 2C 2D • Materials and Methods • Vegetables were collected from three chains of grocery stores: four Harris Teeter stores, four Food Lion stores, and three Wal-Mart stores. Vegetables selected for the study were broccoli, cucumber, mustard green and rutabaga. Two samples of each vegetable were selected from each store and total of 88 samples were collected in this study. • The surface of the vegetables were swabbed with a sterile cotton Q-tip pre-dipped in phosphate buffer saline. Samples were stored in LB broth at 4ºC overnight. • Ten µl was taken from the stock and, with 1:100 dilution, plated on a nutrient agar plates. Plates were incubated at 37°C for 48 hours. After incubation, the bacterial colonies were quantified. • Representative colonies of different morphologies were selected and analyzed by Gram stain (Holt et al. 1994; Ryan et al. 2004). Digital images were made at 1000x magnification with a light compound microscope using the software, Axio Vision Rel 4.8. Acknowledgements 2F 2E This study is supported by Interdisciplinary Training for Undergraduates in Biological and Mathematical Sciences (UBM) Program (Grant 0634598 and 1029426 from the National Science Foundation) • Iron an essential micronutrient bacteria need to survive, and must acquire from their environments or hosts (Skaar 2010). Food-borne bacterial pathogens have been found on both iron-poor and iron-rich vegetables as shown in Table 2. There may be some association between the type of pathogen and availability of iron for different types of vegetables. Only 6 of the 19 reports of Gram positive pathogens (32%) occurred on iron-poor vegetables while 19 of 39 reports of Gram negative pathogens (49%) occurred on iron-poor vegetables. Figure 2 Bacteria identified by Gram stain. 2A: gram positive, long rods (bacilli), in chain arrangements from bacteria found in broccoli sample. 2B: gram positive, short rods, sporadically arranged bacteria found from mustard green samples. 2C: gram negative, rounded spheres (cocci) as well as short rods (bacilli) isolated fromrutabaga samples. 2D: gram negative, short rods (bacilli) with rounded ends; bacteria isolated from rutabaga. 2E: gram positive, spheres (coccus), and tightly clustered bacteria found from broccoli samples (possibly genus Staphylococcus). 2F: gram positive, slightly elongated spheres (cocci), and tightly clustered bacteria found from mustard greens samples (possibly genus Staphylococcus).