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Title: Inactivation of E. coli O157:H7 attached to spinach harvester blade using bacteriophage

Author
item Patel, Jitu
item Sharma, Manan
item Millner, Patricia
item CALAWAY, TODD - US Department Of Agriculture (USDA)
item SINGH, MANPREET - University Of Auburn

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2010
Publication Date: 4/1/2011
Citation: Patel, J.R., Sharma, M., Millner, P.D., Calaway, T., Singh, M. 2011. Inactivation of E. coli O157:H7 attached to spinach harvester blade using bacteriophage. Foodborne Pathogens and Disease. 8(4):541-546.

Interpretive Summary: A large recent outbreak of E. coli O157:H7 infections associated with spinach has focused attention on potential mechanisms of contamination in the field, and possible mitigation strategies. Spinach contamination is of particular concern since there is not a disinfection or pathogen 'kill' step during the processing. One potential source of pathogen contamination is the band saw blade used for harvesting spinach in the field. We determined biofilm formation (a mass of bacterial growth) by E. coli O157:H7 on spinach harvester blades. Pieces of a spinach harvester blade were immersed in tubes containing spinach extract and E. coli O157:H7; the tubes were incubated at a constant room temperature (22°C) or a fluctuating temperature (30°C-day, 20°C-night). Adherence of E. coli O157:H7 to spinach blades was higher when incubated at a fluctuating temperature. The condition of the blade (new vs. rusty) did not influence E. coli O157:H7 adherence. Some blades were sprayed with E. coli O157-specific bacteriophages to determine the killing effect of bacteriophages. Application of bacteriophages reduced E. coli O157:H7 populations by 2.5 orders of magnitude after 10 min and 4 orders of magnitude after 120 min. Our study demonstrates that E. coli O157:H7 can attach and produce biofilms on spinach harvester blades; however, bacteriophages can reduce E. coli O157:H7 populations on spinach harvesting equipment. This research is of interest to food scientists and the leafy greens fresh cut industry.

Technical Abstract: Outbreaks associated with leafy greens have focused attention on the transfer of human pathogens to leafy greens during harvest with commercial equipment. Bacteriophages can kill bacteria and are suitable candidates for biocontrol of these pathogens. We determined biofilm formation by Escherichia coli O157:H7 on a spinach harvester blade immersed in spinach extract. Bacteriophages specific for E. coli O157:H7 were evaluated to kill cells in these biofilms. A cocktail of five nalidixic acid (NA)-resistant E. coli O157:H7 isolates was transferred in 25-ml 10 percent TSB or spinach extract. A piece of sterilized spinach harvester blade (2 x 1") was placed in a tube and incubated at room (22°C) or dynamic (30°C-day, 20°C-night) temperature. Bacterial populations in biofilms were determined by scraping the blade with a Teflon spatula, followed by spiral plating on SMAC containing 50 µg/ml NA (SMAC-NA). Two blades were sprayed on each side with a cocktail of E. coli O157-specific bacteriophages before scraping the blade, and subsequent plating on SMAC-NA. When inoculated at 1 log CFU/ml, E. coli O157:H7 adherence to blades after 24 and 48 h incubation at dynamic temperature (6.09 and 6.37 log CFU/ml) was significantly higher than when incubated at 22°C (4.84 and 5.68 log CFU/ml), respectively. E. coli O157:H7 populations recovered from blades after 24 and 48 h were similar (P>0.05) when inoculated at 4 log CFU/ml, regardless of incubation conditions. Application of bacteriophages reduced E. coli O157:H7 populations by 2.5 and 4 log CFU on blades after 10 and 120 min, respectively. Our study demonstrates that E. coli O157:H7 can attach and proliferate on spinach harvester blades under static and dynamic temperature conditions. Bacteriophages can reduce E. coli O157:H7 populations on spinach harvesting equipment.