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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL ECOLOGY AND SAFETY OF FRESH ON-FARM ORGANICALLY GROWN PRODUCE Title: Persistence of somatic and F-specific coliphages, potential indicators of fecal contamination, on spinach foliar tissue

Authors
item Lilly, Allison -
item Sapkota, Amy -
item Roberts, Cheryl
item Ferguson, Sean
item Shelton, Daniel
item Ingram, David
item Sharma, Manan

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: March 21, 2011
Publication Date: April 27, 2011
Citation: Lilly, A., Sapkota, A., Roberts, C.L., Ferguson, S.E., Shelton, D.R., Ingram, D.T., Sharma, M. 2011. Persistence of somatic and F-specific coliphages, potential indicators of fecal contamination, on spinach foliar tissue. [abstract]. BARC Poster Day. .

Technical Abstract: Recent outbreaks of foodborne illness have been linked to consumption of fresh leafy greens. E. coli is commonly used as the indicator microorganism for fecal contamination on leafy greens. However, alternative microorganisms, such as coliphages, may deserve consideration as indicators of fecal contamination on produce. The objective was to determine if the recovery of somatic and F-specific coliphages was possible from inoculated spinach plants, and which of three methods was most sensitive in their detection. The persistence of coliphages on spinach plants was also evaluated. For the optimization of recovery methods, somatic (phiX174) and F-specific (MS2) coliphages were prepared and spot-inoculated on 4-week old spinach plants in growth chambers at 105 PFU/plant. Foliar tissue was aseptically harvested and homogenized in either sterile water, sterile magnesium (SM) buffer or 3% beef extract (BE). A soft agar (0.60% LB agar) overlay method with E. coli host strains CN13 (for phiX174) and F-amp (for MS2) was used to determine phage recovery from spinach plants five times over 20 days. On each day of analysis, three plants inoculated with each coliphage were homogenized in each diluent. MS2 recovery was 3.42 log PFU/plant on day 0. After 4 days, MS2 phage declined by 1.78 log PFU/plant to below the level of detection (1.65 log PFU/plant), but was recovered by enrichment for two weeks. Somatic coliphages (phiX174) persisted on spinach plants for 20 days, declining by 2.22 log PFU/plant from 5.42 log PFU/plant on day 0. All diluents recovered both coliphages from spinach. Overall, BE was significantly more (p < 0.05) effective than water or SM buffer in recovering phiX174 from spinach leaves. The persistence at varying inoculation levels of phiX174 and MS2 coliphages at 105 PFU/plant and 102 PFU/plant was then determined. Phage recovery from spinach plants was analyzed six times over 22 days with three plants inoculated with each coliphage at each inoculation level. During the evaluation of persistence at varying inoculation levels, MS2 recovery for the low inoculation on day 0 was 3.37 log PFU/plant for MS2 and 2.43 log PFU/plant for phiX174. After 22 days, neither MS2 nor phiX174 were detectable from plants inoculated with 102 PFU/plant. For plants inoculated with 105 PFU/plant, phiX174 coliphages persisted on spinach plants for 22 days, declining by 3.27 log PFU/plant from 4.57 log PFU/plant on day 0. For MS2, recovery was 5.88 log PFU on day 0 and declined below the level of detection by day 22. . Uninoculated plants did not contain coliphages. The recovery of phiX174 and MS2 with BE after 22 days indicates the potential utility of coliphages as indicators of fecal contamination on leafy greens.

Last Modified: 12/22/2014
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