<OL> <LI> To determine ecological factors influencing attraction of two free-living nematodes, Caenorhabditis elegans and Diploscapter sp., to Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes; <LI> To determine effects of manure, compost, and manure-amended soil inoculated with foodborne pathogens on growth and reproduction of free-living nematodes; <LI> To determine dispersal and survival of pathogens after death of nematodes; <LI> To determine effectiveness of produce sanitizers in killing foodborne pathogens internalized by free-living nematodes.
APPROACH: Cells of pathogenic bacteria tagged with GFP (several strains each of Salmonella, E. coli 0157:H7, and L. monocytogenes) will be used in studies to monitor their dispersal by C. elegans. Studies will be extended to include another free-living soil nematode, Diploscapter sp. Nematode movement toward colonies, manure/soil, or pieces of fresh produce on agar will be video captured and images analysed at predetermined times over a 16-to 20-min period, followed by a 24 h exam to check their dispersal; and Attracting Activity Index will be calculated for each bacterial strain. A second assay will be used to determine the survival of any ingested bacteria after nematode surfaces are washed and worms are killed.
PROGRESS: 2002/09 TO 2005/08
4d Progress report. This report describes work conducted under a reimbursable agreement with the University of Georgia Center for Food Safety, within the scope of the parent project 1265-32630-002-00D titled "Pathogen Tansport and Dissemination from Manure". A study of the persistence of Escherichia coli O157:H7 and salmonellae in the gut of a free-living nematode, Caenorhabditis elegans, as affected by temperature and relative humidity was completed. This study also examined whether infected worms would transmit Salmonella enterica serotype Newport to progeny and uninfected worms. Worms were fed cells of a non-pathogenic strain of E. coli (OP50), E. coli O157:H7, S. enterica serotype Newport, and S. enterica serotype Poona, followed by incubating at 4, 20, or 37 C for up to 5 days. Initial populations of ingested pathogens significantly increased by up to 2.93 log10 cfu/worm within 1 day at 20 C on K agar and remained constant for an additional 4 days. When worms were placed on Bacto agar, populations of ingested pathogens remained constant at 4 C, decreased significantly at 20 C, and increased significantly at 37 C within 3 days. Worms fed E. coli OP50 or S. Newport were incubated at 4 or 20 C at relative humidities of 33%, 75%, or 98% to determine survival characteristics of ingested bacteria. Fewer cells of the pathogens survived incubation at 33% relative humidity compared to higher relative humidities. Populations of ingested E. coli OP50 and S. Newport decreased by up to 1.65 and 3.44 log10 cfu/worm, respectively, in worms incubated at 20 C and 33% relative humidity. Placement together on K agar of adult worms, labeled with green fluorescent protein (gfp) in the pharynx area, that had ingested gfp-labeled S. Newport and uninfected wild type worms resulted in transfer of the pathogen to gut of wild type worms. S. Newport was isolated from C. elegans two generations removed from exposure to the pathogen. Results of these studies show that C. elegans may serve as a temporary reservoir of foodborne pathogens, and could perhaps be a vector for contaminating preharvest fruits and vegetables, thus potentially increasing the risk of enteric infections associated with consumption of raw produce. A study was undertaken to evaluate the efficacy of cleaners and sanitizers in killing Salmonella Newport in the gut of C. elegans. Adult worms were fed nalidixic acid-adapted cells of Escherichia coli OP50 (control) or S. Newport for 24 h, washed, placed on paper discs, and incubated at 4 or 20°C and relative humidites of 33, 75, or 98% for 24 h. Two commercial cleaners (Enforce and K Foam Lo) and four sanitizers (2% acetic acid, 2% lactic acid, Sanova, and chlorine [50 and 200 µg/ml]) were applied to worms for 0, 2, or 10 min. Populations of E. coli and S. Newport (CFU/worm) in untreated and treated worms were determined by sonicating worms in 0.1% peptone and surface plating suspensions of released cells on tryptic soy agar containing nalidixic acid. Populations of S. Newport in worms exposed to 33 or 75% relative humidity at 4°C or 33% relative humidity at 20°C were significantly (P less than 0. 05) lower than the number surviving exposure to higher relative humidities at respective temperatures. In general, treatment of worms with cleaners and sanitizers was effective in significantly (P less than 0.05) reducing the population of S. Newport in desiccated worms. Results indicate that temperature and relative humidity influence S. Newport survival in the gut of C. elegans but cleaners and sanitizers may not eliminate the pathogen from desiccated worms. Caenorhabditis elegans, a free-living nematode found in soil, has been shown to ingest human enteric pathogens, thereby potentially serving as a vector for preharvest contamination of fruits and vegetables. A study was undertaken to evaluate the efficacy of cleaners and sanitizers in killing Salmonella enterica serotype Newport in the gut of C. elegans. Adult worms were fed nalidixic acid-adapted cells of Escherichia coli OP50 (control) or Salmonella Newport for 24 h, washed, placed on paper discs, and incubated at temperatures of 4 or 20 degrees C and relative humidities of 33 or 98% for 24 h. Two commercial cleaners (Enforce and K Foam Lo) and four sanitizers (2% acetic acid, 2% lactic acid, Sanova, and chlorine [50 and 200 microg/ml]) were applied to worms for 0, 2, or 10 min. Populations of E. coli and Salmonella Newport (CFU per worm) in untreated and treated worms were determined by sonicating worms in 0.1% peptone and surface plating suspensions of released cells on tryptic soy agar containing nalidixic acid. Populations of Salmonella Newport in worms exposed to 33 or 98% relative humidity at 4 degrees or 33% relative humidity at 20 degrees C were significantly (P < or = 0.05) lower than the number surviving exposure to 98% relative humidity at 20 degrees C. In general, treatment of desiccated worms with cleaners and sanitizers was effective in significantly (P < or = 0.05) reducing the number of ingested Salmonella Newport. Results indicate that temperature and relative humidity influence the survival of Salmonella Newport in the gut of C. elegans, and cleaners and sanitizers may not eliminate the pathogen.