INFLUENCE OF COMPOST PARTICLE SIZE ON PATHOGEN SURVIVAL UNDER GREENHOUSE CONDITION

The number of foodborne pathogen outbreaks related to fresh produce has increased significantly in recent decades. Animal waste directly applied to agricultural field is one of the possible contamination sources for fresh produce. Composting is one of the recommended means for waste treatments to eliminate or reduce pathogens in manure on farms. Although pathogens can be eliminated by proper composting process, pathogens are able to survive, recolonize and regrow on compost heap surfaces under certain conditions. Due to the outdoor nature of composting process, bioaerosols with different particle sizes can be generated on compost surfaces which can carry pathogens, travel via air and contaminate fresh produce field nearby. This study was to investigate the survival of Escherichia coli O157:H7 and Salmonella Typhimurium in dairy compost with different particle sizes as affected by initial moisture content and seasonality under greenhouse conditions. Also, in order to understand the pathogen survival strategies, microscopic observations of the interactions between bacterial cells and compost particles were explored using several approaches. The mixture of E. coli O157:H7 and avirulent S. Typhimurium strains were inoculated into the finished composts with initial moisture content of 20, 30 and 40%. Then, the finished compost samples were sieved into three portions with particle sizes of \u3e1000, 500-1000 and \u3c500 \u3eμm, and stored in greenhouse for 30 days. At selected intervals, compost samples were tested for pathogen population. For the microscope study, the morphological and surface characteristics of compost particles with different particle sizes were analyzed by a surface profiler. Then the green fluorescence proteins (GFP)-labeled E. coli O157 inoculated compost samples were observed under a fluorescence microscope, followed by spectral analysis and unmixing. Our next approach was to use immunofluorescence (IF) protocol for enhancing the GFP signal with different fluorescence dyes, such as AlexaFluor 594 and quantum dots. For the greenhouse study, the moisture contents in compost samples dropped rapidly to under 10% within 5 days of storage followed by gradual decline till 30 days in all treatments. For compost with moisture contents of 20 and 30%, the average Salmonella reductions in compost with particle sizes of \u3e1000, 500-1000 and \u3c500 \u3eμm were 2.15, 2.27 and 2.47 log CFU g-1 within 5 days of storage in summer, respectively, as compared with 1.60, 2.03 and 2.26 log CFU g-1 in late fall, and 2.61, 3.33 and 3.67 log CFU g-1 in winter, respectively. For samples with initial moisture content of 40%, the Salmonella reductions in compost with particle sizes of \u3e1000 and \u3c1000 \u3eμm were 2.14 and 3.17 log CFU g-1 within 5 days in summer, respectively, as compared with 3.17 and 3.16 log CFU g-1 in late fall, and 2.93 and 3.36 log CFU g-1 in winter, respectively. For compost with moisture contents of 20 and 30%, the average E. coli O157 reductions in compost with particle sizes of \u3e1000, 500-1000 and \u3c500 \u3eμm were 1.98, 2.30 and 2.54 log CFU g-1 within 5 days of storage in summer, respectively, as compared with 1.70, 2.56 and 2.90 log CFU g-1 in winter, respectively. For samples with initial moisture content of 40%, the E. coli O157 reductions in compost with particle sizes of \u3e1000 and \u3c1000 \u3eμm were 2.08 and 2.48 log CFU g-1 within 5 days in summer, respectively, as compared with 2.20 and 2.84 log CFU g-1 in winter, respectively. In fluorescence microscope observation, the sensitivity is negatively affected by broad spectra and high intensity from autofluorescence of compost matrix. For spectra analysis, the emission wavelength and intensity of both GFP and autofluorescence from compost were measured and compared statistically, however the method could not provide information about location of individual cells in compost matrix visually. Additionally, IF results showed that GFP in E. coli O157: H7 and autofluorescence of compost can be differentiated both visually and quantitatively according to the signal intensity. However, Qdots results showed that it is an more effective treatment for enhancing signals of GFP-labeled E. coli O157 in compost matrix compared with AlexaFluor based IF method. Our results revealed that compost with larger particle size supports pathogen survival better than the compost with small particle size, and the initial rapid moisture loss in compost may contribute to fast inactivation of pathogens in the finished compost. Green fluorescence protein (GFP) expressed by bacterial cells is not sufficient to differentiate target cells from background autofluorescence in compost. Spectra analysis can separate overlap emissions from GFP-labeled pathogen and compost statistically, however, amplification of GFP signal by immunofluorescence methods, esp. Qdots, can improve the detection of target cells