Patricia D. Millner

Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water

Outbreaks of enterohemorrhagic Escherichia coli O157:H7 infections associated with lettuce and other leaf crops have occurred with increasing frequency in recent years. Contaminated manure and polluted irrigation water are probable vehicles for the pathogen in many outbreaks. In this study, the occurrence and persistence of E. coli O157:H7 in soil fertilized with contaminated poultry or bovine manure composts or treated with contaminated irrigation water and on lettuce and parsley grown on these soils under natural environmental conditions was determined. Twenty-five plots, each 1.8 by 4.6 m, were used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but treated with contaminated water) and five replication plots for each treatment. Three different types of compost, PM-5 (poultry manure compost), 338 (dairy manure compost), and NVIRO-4 (alkaline-stabilized dairy manure compost), and irrigation water were inoculated with an avirulent strain of E. coli O157:H7. Pathogen concentrations were 10(7) CFU/g of compost and 10(5) CFU/ml of water. Contaminated compost was applied to soil in the field as a strip at 4.5 metric tons per hectare on the day before lettuce and parsley seedlings were transplanted in late October 2002. Contaminated irrigation water was applied only once on the plants as a treatment in five plots for each crop at the rate of 2 liters per plot 3 weeks after the seedlings were transplanted. E. coli O157:H7 persisted for 154 to 217 days in soils amended with contaminated composts and was detected on lettuce and parsley for up to 77 and 177 days, respectively, after seedlings were planted. Very little difference was observed in E. coli O157:H7 persistence based on compost type alone. E. coli O157:H7 persisted longer (by > 60 days) in soil covered with parsley plants than in soil from lettuce plots, which were bare after lettuce was harvested. In all cases, E. coli O157:H7 in soil, regardless of source or crop type, persisted for > 5 months after application of contaminated compost or irrigation water.

Fate of Salmonella enterica Serovar Typhimurium on Carrots and Radishes Grown in Fields Treated with Contaminated Manure Composts or Irrigation Water

ABSTRACT Three different types of compost, PM-5 (poultry manure compost), 338 (dairy cattle manure compost), and NVIRO-4 (alkaline-pH-stabilized dairy cattle manure compost), and irrigation water were inoculated with an avirulent strain of Salmonella enterica serovar Typhimurium at 107 CFU g−1 and 105 CFU ml−1, respectively, to determine the persistence of salmonellae in soils containing these composts, in irrigation water, and also on carrots and radishes grown in these contaminated soils. A split-plot block design plan was used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but with contaminated water applied) and five replicates for a total of 25 plots for each crop, with each plot measuring 1.8 × 4.6 m. Salmonellae persisted for an extended period of time, with the bacteria surviving in soil samples for 203 to 231 days, and were detected after seeds were sown for 84 and 203 days on radishes and carrots, respectively. Salmonella survival was greatest in soil amended with poultry compost and least in soil containing alkaline-pH-stabilized dairy cattle manure compost. Survival profiles of Salmonella on vegetables and soil samples contaminated by irrigation water were similar to those observed when contamination occurred through compost. Hence, both contaminated manure compost and irrigation water can play an important role in contaminating soil and root vegetables with salmonellae for several months.

Persistence of Salmonella enterica Serovar Typhimurium on Lettuce and Parsley and in Soils on Which They Were Grown in Fields Treated with Contaminated Manure Composts or Irrigation Water

There are many sources of pathogen contamination of vegetable crops in the field that include manure used as fertilizer and irrigation water. An avirulent strain of Salmonella enterica serovar Typhimurium was added to three different types of composts-PM-5 (poultry manure compost), 338 (dairy manure compost), and NVIRO-4 (alkaline stabilized dairy manure compost)-and irrigation water at 10(7) colony forming units (cfu)/g and 10(5) cfu/mL, respectively, to determine under field conditions the persistence of salmonellae in soils treated with these composts or irrigation water, and also on leaf lettuce and parsley grown on such treated soil. Contaminated compost was applied to soil in the field as a strip at a rate of 4.5 metric tons/hectare on the day before lettuce and parsley seedlings were transplanted. Contaminated irrigation water was applied only once on the plants at the rate of 2 liters per plot on the same day after the seedlings were transplanted. Twenty-five plots, each measuring 1.8 x 4.6 meters, were used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but applied with contaminated water) and five replication plots for each treatment. Salmonella persisted for 161 and up to 231 days in soils amended with contaminated composts on which lettuce and parsley, respectively, were grown, and was detected for up to 63 days and 231 days on lettuce and parsley, respectively. The type of contaminated compost had minimal effect on the persistence of S. Typhimurium in soil. Occurrence of Salmonella on vegetables and survival in soil on which these vegetables were grown, irrespective of source of contamination through irrigation water or compost, were similar, suggesting both contaminated manure compost and irrigation water can play important roles in contaminating soil and vegetables with Salmonella for an extended period of time.

Bioaerosols associated with animal production operations.

Air emissions from animal housing and manure management operations include a complex mixture of biological, microbial, and inorganic particulates along with odorous volatile compounds. This report highlights the state of current issues, technical knowledge, and remaining challenges to be addressed in evaluating the impacts of airborne microorganisms, dusts, and odorants on animals and workers at animal production facilities and nearby communities. Reports documenting bioaerosol measurements illustrate some of the technical issues related to sample collection, analysis, as well as dispersion and transport to off-farm locations. Approaches to analysis, mitigation and modeling transport are discussed in the context of the risk reduction and management of airborne spread of bioaerosols from animal operations. The need for standardization and validation of bioaerosol collection and analytical techniques for indoor as well as outdoor animal agriculture settings is critical to evaluation of health effects from modern animal production systems that are increasingly situated near communities.

Development of a second-generation environmentally superior technology for treatment of swine manure in the USA

New swine waste management systems in North Carolina need to meet high performance standards of an environmentally superior technology (EST) regarding nitrogen, phosphorus, heavy metals, pathogens, ammonia and odor emissions, and remain affordable and simple to operate. The objective of this study was to develop a second-generation treatment system that can achieve high EST standards at reduced costs. The system used solids separation, nitrification/denitrification and phosphorus removal/disinfection, and was demonstrated at full-scale on a 5145-head swine farm during three production cycles (15-months). Removal efficiencies were: 98% suspended solids, 97% ammonia, 95% phosphorus, 99% copper and zinc, 99.9% odors, and 99.99% pathogens. The system met EST standards at 1/3 the cost of the previous version. Animal health and productivity were enhanced; hog sales increased 32,900 kg/cycle (5.6%). These results demonstrated that: (1) significant cost reductions were achieved by on-farm implementation and continued engineering improvements, and (2) the new waste management system substantially benefited livestock productivity.

A novel approach to investigate the uptake and internalization of Escherichia coli O157:H7 in spinach cultivated in soil and hydroponic medium.

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. A Tn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein-labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 10(3) or 10(7) CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 10(7) CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

A pilot plant scale evaluation of a new process aid for enhancing chlorine efficacy against pathogen survival and cross-contamination during produce wash

Developing food safety intervention technology that can be readily adopted by the industry often requires test conditions that match as closely as possible to those of commercial food processing operations; yet biosafety risks inherent in pathogen studies constrain most experiments to laboratory settings. In this study, we report the first semi-commercial pilot-scale evaluation of a new process aid, T128, for its impact on enhancing the antimicrobial efficacy of chlorinated wash water against pathogen survival and cross-contamination. A non-pathogenic, BSL-1, strain of Escherichia coli O157:H7 was inoculated onto freshly harvested baby spinach leaves and washed with large amounts of freshly cut un-inoculated iceberg lettuce shreds in wash water with free chlorine periodically replenished, in the presence or absence of T128. Changes in water quality and pathogen survival and cross-contamination were monitored at every 2 min intervals for up to 36 min for each treatment during the wash operation. Results indicated that the use of T128 did not significantly (P>0.05) influence the rate of wash water deterioration, nor the pathogen populations remaining on the inoculated spinach leaves. However, in the absence of T128 (control), survival of E. coli O157:H7 in wash water and cross-contamination of un-inoculated lettuce frequently occurred when free chlorine in solution dropped below 1mg/l during the wash process. In contrast, the use of T128 significantly reduced the occurrence of E. coli O157:H7 surviving in wash water and of cross-contamination to un-inoculated shredded iceberg lettuce under the same operational conditions, suggesting that the application of T128 in a chlorine-based fresh produce sanitization system could increase the safety margin of process control on fresh-cut operations.

Colonization and Internalization of Salmonella enterica in tomato plants

ABSTRACT The consumption of fresh tomatoes has been linked to numerous food-borne outbreaks involving various serovars of Salmonella enterica. Recent advances in our understanding of plant-microbe interactions have shown that human enteric pathogenic bacteria, including S. enterica, are adapted to survive in the plant environment. In this study, tomato plants (Solanum lycopersicum cv. Micro-Tom) grown in sandy loam soil from Virginias eastern shore (VES) were inoculated with S. enterica serovars to evaluate plausible internalization routes and to determine if there is any niche fitness for certain serovars. Both infested soil and contaminated blossoms can lead to low internal levels of fruit contamination with Salmonella. Salmonella serovars demonstrated a great ability to survive in environments under tomato cultivation, not only in soil but also on different parts of the tomato plant. Of the five serovars investigated, Salmonella enterica serovars Newport and Javiana were dominant in sandy loam soil, while Salmonella enterica serovars Montevideo and Newport were more prevalent on leaves and blossoms. It was also observed that Salmonella enterica serovar Typhimurium had a poor rate of survival in all the plant parts examined here, suggesting that postharvest contamination routes are more likely in S. Typhimurium contamination of tomato fruit. Conversely, S. Newport was the most prevalent serovar recovered in both the tomato rhizosphere and phyllosphere. Plants that were recently transplanted (within 3 days) had an increase in observable internalized bacteria, suggesting that plants were more susceptible to internalization right after transplant. These findings suggest that the particular Salmonella serovar and the growth stage of the plant were important factors for internalization through the root system.

Dynamic Effects of Free Chlorine Concentration, Organic Load, and Exposure Time on the Inactivation of Salmonella, Escherichia coli O157:H7, and Non-O157 Shiga Toxin–Producing E. coli†

This study evaluated the dynamic effects of free-chlorine (FC) concentration, contact time, and organic load on the inactivation of Salmonella, Escherichia coli O157:H7, and non-O157 Shiga toxin-producing E. coli (STEC) in suspension. Bacterial cells from four strains each of Salmonella, E. coli O157:H7, and non-O157 STEC were inoculated separately or as a multistrain cocktail into solutions with varying FC concentrations. Lettuce or tomato extract was used to simulate the organic matter present during commercial fresh and fresh-cut produce wash operations. After exposure to FC for various lengths of time, the bacterial survival and water-quality changes were determined. In the absence of organic matter in a wash solution, pathogen inactivation is primarily a function of initial FC concentration (P < 0.0001), exposure time (P < 0.0001), and pathogen strains (P < 0.0001). In general, an over 4.5-log CFU/ml pathogen reduction was found after exposure to >0.5 mg/liter FC for over 30 s, or to >1.0 mg/liter FC for over 5 s. When the combination of FC concentration and contact time were less than or equal to the above conditions, survival of pathogens was strain dependant and ranked as: Salmonella > E. coli O157:H7 > non-O157 STEC. When organic matter was present in the wash solution, pathogen inactivation efficacy was specifically dependent on the residual FC concentration, which directly relates to both the initial FC concentration and the organic load. Prevention of pathogen survival in chlorinated produce wash solutions can be achieved by maintaining sufficient FC concentration and reducing the accumulation of organic matter.

Analysis of fungal communities by sole carbon source utilization profiles.

A simple method for characterization of fungal communities in environmental samples was developed. Dilute suspensions of samples in 0.2% agar containing three different antibiotics were pipetted into 96-well plates (Biolog SF-N) containing a diverse collection of 95 different carbon sources. The plates were incubated for 4-12 days at 22 degrees C and the absorbance measured at 650 nm. Canonical variates analysis was then used to analyze the multivariate data. This method allowed fungal communities in rhizosphere soil of corn and soybean to be distinguished according to soil and plant type. Data taken at a single time-point, which varied greatly in total absorbance of the plate, separated rhizosphere samples primarily by soil type. When multiple time-points were combined to keep the total absorbance constant, differences in substrate utilization patterns due to different plant types could be distinguished. The method was also applicable to analysis of phylloplane and compost fungal communities. This method is readily applied to large numbers of samples and should be useful for community analysis in a variety of agricultural and ecological studies.