The proportion of foodborne illnesses associated with fresh produce has increased over the last decade.Economically effective and practical programs to reduce the risk of contamination are limited by gaps in our knowledge of the microbiological quality and safety of produce.In an effort to address this concern, the objectives of the first three studies were (i) to determine the quality and safety of produce sampled from the farm and packing sheds by enumerating microbial indicator organisms and testing for various pathogens; (ii) to examine the routes of microbial contamination in domestic and imported produce, and identify areas of potential contamination throughout production and processing; and (iii) to characterize the antimicrobial resistance profiles of Enterococcus spp. among fresh produce.Furthermore, in response to recent governmental testing recommendations for the seed sprout industry, the final study seeks to improve rapid detection methods of foodborne pathogens associated with raw sprouts. For the first study, produce samples were collected from the southern United States throughout production and processing and assayed by enumerative tests for total aerobic bacteria, total coliforms, total Enterococcus, and E. coli.These samples were also analyzed for the presence of Salmonella, Listeria monocytogenes, and E. coli O157:H7.Microbial levels remained relatively constant throughout processing for most of these products; however, for cilantro and parsley, total coliform levels increased during the packing process.For cantaloupe, microbial levels significantly increased from field through processing.Only Salmonella was detected among the produce samples. The second study analyzed the antibiotic resistance profiles of Enterococcus faecium and E. faecalis isolated from a subset of the product samples collected during the first study, all harvested in the southern U.S.E. faecium and E. faecalis isolates were screened for antibiotic resistance profiles using a panel of seventeen antibiotics.Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis.E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both human and animal clinical relevance.Thirty-four percent of the isolates had multiple drug resistance patterns, excluding intrinsic resistance. The third study evaluated the microbiological quality and safety of domestic and imported produce samples collected from packing sheds in the southern U.S., including microbial loads on environmental surface swabs.Antibiotic resistance profiles of E. faecium and E. faecalis strains isolated from the produce samples was also analyzed.Enumerative tests included total aerobic bacteria (APC), total coliforms, total Enterococcus, and E. coli.Produce samples were also analyzed for the presence of Salmonella, Listeria monocytogenes, Shigella, and E. coli O157:H7.The levels of microbial indicators remained constant in most cases, with increases in several commodities, throughout the wash process within the packing sheds.The degree of environmental contamination from swabs was similar to that found for fresh produce.No Salmonella, Shigella, or E. coli O157:H7 were detected from the produce samples.However, three samples were found positive for L. monocytogenes.Overall, a low degree of antibiotic resistance was found among Enterococcus isolates. The purpose of final study was to develop a simple method to pre-concentrate pathogens from sprouts and spent irrigation water to facilitate the direct (without prior cultural enrichment) detection of pathogens using the polymerase chain reaction (PCR).Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 in the range of 10⁻¹ to 10⁶ CFU/g or ml.The samples were then pre-concentrated by centrifugation, and the resulting precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization.Using primers targeting the inv A gene for serovar Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding levels as low as 10¹ CFU/g.PCR detection limits for both pathogens from spent irrigation water were 10⁻¹ CFU/ml, the equivalent of 10² CFU/liter. Taken together, this research contributes to a body of knowledge that will assist in the design of effective intervention strategies for the lowering the risk of foodborne disease associated with fresh produce.
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