Karl R. Matthews

Transmission of Escherichia coli O157:H7 from Contaminated Manure and Irrigation Water to Lettuce Plant Tissue and Its Subsequent Internalization

ABSTRACT The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants. E. coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that E. coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.

Vesicle-mediated transfer of virulence genes from Escherichia coli O157:H7 to other enteric bacteria.

ABSTRACT Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, andgfp), chromosomal DNA (uidA andeaeA), and phage DNA (stx1 andstx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 103 transformants μg of DNA−1) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.

A reverse transcriptase‐polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes

Aims: To determine suitable target genes for detection of the pathogen Escherichia coli O157:H7 by reverse transcriptase‐polymerase chain reaction (RT‐PCR).

Effect of Irrigation Method on Transmission to and Persistence of Escherichia coli O157:H7 on Lettuce

In this study, the transmission of Escherichia coli O157:H7 to lettuce plants through spray and surface irrigation was demonstrated. For all treatments combined, the number of plants testing positive following a single exposure to E. coli O157: H7 through spray irrigation (29 of 32 plants) was larger than the number testing positive following surface irrigation (6 of 32 plants). E. coli O157:H7 persisted on 9 of 11 plants for 20 days following spray irrigation with contaminated water. Immersion of harvested lettuce heads for 1 min in a 200 ppm chlorine solution did not eliminate all E. coli O157:H7 cells. The results of this study suggest that regardless of the irrigation method used, crops can become contaminated; therefore, the irrigation of food crops with water of unknown microbial quality should be avoided.

Use of green fluorescent protein expressing Salmonella Stanley to investigate survival, spatial location, and control on alfalfa sprouts.

Laser scanning confocal microscopy (LSCM) was used to observe the interaction of Salmonella Stanley with alfalfa sprouts. The green fluorescent protein (gfp) gene was integrated into the chromosome of Salmonella Stanley for constitutive expression, thereby eliminating problems of plasmid stability and loss of signal. Alfalfa seeds were inoculated by immersion in a suspension of Salmonella Stanley (ca. 10(7) CFU/ml) for 5 min at 22 degrees C. Epifluorescence microscopy demonstrated the presence of target bacteria on the surface of sprouts. LSCM demonstrated bacteria present at a depth of 12 microm within intact sprout tissue. An initial population of ca. 10(4) CFU/g seed increased to 7.0 log CFU/g during a 24-h germination period and then decreased to 4.9 log CFU/g during a 144-h sprouting period. Populations of Salmonella Stanley on alfalfa seeds decreased from 5.2 to 4.1 log CFU/g and from 5.2 to 2.8 log CFU/g for seeds stored 60 days at 5 and 22 degrees C, respectively. The efficacy of 100, 200, 500, or 2,000 ppm chlorine in killing Salmonella Stanley associated with sprouts was determined. Treatment of sprouts in 2,000 ppm chlorine for 2 or 5 min caused a significant reduction in populations of Salmonella Stanley. Influence of storage on Salmonella Stanley populations was investigated by storing sprouts 4 days at 4 degrees C. The initial population (7.76 log CFU/g) of Salmonella Stanley on mature sprouts decreased (7.67 log CFU/g) only slightly. Cross-contamination during harvest was investigated by harvesting contaminated sprouts, then directly harvesting noncontaminated sprouts. This process resulted in the transfer of ca. 10(5) CFU/g Salmonella Stanley to the noncontaminated sprouts.

The synergistic effect of nisin and lactoferrin on the inhibition of Listeria monocytogenes and Escherichia coli O157:H7

Aims:  The goal of this study was to determine whether nisin and lactoferrin would act synergistically to inhibit the growth of Listeria monocytogenes and Escherichia coli O157:H7.

Transfer of Escherichia coli O157:H7 from soil, water, and manure contaminated with low numbers of the pathogen to lettuce plants.

The sources of contamination of leafy greens remain unclear, but it is evident that contaminated water, soil amendments, and wildlife likely contribute. The objective of the present study was to determine transfer of low numbers of Escherichia coli O157:H7 from soil, manure-amended soil, and water to growing lettuce plants. Lettuce plants, young (12 days of age) or mature (30 days of age), were grown in soil, manure-amended soil, or irrigated with water containing 10(1), 10(2), 10(3), or 10(4) CFU E. coli O157:H7 per g or ml. Harvested plants were processed to determine whether E. coli O157:H7 was associated with the entire plant or within internal locations. Young plants (12 days) were harvested at 1, 10, 20, and 30 days postexposure. No samples were positive for E. coli O157:H7 after direct plating of serial dilutions. Enrichment of all samples from young plants exposed to contaminated soil, manure-amended soil, and irrigation water demonstrated that approximately 21% (113 of 552) of plants were positive for E. coli O157:H7. Approximately 30% (36 of 120) of the mature plants initially irrigated with or grown in contaminated soil (including manure-amended soil) for 15 days were positive for E. coli O157:H7. Based on sterilization of surface tissue, E. coli O157:H7 was in protected locations of lettuce tissue. The results suggest that lettuce exposed to, and grown in the presence of, low numbers of E. coli O157:H7 may become contaminated and thus present a human health risk.

Antibacterial activity of pepsin-digested lactoferrin on foodborne pathogens in buffered broth systems and ultra-high temperature milk with EDTA

Aims: To evaluate the antimicrobial activity in peptone yeast extract glucose (PYG) broth and ultra‐high temperature (UHT) milk of bovine lactoferrin hydrolysate (LFH) with pepsin against the foodborne pathogens Salmonella Stanley, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.

Efficacy of chlorine and calcinated calcium treatment of alfalfa seeds and sprouts to eliminate Salmonella

The efficacy of a 20,000 ppm calcium hypochlorite treatment of alfalfa seeds artificially contaminated with Salmonella was studied. Salmonella populations reached >7.0 log on sprouts grown from seeds artificially contaminated with Salmonella and then treated with 20,000 ppm Ca(OCl)(2). The efficacy of spray application of chlorine (100 ppm) to eliminate Salmonella during germination and growth of alfalfa was assessed. Alfalfa seed artificially contaminated with Salmonella was treated at germination, on day 2 or day 4, or for the duration of the growth period. Spray application of 100 ppm chlorine at germination, day 2, or day 4 of growth was minimally effective resulting in approximately a 0.5-log decrease in population of Salmonella. Treatment on each of the 4 days of growth reduced populations of Salmonella by only 1.5 log. Combined treatment of seeds with 20,000 ppm Ca(OCl)(2) and followed by 100 ppm chlorine or calcinated calcium during germination and sprout growth did not eliminate Salmonella.

Effect of the food production chain from farm practices to vegetable processing on outbreak incidence.

The popularity in the consumption of fresh and fresh‐cut vegetables continues to increase globally. Fresh vegetables are an integral part of a healthy diet, providing vitamins, minerals, antioxidants and other health‐promoting compounds. The diversity of fresh vegetables and packaging formats (spring mix in clamshell container, bagged heads of lettuce) support increased consumption. Unfortunately, vegetable production and processing practices are not sufficient to ensure complete microbial safety. This review highlights a few specific areas that require greater attention and research. Selected outbreaks are presented to emphasize the need for science‐based ‘best practices’. Laboratory and field studies have focused on inactivation of pathogens associated with manure in liquid, slurry or solid forms. As production practices change, other forms and types of soil amendments are being used more prevalently. Information regarding the microbial safety of fish emulsion and pellet form of manure is limited. The topic of global climate change is controversial, but the potential effect on agriculture cannot be ignored. Changes in temperature, precipitation, humidity and wind can impact crops and the microorganisms that are associated with production environments. Climate change could potentially enhance the ability of pathogens to survive and persist in soil, water and crops, increasing human health risks. Limited research has focused on the prevalence and behaviour of viruses in pre and post‐harvest environments and on vegetable commodities. Globally, viruses are a major cause of foodborne illnesses, but are seldom tested for in soil, soil amendments, manure and crops. Greater attention must also be given to the improvement in the microbial quality of seeds used in sprout production. Human pathogens associated with seeds can result in contamination of sprouts intended for human consumption, even when all appropriate ‘best practices’ are used by sprout growers.