Efficacy of post-harvest rinsing and bleach disinfection of E. coli O157:H7 on spinach leaf surfaces

Protecting fresh-packed produce microbiological safety against pre- and post-harvest microbial pathogen contamination requires innovative antimicrobial strategies. Although largely ignored in the scientific literature, there exists the potential for gross failure in food safety protection of fresh fruits and vegetables leading to opportunity for multiple produce contamination events to occur during production and post-harvest handling of food crops. The primary objective of this research was to determine the efficacy of plant-derived antimicrobial-loaded nanoparticles to reduce Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium on spinach leaf surfaces whilst simulating multiple pathogen contamination events (pre-harvest and post-harvest). Spinach samples were inoculated with a blend of E. coli O157:H7 and S. Typhimurium, each diluted to ~8.0 log10 CFU/mL. The inoculated samples were then submerged in solutions containing nanoparticles loaded with geraniol (GPN; 0.5 wt.% geraniol), unencapsulated geraniol (UG; 0.5 wt.%), or 200 ppm chlorine (HOCl; pH 7.0), with untreated samples serving for controls. Following antimicrobial treatment application, samples were collected for surviving pathogen enumeration or were placed under refrigeration (5°C) for up to 10 days, with periodic enumeration of pathogen loads. After 3 days of refrigerated storage, all samples were removed, aseptically opened and subjected to a second inoculation with both pathogens. Treatment of spinach surfaces with encapsulated geraniol reduced both pathogens to non-detectable numbers within 7 days of refrigerated storage, even with a second contamination event occurring 3 days after experiment initiation. Similar results were observed with the UG treatment, except that upon recontamination at day 3, a higher pathogen load was detected on UG-treated spinach vs. GPN-treated spinach. These data fill a research gap by providing a novel tool to reduce enteric bacterial pathogens on spinach surfaces despite multiple contamination events, a potential food safety risk for minimally processed edible produce.

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Inhibition of Escherichia coli O157:H7 and Salmonella enterica Isolates on Spinach Leaf Surfaces Using Eugenol-Loaded Surfactant Micelles

Foods ◽

10.3390/foods8110575 ◽

2019 ◽

Vol 8 (11) ◽

pp. 575

Author(s):

Songsirin Ruengvisesh ◽

Chris R. Kerth ◽

T. Matthew Taylor

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Limit Of Detection ◽

Sodium Dodecyl ◽

Microbial Pathogens ◽

Escherichia Coli O157 ◽

Human Pathogens ◽

Surfactant Micelles ◽

Leaf Surfaces ◽

Spinach Leaf

Spinach and other leafy green vegetables have been linked to foodborne disease outbreaks of Escherichia coli O157:H7 and Salmonella enterica around the globe. In this study, the antimicrobial activities of surfactant micelles formed from the anionic surfactant sodium dodecyl sulfate (SDS), SDS micelle-loaded eugenol (1.0% eugenol), 1.0% free eugenol, 200 ppm free chlorine, and sterile water were tested against the human pathogens E. coli O157:H7 and Salmonella Saintpaul, and naturally occurring microorganisms, on spinach leaf surfaces during storage at 5 °C over 10 days. Spinach samples were immersed in antimicrobial treatment solution for 2.0 min at 25 °C, after which treatment solutions were drained off and samples were either subjected to analysis or prepared for refrigerated storage. Whereas empty SDS micelles produced moderate reductions in counts of both pathogens (2.1–3.2 log10 CFU/cm2), free and micelle-entrapped eugenol treatments reduced pathogens by >5.0 log10 CFU/cm2 to below the limit of detection (<0.5 log10 CFU/cm2). Micelle-loaded eugenol produced the greatest numerical reductions in naturally contaminating aerobic bacteria, Enterobacteriaceae, and fungi, though these reductions did not differ statistically from reductions achieved by un-encapsulated eugenol and 200 ppm chlorine. Micelles-loaded eugenol could be used as a novel antimicrobial technology to decontaminate fresh spinach from microbial pathogens.

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Effects of Water Quality and Post-Harvest Handling on Microbiological Contamination of Lettuce at Urban and Peri-Urban Locations of Ouagadougou, Burkina Faso

Foods ◽

10.3390/foods7120206 ◽

2018 ◽

Vol 7 (12) ◽

pp. 206 ◽

Cited By ~ 6

Author(s):

Juliane Dao ◽

Kathrin Stenchly ◽

Oumar Traoré ◽

Philip Amoah ◽

Andreas Buerkert

Keyword(s):

Water Quality ◽

Irrigation Water ◽

World Health ◽

Vegetable Production ◽

Salmonella Spp ◽

Total Coliforms ◽

E Coli ◽

Post Harvest ◽

Health Organization ◽

Production Areas

Vegetable production in urban gardens of Ouagadougou contributes to food security, but water for irrigation is often of low quality. This is particularly acute if irrigation water is taken from wastewater polluted channels. This study aimed at (i) verifying to what degree irrigation water quality is correlated with contamination of lettuce with Escherichia coli, total coliforms, and Salmonella spp., and (ii) assessing effects of post-harvest handling on pathogen development during the trade chain. We tested pathogen removal efficiency on lettuce by applying post-harvest washing. Irrigation water of production areas in Ouagadougou (n = 10) showed a mean E. coli load of 2.1 × 105 CFU 100 mL−1. In 60% of the cases, irrigation water did not meet the standards of the World Health Organization (WHO) for safe irrigation water, and in 30% of the cases, irrigation water was contaminated with Salmonella spp. Loads of total coliforms on lettuce leaves ranged from 2.9 × 103 CFU g−1 to 1.3 × 106 CFU g−1, while E. coli averaged 1.1 × 102 CFU g−1. Results on post-harvest handling revealed that microbial loads increased along the trade chain. Overall, half of all lettuce samples (n = 60) were tested positively for Salmonella spp. The experiment showed that appropriate post-harvest handling could prevent the increase of total coliforms.

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Lack of Internalization of Escherichia coli O157:H7 in Lettuce (Lactuca sativa L.) after Leaf Surface and Soil Inoculation

Journal of Food Protection ◽

10.4315/0362-028x-72.10.2028 ◽

2009 ◽

Vol 72 (10) ◽

pp. 2028-2037 ◽

Cited By ~ 52

Author(s):

GUODONG ZHANG ◽

LI MA ◽

LARRY R. BEUCHAT ◽

MARILYN C. ERICKSON ◽

VANESSA H. PHELAN ◽

...

Keyword(s):

Escherichia Coli ◽

Leaf Surface ◽

Escherichia Coli O157 ◽

Adaxial Side ◽

Abaxial Side ◽

E Coli ◽

Lactuca Sativa L ◽

Leaf Surfaces ◽

Leaves And Roots ◽

Leaf Lettuce

Survival and internalization characteristics of Escherichia coli O157:H7 in iceberg, romaine, and leaf lettuce after inoculation of leaf surfaces and soil were determined. A five-strain mixture of E. coli O157:H7 in water and cow manure extract was used as an inoculum for abaxial and adaxial sides of leaves at populations of 6 to 7 log and 4 log CFU per plant. The five strains were individually inoculated into soil at populations of 3 and 6 log CFU/g. Soil, leaves, and roots were analyzed for the presence and population of E. coli O157:H7. Ten (4.7%) of 212 samples of leaves inoculated on the adaxial side were positive for E. coli O157:H7, whereas 38 (17.9%) of 212 samples inoculated on the abaxial side were positive. E. coli O157:H7 survived for at least 25 days on leaf surfaces, with survival greater on the abaxial side of the leaves than on the adaxial side. All 212 rhizosphere samples and 424 surface-sanitized leaf and root samples from plants with inoculated leaves were negative for E. coli O157:H7, regardless of plant age at the time of inoculation or the location on the leaf receiving the inoculum. The pathogen survived in soil for at least 60 days. Five hundred ninety-eight (99.7%) of 600 surface-sanitized leaf and root samples from plants grown in inoculated soil were negative for E. coli O157:H7. Internalization of E. coli O157:H7 in lettuce leaves and roots did not occur, regardless of the type of lettuce, age of plants, or strain of E. coli O157:H7.

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Transfer of Escherichia coli O157:H7 to Romaine Lettuce due to Contact Water from Melting Ice

Journal of Food Protection ◽

10.4315/0362-028x-71.2.252 ◽

2008 ◽

Vol 71 (2) ◽

pp. 252-256 ◽

Cited By ~ 20

Author(s):

JIN KYUNG KIM ◽

MARK A. HARRISON

Keyword(s):

Escherichia Coli ◽

Tap Water ◽

Sampling Site ◽

Contaminated Water ◽

Escherichia Coli O157 ◽

Romaine Lettuce ◽

E Coli ◽

Ice Melt ◽

Leaf Surfaces ◽

Shipping Containers

Ice can be used to chill romaine lettuce and maintain relative humidity during transportation. Escherichia coli O157:H7 may contaminate water used for ice. The objective of this study was to determine the potential for E. coli O157:H7 contamination of romaine lettuce from either ice contaminated with the pathogen or by transfer from lettuce surfaces via melting ice. In experiment 1, lettuce was spot inoculated with E. coli O157:H7 and chilled with ice prepared from uncontaminated tap water. In experiment 2, water inoculated with this pathogen was frozen and used to ice lettuce. Three heads of lettuce were stacked in each container and stored at 4 or 20°C. After the ice melted, E. coli O157:H7 attachment to and recovery from the lettuce leaves were determined. For experiment 1, the population of E. coli O157:H7 attached to inoculated sites averaged 3.8 and 5.5 CFU/cm2 at 4 and 20°C, respectively. Most of the uninoculated sites became contaminated with the pathogen due to ice melt. For experiment 2, 3.5 to 3.8 log CFU E. coli O157:H7 per cm2 was attached to the top leaf on the first head. After rinsing with chlorinated water (200 μg/ml), E. coli O157:H7 remained on the surface of the top head (1.8 to 2.0 log CFU/cm2). There was no difference in numbers of E. coli O157:H7 recovered from each sampling site at 4 and 20°C. Results show that E. coli O157:H7 can be transferred onto other produce layers in shipping containers from melted ice made of contaminated water and from contaminated to uncontaminated leaf surfaces.

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Impact of Preinoculation Culture Conditions on the Behavior of Escherichia coli O157:H7 Inoculated onto Romaine Lettuce (Lactuca sativa) Plants and Cut Leaf Surfaces

Journal of Food Protection ◽

10.4315/0362-028x-72.7.1553 ◽

2009 ◽

Vol 72 (7) ◽

pp. 1553-1559 ◽

Cited By ~ 24

Author(s):

CHRISTOPHER G. THEOFEL ◽

LINDA J. HARRIS

Keyword(s):

Escherichia Coli ◽

High Standard ◽

Culture Conditions ◽

Escherichia Coli O157 ◽

Late Stationary Phase ◽

Romaine Lettuce ◽

Preparation Methods ◽

E Coli ◽

Leaf Surfaces ◽

Inoculum Preparation

Inoculum preparation methods can impact growth or survival of organisms inoculated into foods, thus complicating direct comparison of results among studies. The objective of this study was to evaluate preinoculation culture preparation for impact on Escherichia coli O157:H7 inoculated onto leaves of romaine lettuce plants and cut leaf surfaces. E. coli O157:H7 was grown quiescently or shaken at 15, 25, or 37°C to different growth phases in tryptic soy or M9 minimal salts broth or agar. Cells were harvested, washed, and suspended in 0.1% peptone, Milli Q water, or well water and refrigerated for 0 or 18 h. Prepared inoculum was spotted onto cut romaine lettuce (10 μl; 3 × 104 CFU/10 g) or onto romaine lettuce plants (20 μl; 3 × 106 CFU per leaf). Cut lettuce was sealed in 100-cm2 bags (made from a commercial polymer film) and incubated at 5 or 20°C. Lettuce plants were held at 23°C for 24 h. For all tested conditions, levels of E. coli O157:H7 increased at 20°Concut lettuce and decreased on cut lettuce stored at 5°C or on leaves of lettuce plants. At 20°C, preinoculation culture conditions had little impact on growth of E. coli O157:H7 on cut lettuce. However, survival at 5°C was significantly better (P < 0.05) for cultures grown at 15 or 37°C in minimal medium and to late stationary phase. Impact of preinoculation handling on survival on lettuce plants was less clear due to relatively high standard deviations observed among samples.

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Transcriptional Responses of Escherichia coli K-12 and O157:H7 Associated with Lettuce Leaves

Applied and Environmental Microbiology ◽

10.1128/aem.07454-11 ◽

2012 ◽

Vol 78 (6) ◽

pp. 1752-1764 ◽

Cited By ~ 64

Author(s):

Ryan C. Fink ◽

Elaine P. Black ◽

Zhe Hou ◽

Masayuki Sugawara ◽

Michael J. Sadowsky ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Leaf Surface ◽

Short Term ◽

Transcriptional Responses ◽

Content Type ◽

E Coli ◽

Leaf Surfaces ◽

K 12

ABSTRACTAn increasing number of outbreaks of gastroenteritis recently caused byEscherichia coliO157:H7 have been linked to the consumption of leafy green vegetables. Although it is known thatE. colisurvives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identifyE. coligenes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparingE. coliK-12, a model system, andE. coliO157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, includingtnaA(33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsAandybiM) and curli production (csgAandcsgB) were significantly upregulated inE. coliK-12 and O157:H7. BothcsgAandbhsA(ycfR) mutants were impaired in the long-term colonization of the leaf surface, but onlycsgAmutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction ofE. coliK-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.

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