Francisco Diez-Gonzalez

Preharvest Evaluation of Coliforms, Escherichia coli, Salmonella, and Escherichia coli O157:H7 in Organic and Conventional Produce Grown by Minnesota Farmers

Microbiological analyses of fresh fruits and vegetables produced by organic and conventional farmers in Minnesota were conducted to determine the coliform count and the prevalence of Escherichia coli, Salmonella, and E. coli O157:H7. A total of 476 and 129 produce samples were collected from 32 organic and 8 conventional farms, respectively. The samples included tomatoes, leafy greens, lettuce, green peppers, cabbage, cucumbers, broccoli, strawberries, apples, and seven other types of produce. The numbers of fruits and vegetables was influenced by their availability at participating farms and varied from 11 strawberry samples to 108 tomato samples. Among the organic farms, eight were certified by accredited agencies and the rest reported the use of organic practices. All organic farms used aged or composted animal manure as fertilizer. The average coliform counts in both organic and conventional produce were 2.9 log most probable number per g. The percentages of E. coli-positive samples in conventional and organic produce were 1.6 and 9.7%, respectively. However, the E. coli prevalence in certified organic produce was 4.3%, a level not statistically different from that in conventional samples. Organic lettuce had the largest prevalence of E. coli (22.4%) compared with other produce types. Organic samples from farms that used manure or compost aged less than 12 months had a prevalence of E. coli 19 times greater than that of farms that used older materials. Serotype O157:H7 was not detected in any produce samples, but Salmonella was isolated from one organic lettuce and one organic green pepper. These results provide the first microbiological assessment of organic fruits and vegetables at the farm level.

Longitudinal microbiological survey of fresh produce grown by farmers in the upper midwest.

Microbiological analyses of fruits and vegetables produced by farms in Minnesota and Wisconsin were conducted to determine coliform and Escherichia coli counts and the prevalence of E. coli, Salmonella, and E. coli O157:H7. During the 2003 and 2004 harvest seasons, 14 organic farms (certified by accredited organic agencies), 30 semiorganic farms (used organic practices but not certified), and 19 conventional farms were sampled to analyze 2,029 preharvest produce samples (473 organic, 911 semiorganic, and 645 conventional). Produce varieties included mainly lettuces, leafy greens, cabbages, broccoli, peppers, tomatoes, zucchini, summer squash, cucumber, and berries. Semiorganic and organic farms provided the majority of leafy greens and lettuces. Produce samples from the three farm types had average coliform counts of 1.5 to 2.4 log most probable number per g. Conventional produce had either significantly lower or similar coliform populations compared with the semiorganic and organic produce. None of the produce samples collected during the 2 years of this study were contaminated with Salmonella or E. coli O157:H7. E. coli contamination was detected in 8% of the samples, and leafy greens, lettuces, and cabbages had significantly higher E. coli prevalence than did all the other produce types in both years for the three farm types. The prevalence of E. coli contamination by produce type was not significantly different between the three farm types during these 2 years, with the exception of organic leafy greens, in which E. coli prevalence was one-third that of semiorganic leafy greens in 2003. These results indicate that the preharvest microbiological quality of produce from the three types of farms was very similar during these two seasons and that produce type appears to be more likely than farm type to influence E. coli contamination.

Reduction of Escherichia coli O157:H7 Populations in Cattle by Addition of Colicin E7-Producing E. coli to Feed

ABSTRACT A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 107 CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log10 CFU/g was observed, with a maximum decrease of 1.8 log10 CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment groups intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 108 CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.

Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major foodborne pathogen responsible for frequent gastroenteritis outbreaks. Phages and essential oils can be used as a natural antimicrobial method to reduce bacterial pathogens from the food supply. The objective of this study was to determine the effect of a bacteriophage cocktail, BEC8, alone and in combination with the essential oil trans-cinnameldehyde (TC) on the viability of a mixture of EHEC O157:H7 strains applied on whole baby romaine lettuce and baby spinach leaves. The EHEC O157:H7 strains used were Nal(R) mutants of EK27, ATCC 43895, and 472. Exponentially growing cells from tryptic soy (TS) broth cultures were spot inoculated on leaves and dried. EHEC cells were placed at low, medium, and high inoculum levels (10(4), 10(5), and 10(6) CFU/mL, respectively). Appropriate controls, BEC8 (approx. 10(6) PFU/leaf), and TC (0.5% v/v) were applied on treated leaves. The leaves were incubated at 4, 8, 23, and 37 °C in Petri dishes with moistened filter papers. EHEC survival was determined using standard plate count on nalidixic acid (50 μg/mL) Sorbitol MacConkey agar. No survivors were detected when both leaves were treated with BEC8 or TC individually at low inoculum levels after 24 h at 23 and 37 °C. When the EHEC inoculum size increased and/or incubation temperature decreased, the efficacy of BEC8 and TC decreased. However, when the two treatments were combined, no survivors were detected after 10 min at all temperatures and inoculum levels on both leafy greens. These results indicated that the BEC8/TC combination was highly effective against EHEC on both leafy greens. This combination could potentially be used as an antimicrobial to inactivate EHEC O157:H7 and reduce their incidence in the food chain.

Detection of a foreign protein in milk using surface-enhanced raman spectroscopy coupled with antibody-modified silver dendrites

Herein we developed a rapid and simple method which used surface-enhanced Raman spectroscopy (SERS) coupled with antibody-modified silver dendrites to detect ovalbumin (OVA), the egg white protein, introduced into whole milk. OVA was first captured out of milk by use of antibody-modified silver dendrites and then directly measured on the silver dendrites by Raman spectroscopy. Results show that this method is capable of detecting OVA at 0.1 μg/mL in phosphate buffered saline (PBS) and 5 μg/mL in milk within 30 min based on the principal component analysis. This method has the potential for wide use in areas such as allergenic protein detection and bioterrorism agent detection in complex matrixes.

Selection of recently isolated colicinogenic Escherichia coli strains inhibitory to Escherichia coli O157:H7.

Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E. coli per animal species ranged from 14% for horse isolates to 64% for sheep strains. Those isolates that inhibited E. coli K12 were screened against E. coli O157:H7, and 42 strains were found to be capable of inhibiting all 22 pathogenic strains tested. None of these 42 strains produced bacteriophages, and only 24 isolates inhibited serotype O157:H7 in liquid culture. The inhibitory activity of these strains was completely eliminated by treatment with proteinase K. When mixtures of these 24 colicinogenic strains were grown in anaerobic continuous culture, the four-strain E. coli O157:H7 population was reduced at a rate of 0.25 log10 cells per ml per h, which was fivefold faster than the washout rate. Two strains originally isolated from cat feces (F16) and human feces (H30) were identified by repetitive sequences polymerase chain reaction as the predominant isolates in continuous cultures. The results of this work indicate that animal species other than cattle can be sources of anti-O157 colicinogenic strains, and these results also lead to the identification of at least two isolates that could potentially be used in preharvest control strategies.

Aptamer-based surface-enhanced Raman scattering detection of ricin in liquid foods

A “two-step” aptamer-based surface-enhanced Raman scattering (SERS) detection assay was developed for ricin in liquid foods. Ricin B chain was first captured out of food matrices by aptamer-conjugated silver dendrites and then the spectrum was directly read on the silver dendrites. Aptamer use in this assay promotes ease of manipulation as well as improved sensitivity compared to antibody-based approaches. The limit of detection for ricin B chain was 10 ng mL−1 in phosphate buffered saline (PBS), 50 ng mL−1 in orange juice, and 100 ng mL−1 in milk based on principal component analysis (PCA) of measured spectra. This assay shows great promise as a rapid (< 40 min), sensitive, and simple “Yes/No” method to detect bio-weapons like ricin in liquid foods.

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

ABSTRACT An increasing number of outbreaks of gastroenteritis recently caused by Escherichia coli O157:H7 have been linked to the consumption of leafy green vegetables. Although it is known that E. coli survives 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 identify E. coli genes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparing E. coli K-12, a model system, and E. coli O157: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, including tnaA (33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsA and ybiM) and curli production (csgA and csgB) were significantly upregulated in E. coli K-12 and O157:H7. Both csgA and bhsA (ycfR) mutants were impaired in the long-term colonization of the leaf surface, but only csgA mutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction of E. coli K-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.

Soil survival of Escherichia coli O157:H7 acquired by a child from garden soil recently fertilized with cattle manure.

Aims:  This investigation was conducted to determine the survival of a naturally occurring Escherichia coli O157:H7 in garden soil linked to a sporadic case of E. coli O157 infection in Minnesota.

Reduction of Escherichia coli O157: H7 viability on hard surfaces by treatment with a bacteriophage mixture

This study determined the effect of a previously characterized phage mixture, referred as BEC8 on enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains applied on materials typically used in food processing surfaces. Sterile stainless steel chips (SSC), ceramic tile chips (CTC), and high density polyethylene chips (HDPEC) were used. Cultures of EHEC O157:H7 strains EK27, ATCC 43895, and 472 were combined, spot inoculated on surfaces, and dried. Chips were inoculated with 10(6), 10(5), and 10(4)CFU/chip, to obtain 1, 10 and 100 multiplicity of infection (MOI) values, respectively. Controls and BEC8 (approx. 10(6) PFU/chip) were applied on inoculated surfaces and incubated at 4, 12, 23, and 37 °C. EHEC survival was determined using standard plate count on tryptic soy agar. At 37 °C and 12 °C on SSC, no survivors were detected (detection limit 10CFU/chip) after BEC8 treatment at MOI of 100 after 10 min and at 23 °C after 1h on SSC. A similar result was obtained on CTC at 37 °C after 10 min, and after 1h at 23 °C. These results indicated that the phage cocktail was effective within an hour against low levels of the EHEC mixture at above room temperature on all 3 hard surfaces.