Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: Activities: This multi-state project has several objectives and the efforts at the University of Minnesota have been focused to develop or improve methods for control or elimination of pathogens in pre harvest environments investigating the prevalence of E. coli O157 in cattle populations and by studying the relationship of this pathogen with plant tissues. Recent reports indicated that feeding distiller's grains solids (DGS) to cattle increased fecal shedding and prevalence of E. coli O157:H7. In Minnesota, the use of DGS as feed for livestock is extensive, but there was no previous report about the prevalence of E. coli O157 in beef cattle in the State. In a follow up experiment a taxonomic study was conducted to find relatedness among enterohemorragic E. coli strains. Experimental work was also conducted to identify spectral changes in E. coli O157 as it interacts with lettuce roots. Dissemination: Researchers attended the American Society for Microbiology annual meeting in San Francisco, CA to present the most relevant findings of the phylogenetic analysis of E. coli isolates from May 18-20 as posters, and at the IAFP Annual Meeting July 22-25, Providence, RI. Researchers discussed their findings with audiences as well as distributed copies of the poster to attendees. PARTICIPANTS: From the Department of Food Science and Nutrition: PI and Professor Francisco Diez-Gonzalez, Research Associate Ryan Fink, Post-doctoral Research Associate Lili He, Ph. D. candidates Zhe Hou and Hongshun Yang. Undergraduate students Alex Amman and Christie Radtke. From the Dept. of Animal Science: Co-PI and Professor Alfredo DiCostanzo. From the Biotechnology Institute: Professor Michael Sadowsky. TARGET AUDIENCES: Cattlemen, meat producers and meat industry, fresh vegetable industry as well as the scientific community involved in food safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: For the project on Enterohemorhagic E. coli (EHEC) in cattle populations: The population structure and diversity of 293 E. coli O157 isolates collected in from the Upper Midwest was determined by using the horizontal, fluorophore-enhanced, repetitive-PCR (HFERP) DNA fingerprinting technique. The fingerprinting analysis determined that the 293 isolates could be clustered into 47 clonal groups with a very low overall diversity index (0.16). MDS analysis did not reveal any significant environmental variable influencing the selection of different genotypes. H' and J' indices indicated a higher genetic diversity within farm (H' = 2.05, J' = 0.40) than in abattoir (H' = 0.87, J' = 0.18) isolates and a much higher diversity in the spring (H' = 2.22, J' = 0.53) compared to the other seasons. There was no correlation between the presence of the virulence genes stx1 and 2 and specific genotypes. The low overall genomic diversity may support the hypothesis that the emergence of E. coli O157 is a recent event. The distribution of the virulence factors stx1 and 2 did not match this genetic homogeneity, probably because these factors are located on prophages and can be acquired by horizontal transfer. The higher diversity in the farm environment and during spring might indicate a reduced selective pressure. For the project on interactions of E. coli O157 with lettuce roots: The spectra of E. coli K12 and O157 exposed to the HS showed some differences in the nucleic acid, protein, and lipid regions compared with controls. The spectra of E. coli K12 HS cells exhibited significant differences compared to spectra from E. coli O157 HS cells in the RNA and protein regions. The overall band intensity of amide regions declined for E. coli O157 HS cells, while it increased for E. coli K12 HS cells. The intensity of the RNA bands of E. coli K12 HS cells were also found much higher than those of E. coli O157 HS cells. These findings were in agreement to our previous Microarray data that studied the interactions of E. coli and lettuce at the gene expression level. For the project on use of neutral electrochemical activated water to treat Listeria monocytogenes: Isogenic inlA and sigB mutants were able to form biofilms. After NECAW treatment, both gene expressions increased for the WT. While sigB gene expression of ΔinlA strain increased comparable to the WT, inlA gene expression of ΔsigB strain did not significantly increase. Both genes were expressed more in biofilms than in liquid cultures. The level of inlA gene expression in WT increased by 4.28 and 5.51-fold with treatment with 4 mg/L NECAW for 10 min in liquid cultures and biofilms, respectively, while the corresponding values were 5.91 and 10.05-fold for sigB gene. Mutant strains were more sensitive to NECAW treatment than WT strain. For liquid culture, 10 mg/L NECAW for 10 min resulted in 0.79 and 1.17 more log CFU/ml reductions for ΔinlA and ΔsigB strains, respectively than WT strain while there was no significant difference in biofilms under the same condition
Publications
- Diez-Gonzalez, F., J. Ferguson, K. R. Christensen, M. J. Sadowsky, and R. C. Fink. 2012. Structure and diversity of Escherichia coli O157 isolated from cattle in the upper Midwest. ASM Annual Meeting, June 18-20, San Francisco, CA.
- Hou, Z., L. He, R. C. Fink, T. P. Labuza, M. J. Sadowsky and F. Diez-Gonzalez. 2012. Analysis of Escherichia coli K12 and O157:H7 interaction with lettuce roots using surface enhanced Raman spectroscopy. ASM Annual Meeting, June 18-20, San Francisco, CA.
- Yang, H., J. Feritag, and F. Diez-Gonzalez. 2012. Role of sigB and inlA genes in biofilm formation and antimicrobial efficacy of neutral electrochemically activated water on Listeria monocytogenes. IAFP Annual Meeting, July 22-25, Providence, RI.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities: This multi-state project has several objectives and the efforts at the University of Minnesota have been focused on developing or improving methods for control or elimination of pathogens in pre harvest environments investigating the prevalence of E. coli O157 in cattle populations and by studying the relationship of this pathogen with plant tissues. Recent reports indicated that feeding distiller's grains solids (DGS) to cattle increased fecal shedding and prevalence of E. coli O157:H7. In Minnesota, the use of DGS as feed for livestock is extensive, but there have been no previous reports about the prevalence of E. coli O157 in beef cattle in the State. The second year of a longitudinal study collected fecal samples from farms and an abattoir packing plant. Experimental work was also conducted to identify important genes in E. coli O157 as it interacts with lettuce roots and a survey of commercial lettuce samples was conducted to assess the potential for internalization of bacteria inside of leaves. A Ph. D. and a M.S. students participated in these projects as part of their theses, and two undergraduate students were taught experimental research techniques. Events: researchers attended the American Society for Microbiology annual meeting in New Orleans, LA to present the most relevant findings of the presence of enterohemorrhagic E. coli among cattle feedlots and on plants. Dissemination: two posters were presented at the ASM Annual Meeting May 21-24, New Orleans, and one oral presentation at the IAFP Annual Meeting July 31-August 3, Milwaukee, WI. Researchers discussed their findings with audiences as well as distributed copies of the poster to attendees. PARTICIPANTS: From the Department of Food Science and Nutrition: PI and Professor Francisco Diez-Gonzalez, Research Associate Ryan Fink, Ph. D. candidate, Zhe Hou, Undergraduate students Alex Amman and Christie Radtke. From both the Dept. of Animal Science and the Dept. of Food Science and Nutrition: Asst. Professor Ryan Cox From the Department of Animal Science: Co-PI and Professor Alfredo DiCostanzo, Graduate Research Assistants Jackie Popowski and Jeff Jaderborg From the U of Minnesota Extension Service: Assistant Extension Professor Grant Crawford TARGET AUDIENCES: Cattlemen, meat producers and meat industry, fresh vegetable industry as well as the scientific community involved in food safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: For the project on Enterohemorhagic E. coli (EHEC) in cattle populations: The overall annual EHEC O157 prevalence was 14.8 % when averaged across all samples. However, feedlots and abattoir had different prevalence levels with 12.1% and 18.1% respectively. The prevalence of EHEC O157 in the abattoir in samples from cattle fed DGS was lower (P<0.0001) than in cattle not fed DGS and had no correlation with seasonality. In the feedlot samples, the presence of EHEC O157 in cattle fed high (> 25%) and low (< 25%) dietary inclusions of DGS was not significantly different (P > 0.27), but its prevalence appeared to be slightly correlated with the season cycle. The EHEC O157 prevalence in Minnesota feedlots was lower than the one determined in the abattoir, indicating a lower specific prevalence in the three farms investigated than in the general Northern Midwest area. These findings suggest that DGS feeding has little or no influence increasing the E. coli O157:H7 prevalence. This is the first longitudinal study of E. coli O157:H7 prevalence in Minnesota For the project on interactions of E. coli O157 with lettuce roots: As many as 30 genes involved in stress responses and biofilm formation were significantly up-regulated in cells recovered from both leaves and roots. In contrast, 40 genes in roots and 34 genes in leaves involved in protein synthesis were down-regulated. The phage shock operon (pspABCDE) was induced more than 5-fold on the leaves. Genes involved in biofilm formation, ycfR and ybiM, were highly up-regulated and mutants lacking ycfR displayed a reduced capacity to colonize lettuce leaves and roots as compared to wild type strain. Similar gene expression was observed between EHEC grown on lettuce leaves and roots. Genes involved in curli and biofilm formation appeared to be important in the attachment and colonization of EHEC to lettuce plants. For the project on bacterial internalization in lettuce leaves: One hundred romaine and 100 red leaf lettuce leaves were tested for the presence of internalized bacteria. A total of 20 controls for each type of lettuce had no growth after 3 days. Isolates were obtained from 39 romaine and 39 red leaf lettuce samples. Bacillus species were identified in 30 romaine and in 21 red leaf samples, and Pseudomonas spp. were found in 2 and 14 leaves, respectively. Three Erwinia isolates were also found in romaine leaves. Pantoea, and Staphylococcus were detected in red leaf lettuce. Spore-forming bacteria and epiphytic bacterial genera were predominantly found in commercial lettuce. None of the bacterial genera belonged to groups closely related to Gram-negative pathogenic bacteria transmitted by leafy greens such as E. coli O157:H7 and Salmonella.
Publications
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities: This multi-state project has several objectives and the efforts at the University of Minnesota have been focused to develop or improve methods for control or elimination of pathogens in pre harvest environments investigating the prevalence of E. coli O157 in cattle populations. Recent reports indicated that feeding distiller's grains solids (DGS) to cattle increased fecal shedding and prevalence of E. coli O157:H7. In Minnesota, the use of DGS as feed for livestock is extensive, but there was no previous report about the prevalence of E. coli O157 in beef cattle in the State. A longitudinal study collected fecal samples from farms and an abattoir packing plant. Findings were presented as a poster at the 2010 American Society for Microbiology annual meeting, May 23-27, San Diego, CA. An M.S. student participated in this project as part of her thesis, and two undergraduate students were taught experimental research techniques. Events: researchers attended the American Society for Microbiology annual meeting in San Diego, CA to present the most relevant findings of the presence of enterohemorrhagic E. coli among cattle feedlots. Dissemination: one poster was presented at the ASM meeting. Researchers discussed their findings with audiences as well as distributed copies of the poster to attendees. PARTICIPANTS: From the Department of Food Science and Nutrition: PI and Associate Professor Francisco Diez-Gonzalez, Post-doctoral Research Associate Ryan Fink, Undergraduate Students Johanna L. Dahlberg and Hannah Thompson. From both the Dept. of Animal Science and the Dept. of Food Science and Nutrition: Asst. Professor Ryan Cox From the Department of Animal Science: Co-PI and Professor Alfredo DiCostanzo, Graduate Research Assistants Jackie Popowski and Jeff Jaderborg From the U of Minnesota Extension Service: Assistant Extension Professor Grant Crawford TARGET AUDIENCES: Cattlemen, meat producers and meat industry as well as the scientific community involved in food safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: Overall, E. coli O157:H7 was confirmed in 8% and 9.4% of farm and packing plant samples, respectively. Both prevalences declined from approx. 14% in October to less than 3% in December. The farm samples contained mainly isolates carrying the single form of Stx2 or the combination of Stx1/Stx2 (44 and 51% respectively). Isolates carrying the combination Stx1/Stx2 were predominant in the plant samples (more than 77%) with the carriers of the single forms of the genes equally less frequent (11% each). Conclusions: The seasonal changes in prevalence appear to be similar to those previously reported. These results suggested that there was a marked difference of the pathogenic populations present in the farms and in the abattoir. Since 50% of the animals from the packing plant were not fed DGS, it seemed that this feed may not have a significant influence on natural prevalence of this pathogenic bacterium.
Publications
- Poster: Fink, R.C., Popowski, J.M., Jaderborg, J.P., Crawford, G., DiCostanzo, A., Cox, R.B. and Diez-Gonzalez, F. 2010. Cattle prevalence of Escherichia coli O157 in Minnesota and its association with distillers grains feeding. ASM annual meeting, May 23-27, San Diego, CA.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This multi-state project has several objectives and the efforts at the University of Minnesota have been focused to develop or improve methods for control or elimination of pathogens in post harvest environments including Hispanic cheeses and deli meats. The findings regarding one of the objectives of this project (Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments) was presented as a poster at the 2009 annual meeting of the International Association for Food Protection. The findings of this project have been presented to the Minnesota Dairy Association, the organization that has provided most of the funding during this period, and at the North Central Cheese Industries Association meeting last October. PARTICIPANTS: PI: Francisco Diez-Gonzalez; Graduate Research Assistants: Mary Beth Kamnetz and Camila Gadotti. TARGET AUDIENCES: Dairy and meat industries, as well as the scientific community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
CHANGE IN KNOWLEDGE: The specific goal of this project was to determine the potential impact on quality and sensory characteristics of a mixture of antimicrobial ingredients that had proven to inhibit Listeria monocytogenes previously. The antimicrobial combinations of nisin 0.5 g/kg and caprylic acid (CA) 0.73 g/kg along with nisin 0.5 g/kg and CA 1.46 g/kg were used to determine their effect on naturally present bacterial populations. The treatments caused some slowing of growth as compared to the control for all bacterial counts including lactic acid bacteria (LAB), aerobic plate count, and psychrotrophic bacteria. These results were significant (p <0.005) for both treatment levels for the aerobic bacteria, lactic acid bacteria, and psychrotrophic bacteria counts during 3 week storage. A 3-alternative forced choice test (3-AFC) protocol with 31 volunteers was used to detect sensory differences between samples treated with the nisin and CA treatments. Sixty-seven Hispanic cheese consumers rated their liking of treated cheeses. In the first panel, some sensory differences were detected. They appeared to be due to caprylic acid detection. Nisin/caprylic acid treatments did not cause disliking by consumers and the samples scored very close to controls. The same treatements were also used to treat frankfurter samples to control the growth of Listeria monocytogenes. The combination of nisin and CA were able to reduce at least 2 log CFU/g the concentration of L. monocytogenes after 3 weeks of incubation at refrigeration temperatures. These results indicated that the combination of nisin and CA could be an effective mixture in inhibiting Listeria monocytogenes in ready-to-eat fresh Hispanic cheeses as well as deli meats and may also offer an additional benefit in extending shelf life without noticeable changes in product flavor.
Publications
- Diez-Gonzalez, F., Kamnetz, M.B. and Vickers, Z.M. 2009. Use of nisin and caprylic acid to control Listeria monocytogenes in queso fresco. International Association for Food Protection annual meeting, July 12-15, Dallas, TX.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The findings regarding one of the objectives of this project (Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments) was presented as a poster at the Annual Meeting of the American Society for Microbiology last June in Boston, MA. The findings of this project have been presented to the Minnesota Dairy Association, the organization that has provided most of the funding for that objective. PARTICIPANTS: PI: Francisco Diez-Gonzalez Graduate Research Assistants: Mary Beth Kamnetz and Amit Pal TARGET AUDIENCES: Meat and dairy industries, as well as the scientific community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: Objective 1: Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments including meat, poultry, seafood, fruits and vegetables and nutmeats. The effect of several antimicrobial ingredients to inhibit growth and inactivate Listeria monocytogenes in queso fresco was tested. We first evaluated the effect of individual treatments of antimicrobials on queso fresco batches that had been inoculated with a mixture of five L. monocytogenes and stored at 4 degree C. After that initial evaluation, we tested the combined effect of addition of these food ingredients. Listeria was able to grow and multiply rapidly in queso fresco when no starter culture and no antimicrobial treatment were added Individual treatments with lacticin-producing L. lactis, monolaurin, lactic acid, and combinations of sodium lactate/diacetate had little effect in inhibiting growth. SL/SD mixtures have been used successfully for ready-to-eat meats, but our results indicated that they were not effective in queso fresco. Nisin caused an initial count reduction but was not able to stop growth. Increasing concentrations of caprylic acid slowed growth, and almost no population increase was observed. Several combinations of antimicrobial GRAS ingredients reduced the population and inhibited growth of L. monocytogenes achieving from 2 to 4 log CFU/g count differences compared to controls. However, none of those treatments were as effective as binary mixtures of nisin and caprylic acid. At a nisin concentration of 0.5 g/Kg combined with caprylic acid (0.8 to 1.6 g/Kg), the initial L. monocytogenes population was immediately reduced and remained less or equal to 10 CFU/g during most of the experiments at 4 degree C. Objective 2: Develop and validate mathematical modeling to gain understanding of pathogen behavior in macro and micro-environments. This study compared the performance of four primary mathematical models to study the growth kinetics of Listeria monocytogenes ribotypes grown at low temperature so as to identify the best predictive model. Nineteen food and animal L. monocytogenes isolates with distinct ribotype were grown at 4, 8, and 12 degree C in tryptic soy broth and slurries prepared from cooked uncured sliced turkey breasts (with or without potassium lactate and sodium diacetate, PL/SD) and cooked cured frankfurters (with or without PL/SD). No significant differences were found in the performance of linear and non-linear models, but the Baranyi model had the best fit for most growth curves. The maximum growth rate (MGR) of Listeria strains increased with increases in temperature. Similarly MGR was found to be significantly greater when no antimicrobials were present in the initial formulation of turkey or frankfurter products. The variability in lag times and maximum growth rates in all media as determined by the Baranyi model was not consistent among strains. The fastest growing strains resulting from this study can be recommended for future use in L. monocytogenes challenge studies in delicatessen meat and poultry food matrices, so as to develop conservative pathogen growth predictions.
Publications
- Kamnetz, M. B., and Diez-Gonzalez, F. 2008. Inhibition of Listeria monocytogenes in queso fresco by using GRAS ingredient combinations. ASM Annual Meeting, June 1-6, Boston, MA.
- Pal, A., Labuza, T. P., and Diez-Gonzalez, F. 2008. Comparison of primary predictive models to study the growth of Listeria monocytogenes at low-temperatures in liquid cultures and selection of fastest growing ribotypes in meat and turkey. Food Microbiol. 25: 460-470.
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