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Canada Research Chair in Food Microbiology and Probiotics

Tales of an amateur thermophile: Resistance of Escherichia coli to pathogen intervention treatments in beef processing M. Gänzle, E. Dlusskaya, D. Pierce, L. Ruan, and L. McMullen. Canada Research Chair in Food Microbiology and Probiotics

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Canada Research Chair in Food Microbiology and Probiotics

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  1. Tales of an amateur thermophile: Resistance of Escherichia coli to pathogen intervention treatments in beef processing M. Gänzle, E. Dlusskaya, D. Pierce, L. Ruan, and L. McMullen Canada Research Chair in Food Microbiology and Probiotics Department of Agricultural, Food and Nutritional Science Edmonton, Alberta, Canada www.ualberta.ca

  2. Enterohaemorraghic Escherichia coli (EHEC) on beef Virotype of E. coli(Genes for adhesion to intestinal mucosa and shiga-toxin) Frequent serotypes of EHEC E. coli O157:H7 (O26:H11, O111:H8, O157:NM) Disease: Bloody diarrhea, hemolytic-uraemic syndrome Infectious dose: 10 cells Habitat: Colon of ruminants (up to 107 cfu / g) Frequency: 4 – 7 cases per year and 100,000 population (USA and Canada) – estimated cost of > $60 million annually in Canada Regulation: U.S.A., „zero tolerance“ for E. coli O157:H7 Canada, microbiological criteria for E. coli. George Morris Centre. 2007. Cost of E. coli O157:H7 illness in Canada

  3. Elimination of EHEC on beef: a formidable challenge! EHEC >1010 cells none E. coli >1012 cells < 103 cells / g No pasteurisation or other physical treatment, no preservatives or other additives

  4. Pathogen intervention in beef processing Cattle receiving and holding  Stunning Hide washing  Removal of hide  Evisceration  Lactic acid spray, neck only  Trimming, vacuuming  Steam pasteurization  Lactic acid spray Chilling Photos courtesy of Dell Allen, Cargill Meats

  5. EHEC on beef: an unsolved problem in food processing Current intervention reduce E. coli counts by less than 99% (2 log) => Determine effects of meat matrix on resistance of E. coli to interventions (Dana Pierce) => Determine resistance of > 50 strains of E. coli to intervention treatments (Lena Dlusskaya) => Use of functional genomics to elucidate determinants of heat resistance (Lifang Ruan)

  6. Lab scale intervention treatments Inoculation with ~ 107E. coli (single strain or strain cocktail) Surface treatment with steam (8’’ or 15’’) and lactic acid (4.5%) Meat cylinders (lean or adipose tissue) with 4.9 cm2 surface area surface temperature: >80°C after 2’’ >90°C after 8’’ pH: 4.3 (lean) 3.1 (fat)

  7. Bactericidal effect of intervention on lean and adipose tissue D meat-fat D +/- lactic acid AW 1.7 DM18.3 DM18.3 ATCC 25922 GFP => Treatment effects in keeping with bactericidal effect on carcass => Transformation with fluorescent marker does not influence sensitivity

  8. Use of Green Fluorescent Protein and Red Fluorescent Protein as fluorescent probes eGFP: derivative of GFP (denaturation at 75 - 80°C) RFP: red fluorescent protein, denaturation at 85 - 90°C Distribution of cells on lean and adipose tissue E. coli pRFP on fat E. coli pRFP on meat

  9. 240 µm 475 µm 90 µm 30 µm Z-Scan to determine survival beneath meat surface E. coli DM18.3 with RFP expression plasmid Fluorescence microscopy of untreated lean tissue Focus 0 – 500 µm below surface, 1000 x magnification 0 µm

  10. Distribution of cells of E. coli DM18.3 RFP before and after steam treatment meat and fat ● untreated cells, lean tissue (n= 551 cells) ○ untreated cells on fat tissue (n= 611 cells) ▲ steam-treated cells on lean tissue (n= 89) D steam-treated cells on fat tissue (n= 236)

  11. Bactericidal effect of intervention treatments - interim summary - Penetration of E. coli Penetration of steam and lactic acid treatments Thermal insulating properties of fat mitigate effect of steam treatment Buffering capacity of meat mitigates effect of lactic acid treatment

  12. Are all E. coli the same? – strain variation in resistance 55 strains of E. coli isolated from beef processing plant „historic“ isolate – isolated prior to industrial use of steam and lactic interventions reference isolates from strain collection and live animals Use of strain cocktails instead of single strains

  13. Resistance of strain cocktails to steam and lactic acid intervention treatments # 1 # 2 # 5 # 6

  14. Resistance of E. coli strains to heat Survival at 60°C Survival at 60, 65 and 70°C DM18.3 AW1.7 FUA 1041 GGG10 FUA 1044 K-12

  15. Resistance of E. coli strains to heat: Comparison of D60 values with literature data

  16. Resistance of E. coli strains to lactic acid Survival of E. coli in LB with 4% lactic acid (pH 2.3) GGG10 AW1.7

  17. Survival of E. coli in hamburger patties grilled to internal temperature of 63 or 71°C GGG10, AW 1.7

  18. Heat resistance of cattle and beef isolates of E. coli - interim summary - E. coli isolates from beef processing plants exhibit exceptional heat resistance! >1012 cells – a vast majority are heat sensitive < 103 cells / g – some (most?) are heat resistant Steam and lactic interventions

  19. Determination of gene expression in E. coli AW1.7 and E. coli GGG10 • Use of genome-wide microarray covering three E. coli genomes • Comparison of E. coli AW1.7 and E. coli GGG10 grown at 37°C, and after heat shock at 50°C Red, bold genes are related to transport functions

  20. Validation of microarray data by quantitative reverse-transcription PCR

  21. Validation of microarray data by Proteomics Separation of membrane proteins by SDS-PAGE and protein identification by LC/MS/MS AW1.7 GGG10 Ladder 40kD NmpC and OmpC OmpC

  22. Validation of microarray data by expression of NmpC from E. coli AW1.7 in E. coli GGG10 Expression of nmpC in E. coli GGG10 – YFP served as control Expression of yafQ-dinJ in E. coli AW1.7 – YFP served as control Determination of heat resistance at 60°C

  23. Genetic determinants of heat resistance – interim summary • Many genes overexpressed in heat resistant E. coli related to the transport of amino acids or related compounds • Expression of nmpC increases heat resistance in sensitive strains • Heat resistance is maintained at the expense of acid resistance

  24. Where to go from here? Further work needed to findthe Achilles’ heel of heat resistant E. coli! • Prevalence of heat resistant strains • design interventions that are more effective • Non-thermal technologies • Biocontrol with nonpathogenic organisms

  25. Acknowledgements • Collaborators • Aaron Pleitner and Mandi Socholotuik • Financial support • ALIDF • …..for your attention

  26. Reduction in the numbers of transgenic Escherichia coli inoculated on the surface of lean or fat tissue by treatments with steam (8 s) or treatments with steam, followed by application of 4.5% v/v lactic acid spray. Strains EC Top 10 and DM 18.3 were transformed with a plasmid coding for the expression of RFP. Strain TMW 2.497 caries a plasmid coding for expression of rsGFP. An asterix (*) indicates a significant difference (p < 0.1) between treatments with steam and treatments with steam and lactic acid for the same strain and tissue; an X indicates a significant difference (p < 0.03) between steam treatments on lean and fat tissue for the same strain.

  27. Photograph of E. coli DM 18.3 eGFP inoculated onto lean tissue and counterstained with Syto 17 before (a) and after steam and lactic acid treatment (b). Photographed using the double exposure program with the Rhodamine and GFP filter sets. (a) GFP filter. Exposure time = 171 ms. 1000x magnification. (b) GFP filter. Exposure time = 1027 ms. 1000x magnification.

  28. Use of Green Fluorescent Protein and Red Fluorescent Protein as fluorescent probes Escherichia coli DM 18.3 eGFP on lean tissue, counterstained with propidium iodide. nuclei of meat cells E. coli

  29. Cost of E. coli O157:H7 infections in Canada • Cost to health care: $29.6 million • Market demand loss: $30 million • Recalls: ??? • Total estimate: $60 million + ??? ~ 25% of EHEC infections attributed to meat consumption George Morris Centre. 2007. Cost of E. coli O157:H7 illness in Canada

  30. Genes overexpressed in E. coli GGG10 compared to AW1.7 at 50ºC Ratio Gene Gene function ( GGG10/AW1.7 ) 0.29 chaperone Hsp70, co - chaperone with DnaJ dnaK ydfI 0.29 predicted mannonate dehydrogenase Nfi 0.27 endonuclease V 0.25 30S ribosomal subunit protein S11 rpsK yfiD 0.20 pyruvate formate lyase subunit 0.1 9 30S ribosomal subunit protein S3 rpsC fimA 0.18 major type 1 subunit fimbrin hokD 0.18 small toxin polypeptide 0.16 periplasmic protein combats stress cpxP yccV 0.16 DNA - binding protein, hemimethylated 0.15 50S ribosomal subunit protein L4 rplD 0.1 4 cold shock protein associated with 30S yfiA ribosomal subunit 0.13 50S ribosomal subunit protein L29 rpmC 0.13 30S ribosomal subunit protein S7 rpsG

  31. Use of Green Fluorescent Protein and Red Fluorescent Protein as fluorescent probes GFP, YFP, eGFP: derivatives of GFP (denaturation at 75 - 80°C). RFP: red fluorescent protein, denaturation at 85 - 90°C Transformation did not influence survival after steam / lactic acid treatments

  32. Visualisation of treatment effects by fluorescence microscopy E. coli DM 18.3 on lean meat stained with fluorescein and propidium iodide before after treatment

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