Biocontrol of Salmonella and other Pathogens using a Combination of - PowerPoint PPT Presentation

Biocontrol of Salmonella and other Pathogens using a Combination of Antagonistic Bacteria and Lytic Bacteriophages Keith Warriner Department of Food Science University of Guelph kwarrine@uoguelph.ca

Salmonellosis Outbreaks Linked to Tomatoes Salmonella serotype Year Total cases Javiana 1990 174 Montevideo 1993 84 Baildon 1998 85 Javiana 2002 141 Newport 2002 297 Beranderup 2004 561 Javiana Typhimurium 2006 184 Saint Paul? 2008 1400

Sources of Salmonella  Irrigation water  Rhizosphere  Pesticide solutions  Dump tank water  Rare serovars implicated: Serovar dependent factors?

Salmonella on Post-harvest Tomatoes

Implications  Once internalized Salmonella cannot be removed by washing.  Control Options  Good Agricultural Practice  Sanitizers in irrigation and processing water  Biocontrol

Biocontrol  Established method for controlling plant pests  Bacillus thuringiensis (BT) insecticide  Probiotics  Bacteriophages  Ideal biocontrol agent  Non-pathogenic  Target pathogens  No adverse effects on plant development  Effective  Adapted to the environment

Inoculate flowers (pre- -harvest) harvest) Inoculate flowers (pre 0.1 ml 7 log CFU/ml Salmonella suspension Screen for Screen for 16S rRNA rRNA- -DGGE DGGE 16S Salmonella Salmonella

DGGE gel patterns of tomato samples from Montevideo inoculated plants

Principle Component Analysis ◊ Control Internal ♦ Control Surface ○ Montevideo Internal ● Montevideo Surface □ Typhimurium Internal ■ Typhimurium Surface

Genera identified from 16S rDNA sequencing Bacterium Control Montevideo Pos Neg Cyanobacterium + + + Hefnia + - - Pantoea + + + Bacillus + + + Ps. putida + - + Erwinia + - + Enterobacter + - +

Mung Bean Sprouts/Tomatoes Enterobacter Deferred Assay 16S rRNA Mung Bean Sprouts Tomatoes

Anti- Salmonella Factor • No anti- Salmonella activity recovered in:- – Spent culture medium of E. asburiae – Elution from agar plates on which E. asburiae had been cultured. – Cell membrane fraction Anti- Salmonella factor requires to be elucidated.

monella infecting Bacteriophages  Isolated from pig farms and feedlots  Cocktail of phages which infect broad range of serovars

Lytic Activity of Bacteriophage on Salmonella Serovar Serovar/Phage Serovar/Phage F01 F01 P01 P01 P102 P102 P700 P700 P800 P800 FL 38 FL 38 FL 41 FL 41 Isolate Isolate Javiana + + + + + ND ND Heidelberg + + + + + ND ND Typhymurium + + + + + ND ND Newport + - - + - ND ND Montevideo - - - - - + + + Susceptible - Resistant ND Not determined

In Vitro (Broth) (Broth) Assay Assay In Vitro

Suppression of Salmonella growth in mung bean sprouts Sprouting for Steep beans Steep beans 5 days with In Enterobacter In Salmonella daily irrigation ± Phage suspension Microbiological Analysis

Control of Salmonella on sprouting mung bean sprouts TVC a (Log Treatment Weight of Enterobacter Salmonella Sprouts (g) CFU/g) asburiae JX1 (Log CFU/g) (Log CFU/g) Not Detected b A Negative Control 78.20±7.43A 8.15±0.91A <1.70 A Salmonella 69.98±7.96A 8.15±0.46A <1.70 A 6.72±0.78B 72.43±4.87A 8.48±0.56A 8.14±0.60B 1.16±2.14C Salmonella + E. asburiae Salmonella + phage 70.60±8.27A 8.52±0.44A <1.70 A 3.31±2.48C Positive c D Salmonella + 71.33±9.06A 8.98±0.41A 7.91±0.16B E. asburiae + phage a Total Aerobic Count b Not Dectected: Negative by enrichment c Positive by enrichment

Inoculation of Growing Tomato Plants No Inoculation: Control 0.1 ml 7 log cfu/ml Salmonella 0.1ml 7 log cfu/ml Enterobacter 0.1ml 9 log pfu/ml phage cocktail 7 weeks

Salmonella on and within Tomatoes Treatment Surface a Internal b Positive Negative Positive Negative Control 0 34 0 36 Salmonella 22 (92%) 2 31 (43%) 41 Salmonella + E. asburiae JX1 2 (5%) 38 1 (1%) 83 Salmonella + Phage 25 (83%) 5 11 (19%) 46 Salmonella + Phage + E. asburiae JX1 0 (0%) 38 1 (2%) 56 a One sample consisted of 3 tomatoes b Individual surface disinfected tomatoes were screened for internalized populations

Enterobacter asburiae JX1 on and within tomatoes Treatment Surface a Internal b Positive Negative Positive Negative Control 0 15 0 45 Salmonella 0 24 0 72 Salmonella + 20 0 40 (67%) 20 E. asburiae (100%) JX1 Salmonella + 0 16 0 48 Phage Salmonella + 19 0 38 (67%) 19 Phage + E. (100%) asburiae JX1 a One sample consisted of 3 tomatoes b Individual surface disinfected tomatoes were screened for internalized populations

Control of Salmonella on Tomato Plant rhizosphere .

Control of Salmonella on the surface of Ripened Tomatoes during Post-harvest Storage Salmonella Log CFU/g E. asburiae Log CFU/g Day 0 Day 7 Day 0 Day 7 Salmonella 5.54±0.6 6.98±0.74 ND ND Salmonella + 5.60±0.28 6.93±0.78 1.70±0.46 5.71±0.15 E. asburiae Salmonella + 6.15±0.09 7.04±0.21 3.16±0.80 7.27±0.27 E. asburiae + phages ND: Not Determined

Control of Salmonella on Internal Tissue of Ripened Tomatoes during Post-harvest Storage Salmonella Log CFU/g E. asburiae Log CFU/g Day 1 Day 7 Day 1 Day 7 Salmonella 2.61±1.18 3.46±0.31 ND ND Salmonella + 0.35±0.68 3.42±2.18 ND 1.58±1.27 E. asburiae Salmonella + 1.76±1.53 3.49±0.62 1.08±1.19 4.67±0.22 E. asburiae + phages ND: Not Detected

Does the Same Approach Work with Other Pathogens?

Escherichia coli O157:H7  Three bacteriophage cocktail from culture collection  Enterobacter asburiae JX1  In vitro assay

Growth of E. coli O157:H7 in Broth Culture

Listeria monocytogenes  3 strain cocktail of phages from collection  Antagonistic bacteria  Manure effluent  Soil  Mung bean sprouts

Isolation of Antagonistic ntagonistic Bacteria Bacteria Isolation of A Code Identification Source BA Bacillus pumilus Effluent BB Bacillus lentus Effluent BC Bacillus pumilus Effluent BD Bacillus subtilis Effluent BE Bacillus cereus Soil BF Bacillus megaterium Soil PA Bacillus cereus Soil PB Bacillus cereus Effluent EA Enterobacter asburiae Sprouts

In Vitro Assay 10. 00 C ont rol 8. 00 L LogCFU/ m Phage 6. 00 4. 00 Ant agoni si t i c bact eri a 2. 00 Phage + 0. 00 Ant agoni si t i c bact eri a BA BB BC BD BE BF PA PB EA Ant agoni st i c bact eri a

Conclusions  E. asburiae JX1 exhibited antagonistic activity against Salmonella  Combination of E. asburiae JX1 and lytic bacteriaphage cocktail exhibited synergistic activity against Salmonella on mung bean sprouting seeds.  E. asburiae JX1 or lytic phage cocktail significantly reduced the persistence of Salmonella in the rhizosphere of growing tomato plants.

 Generic approach to control foodborne pathogens although requires further optimization.  Biocontrol preparation that has potential to control Salmonella at the primary production level

Will the Public Accept Biocontrol? CBC Viewers comments “They just can't seem to keep their pesky little fingers out of our food. ” “Where does science get off trying to improve humanity?” “Really ,I think scientists are out to lunch--why don't they make themselves useful on more realistic problems--that are so much more prevalent--- AIDS,STILL CANCER ,DIABETES---and that's a very few to note. For beings scientists ,they're not being very bright!”

 “This may sound as a silly idea to some. As a consumer of one of the healthiest foods on the market and used in so many products, I am happy to see something can be done to protect us. This is no laughing matter. It is our lives. I say to the scientists in Guelph, good work.”

Acknowledgements  OMAFRA Food Safety and Innovation Program  Public Health Agency  Public Health Branch Health Canada, Alberta  Jiaxiong Ye (Graduate student)  Magdalaena Kostrzynska (AAFC)  Kari Dunfield (Land Resource Science, UoG)  Haifeng Wang (Dept Food Science, UoG)