Overview. Michelle D. Danyluk University of Florida. 4/14/14

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1 Michelle D. Danyluk University of Florida Overview Biological Hazards What is the pathogen of concern? Are all strains created equal? What pathogen is the most resistant to the lethal process? Challenge studies What are appropriate organisms? Laboratory studies Processing facility How do I prepare my product? Surrogate organisms What options do I have? How do I validate a surrogate? 1

2 Pathogen of concern Salmonella spp. Non- typhoidal Salmonella enterica (+2,500) Typhimurium Salmonella serotype Pathogen of concern Salmonella spp. Non- typhoidal Salmonella enterica (+2,500) Typhimurium Salmonella serotype Not all Salmonella serotypes are the same 2

3 Reduction during desiccation log CFU/g Salmonella strains selected for evaluation Salmonella serotype Enteritidis PT30 (ATCC BAA- 1045) Enteritidis PT9c Anatum Montevideo Oranienburg Tennessee Enteritids PT30 Enteritidis 9c Anatum Montevideo Oranienburg Tennessee Difference in sensitivity to desiccation Original Source Raw almond isolate from outbreak Clinical isolate from raw almond outbreak Almond survey Pistachios Pecans Clinical isolate from peanut buger outbreak Population log CFU/g Time (days) Similar survival during storage at 23 C Similar data with marked strains Heat resistance of different Salmonella strains Reduction of Salmonella on almonds (4% moisture) exposed to hot oil (121 C/250 F) Treatment time (min) S. Enteritidis PT9c S. Enteritidis PT30 S. Oranienburg S. Anatum log N/N Serotype Slope D- value (min) Enteritidis PT Enteritidis 9c 0.98 Oranienburg 0.81 Anatum 0.75 Rates of decline were not practically significant among Salmonella serotypes evaluated. 3

4 Pathogen of concern Salmonella spp. Non- typhoidal Salmonella enterica (+2,500) Typhimurium Almond Chicken Salmonella serotype Tomato Human Specific Isolates Pathogen of concern Salmonella spp. Non- typhoidal Not all specific isolates (or strains) Salmonella of each enterica (+2,500) serotype are the same Typhimurium Almond Tomato Human Chicken Salmonella serotype Specific Isolates 4

5 Pathogen of concern Salmonella spp. Non- typhoidal Not all specific isolates (or strains) Salmonella of each enterica (+2,500) serotype are the same Select a strain that Typhimurium was isolated from Salmonella your serotype Almond product, or a similar Chicken product Tomato Human (i.e. other dried fruit or nut) Specific Isolates Pathogen of concern Salmonella spp. Non- typhoidal Not all specific isolates (or strains) Salmonella of each enterica (+2,500) serotype are the same Select a strain that Typhimurium was isolated from Salmonella your serotype Almond product, or a similar Chicken product Tomato Human (i.e. other dried fruit or nut) Select a strain that is known to be resistant to your type of lethal process Specific Isolates (i.e. dry vs. wet heat, HPP, gas, etc.) 5

6 What are appropriate organisms? Laboratory Studies - Pathogens Pathogens should be restricted to laboratory use Ideally use a strain or strain(s) isolated from the product If there are known strains that are highly resistant to the lethal process they should be considered, but may not be appropriate What are appropriate organisms? Processing Facilities f - Surrogates Pathogens should NOT be used Unless no surrogate exists (i.e. PPO) and safe use of pathogen (containment) is possible precautions are taken/planned in advance 6

7 What are appropriate organisms? Surrogate should be: Based on pathogen of concern Non-pathogenic Behavior (in your product/process) similar to pathogen Easy to prepare and enumerate Easy to differentiate from background microflora Will not establish itself as a spoilage organism in the processing facility What are appropriate organisms? Single Strain or Cocktail? A cocktail is mixture a number of individual strains together Typically 3 5 different isolates Advantages: Reduces possibility of choosing the wrong strain as the most appropriate strain Disadvantages You may mask subtle nuances between individual strains 7

8 What are appropriate organisms? Single Strain or Cocktail? A cocktail is mixture a number of individual strains together Typically 3 5 different isolates Salmonella serotype Enteritidis PT30 (ATCC BAA- 1045) Enteritidis PT9c Anatum Montevideo Oranienburg Tennessee Original Source Raw almond isolate from outbreak Clinical isolate from raw almond outbreak Almond survey Pistachios Pecans Clinical isolate from peanut buger outbreak How do I prepare my product? Inoculating the product Inoculum Accuracy/Precision of the method Application technique Organism resistance For dried food products recovering the inoculum following growth on a plate, not in broth, is generally recommended (Almond Board of California) Use the lowest possible volume 8

9 Impact of growth conditions on survival during heat treatment (30 sec in 121 C/250 F oil ) Reduction (log CFU/g) Salmonella Enteritidis PT30 (ATCC BAA- 1045) Plate Broth Desiccation resistance of different Salmonella strains at Log CFU/sample C/73 F Salmonella Enteritidis PT30 (ATCC BAA- 1045) Salmonella Oranienberg Time (days) Plate closed Broth - open 9

10 How do I prepare my product? Inoculating the product How many organisms I put on my product? High enough load to be used in calculations At least 2- log higher than background populations Report the complete log reduction Not just complete reduction Include controls Include tests of uninoculated products How do I prepare my product? How do we measure death? Lack of life Lack of ability to grow on media Apply lethal treatment Count survivors over time 10

11 How do I prepare my product? Converting numbers to Log 10 Number Log 10 Number , , , ,000, ,500, ,500, How do I prepare my product? Log reductions 1 log reduction 250 million (8.4 log) to 25 million (7.4 log) Reduced by 225 million 36 million (7.6 log) to 3.6 million (6.6 log) Reduced by 32.4 million 1.2 million (6.1 log) to 120,000 (5.1 log) Reduced by 1.08 million Also called: 90% reduction 10- fold reduction Decimation 11

12 How do I prepare my product? Zero is never technically achieved during log reductions Number Log 10 Number D Reduction 100,000 CFU/g 5 10,000 CFU/g 4 1D 1,000 CFU/g 3 2D 100 CFU/g 2 3D 10 CFU/g 1 4D 1 CFU/g 0 5D 1 CFU/ 10 g - 1 6D 1 CFU/ 100 g - 2 7D 1 CFU/ 1,000 g - 3 8D How do I prepare and test my product? Inoculating the product How should I inoculate my product? After inoculation, dry to original A w Know initial inoculum level, immediately before test is run Retest A w right before process to ensure no significant changes 12

13 Surrogate Organisms What options do I have? What strains have been reported for Salmonella? Surrogate Organism Food Product Reference B. stearothermophilus spores Animal Feed Okelo et al., 2006 B. sterothermophilus Poultry Feed Okelo et al., 2006 Okelo et al., 2008 Enterocooccus faecium NRRL B Pantoea agglomerans SPS2F1 Pediococcus spp. and Pediococcus acidilactici Pediococcus spp. Almonds Dry Roasted Almonds Ground and formed beef jerky Whole- muscle turkey jerky ABC, 2007 b ABC, 2007 d Borowski et al., 2009 Williams et al., 2010 AIOE, 2012 Reduction on almonds during PPO treatment B. stearothermophilus spores vs. Salmonella Enteritidis PT30 Danyluk et al.,

14 Reduction on almonds during PPO treatment B. stearothermophilus spores vs. Salmonella Enteritidis PT30 B. stearothermophilus spores are a BAD surrogate for Salmonella Enteritidis PT30 on almonds during PPO treatment Danyluk et al., 2005 Reduction on almonds in hot oil at 121 C/250 F Log CFU/g Enterococcus faecalis ATCC Salmonella Enteritidis PT30 y = x R 2 = D SE = 0.85 y = x R 2 = D EF = Treatment time (min) SE-TSA EF-TSA Harris, unpublished 14

15 Reduction on almonds in hot oil at 121 C/250 F Log CFU/g Enterococcus faecalis ATCC Salmonella Enteritidis PT30 y = x R 2 = D SE = 0.85 y = x R 2 = D EF = 0.37 Enterococcus faecalis ATCC is a BAD surrogate for Salmonella Enteritidis PT30 on almonds during hot oil treatment Treatment time (min) SE-TSA EF-TSA Harris, unpublished Reduction on almonds in hot oil at 121 C/250 F Log CFU/g Enterococcus faecium NRRL B Salmonella Enteritidis PT30 SE PT30 EF Time (mins) Harris, unpublished 15

16 Reduction on almonds in hot oil at 121 C/250 F Enterococcus 7.0 faecium NRRL B-254 is a GOOD 6.0 surrogate for Salmonella Enteritidis SE PT EF PT30 on almonds during thermal treatment 4.0 Log CFU/g Enterococcus faecium NRRL B Salmonella Enteritidis PT Time (mins) Harris, unpublished How do I validate my surrogate? The story of Enterococcus faecium NRRL B-2354 (ATCC 8459) and the almond industry Organism validated by ABC for use as an appropriate surrogate for thermal treatments applied to almonds Widely adopted by the low moisture food industry Not widely validated as appropriate for use with other low moisture foods Kopit et al., AEM,

17 4/14/14 How do I validate my surrogate? In 2010/2012 strains used for surrogates classified as a BSL 2 organisms (equivalent in biosafety to Salmonella) in 2010/2012 by the American Type Culture Collection and USDA Some strains of E. faecium cause serious hospital acquired infections How do I validate my surrogate? The story of Enterococcus faecium NRRL B-2354 (ATCC 8459) and the almond industry Almond Board funds research at UC Davis to genetically confirmed identification of the surrogate Sequenced and analyze the genome Confirmed absence of important virulence factors Determined antibiotic- resistance profile Confirmed sensitivity to medically- important antibiotics Genetic relationship to other Enterococcus faecalis Most closely related to non- pathogenic isolates Environmental isolates Used in animal feed E. faecalis Used as starter culture E. faecium Kopit et al., AEM,

18 How do I validate my surrogate? The story of Enterococcus faecium NRRL B-2354 (ATCC 8459) and the almond industry E. faecium NRRL B (ATCC 8459) clusters with other community- associated strains Lacks virulence factors Sensitive to antibiotics More tolerant of extreme conditions Kopit et al., AEM, 2013 How do I validate my surrogate? The story of Enterococcus faecium NRRL B-2354 (ATCC 8459) and the almond industry No evidence for safety concerns as a surrogate in thermal process validation E. faecium NRRL B (ATCC 8459) clusters with other community- associated strains Lacks virulence factors Sensitive to antibiotics Strain has returned to BSL1 status by the American More tolerant Type of extreme Culture conditions Collection and USDA Kopit et al., AEM,

19 Summary Biological Hazards Salmonella serotypes/strains respond differently Care should be taken when selecting strains Challenge studies Salmonella should only be used in laboratory studies, validated surrogates should be used in processing facilities Care should be taken when preparing and treating product to mimic actual process conditions Surrogate organisms E. faecium NRRL B (ATCC 8459) has been validated for thermal processes on almonds Other lethal treatments (i.e. PPO) have no validated surrogates identified Do not assume that a surrogate validated for one product/ process will work on another Next up Tom Jones DFA of CA Validating facility sanitation programs environmental monitoring 19