1. Listeria monocytogenes Prevalence, Persistence, and Control Haley F. Oliver, Ph.D. Associate Professor Purdue University

2. Oliver Lab – Listeria and Retail Objective Improve public health through evidence-based interventions to control Listeria monocytogenes. Strategies –Collaborate with retailers & sanitation providers –Locate environmental niches –Determine transient vs. persistent contamination –Test alternative sanitation procedures –Identify best practices –Develop long-term practical controls 2

3. Longitudinal study of deli environments 3

4. LM can be prevalent in retail delis (10-40%) LM can persist in retail delis (observed ~1.5 years) LM does not persist in all stores (only11/30 observed) Non-food contact surfaces have higher LM prevalence than food contact surfaces (NFCS 15% vs. FCS 4%) Conclusions From Longitudinal Study 4

5. Phase 3 -- Intervention SSOP Transfer Points –Deli case handle –Cold room door handle –Slicer adjustment knobs and handles –Scale top & keypad Floors –General floor cleaning –Floor wall juncture –Drains surface. Each retailers will retain current protocols for drain management Single and 3 basin sinks –General floor cleaning –Floor wall juncture Deli Case Food Contact Surfaces –Deli case trays –Opened product (i.e. in-use product) display shelving –Slide and display doors Deli Case Down to the Coils –Entire deli case (not in sections; see FMI protocol) Slicers, Handles, and Knobs –Entire slicer –FCS including blade, guards, and slicer case under the blade Additional Actions/SOPs Squeegee High Pressure Hoses (i.e. spray nozzle/spray gun style) Training Verification/ATP Testing Survey

6. Previous – Longitudinal Study 6 Pre-InterventionPost-Intervention Food Contact63/1595 (4.0%) Transfer Point18/540 (3.3%) Non-Food Contact334/2368 (14.1%)369/2360 (15.6%) Overall425/4512 (9.4%)450/4495 (10.0%) 17/30 stores show low LM prevalence (<5%) 5/30 stores show moderate LM prevalence (5-10%) 8/30 stores show high LM prevalence (≥10%) 11/30 stores show evidence for persistence

7. AprilMayJuneJulyAugustSeptemberOctoberNovemberDecember Food Contact Sites Slicer --------- Deli case NT ------ Deli case near raw meat NT ------ Deli case trays NT ------ 3-basin sink interior NT CU-57,267----- 1-basin sink interior NT CU-258,69---CU-294,321- Cold room rack --------- Cutting board NT -- - Rewrap table NT ------ Counter NT ------ Non-food contact sites 3-basin sink exterior NT ------ Floor/wall junction (3-basin) CU-258,69 CU-8,96LMCU-258,69 1-basin sink exterior NT CU-258,69--LM-CU-258,69 Floor/wall junction (1-basin) NT CU-258,69--LMCU-258,69 Deli drain NT CU-258,69CU-258,333-CU-258,69 Floor adjacent to drain -CU-258,69 -- Deli floor NT CU-258,69--- - Cold room floor NT CU-258,69CU-295,329-CU-258,69 Cold room wall CU-258,69-------- Cold room drain NT CU-258,69 - Standing water NT -- - Squeegee NT CU-258,69 - Cart Wheel --CU-258,69 ----- Hose NT -CU-258,69---- Trash can --CU-258,69- ---- Transfer Points Slicer knob --------- Case handle --------- Scale NT ------

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9. Aggressive Deep Cleans Goal: To adapt and execute manufacturing-based mitigation strategies in retail delis Method:  12-16 hour overnight shutdown  10-20 person crew  Remove products  Disassemble equipment  Clean and sanitize all surfaces 9

10. 10 Overnight Deep Clean Protocol

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12. Interventions by Store 12

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14. Longitudinal Efficacy Reduced average monthly LM prevalence a mean 9.7 percentage points (CI 95 : 2.50, 16.90%; p=0.017) per store. 14

15. Employee-executed deep cleans with training were more effective for long-term control than third party execution of the same protocol.

16. “Whole-genome sequencing shines a new light in this area that helps us find unsuspected gaps in the food safety system… this is a wake-up call to the ice cream industry in general and to the regulators.” ~Robert Tauxe, CDC DOI: 10.1126/science.aac4551 16

18. L. monocytogenes WHOLE GENOME SEQUENCING PROJECT  In the first year of the project, integrating WGS with other Listeria surveillance activities:  Improved detection of listeriosis clusters.  Increased the number of outbreaks solved.  Reduced the average cluster size.  One person’s listeriosis infection was linked to prepackaged lettuce. Prior to WGS it would have been almost impossible to make a link between the contaminated food and one person who was not part of an outbreak.  December 2014: identified caramel apples as source of large listeriosis outbreak. WGS identified the illnesses one week faster than would have occurred with PFGE alone.

19. L. monocytogenes WHOLE GENOME SEQUENCING PROJECT 19

20. Whole Genome Sequencing improves Identification of persistent LM in retail (i)apply single-nucleotide polymorphism (SNP)-based phylogenetics to subtype (ii)to differentiate persistent from repeatedly reintroduced strains (iii) identify genetic determinants of L. monocytogenes persistence 20

21. NGS technologies Illumina MiSeq and HiSeq platforms –Solid state amplification –Single- and paired-end reading (resolve ambiguous alignment) –Use of Solexa chemistry for fluorescent labeling –Read lengths of 300bp (MiSeq) and 125bp (HiSeq) –Read quantity up to 25million (MiSeq) and 2billion (HiSeq) 21

22. WGS Unique to a single retail deli, supporting persistence within the deli Unique to a single state, supporting clonal spread within a state In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis 22

23. PFGE & WGS 23

24. “Now that we’re turning whole-genome sequencing on, we’re identifying outbreak after outbreak…we’re also finding smaller outbreaks that we weren’t able to find before.” ~Brendan Jackson, CDC DOI: 10.1126/science.aac4551 24

25. Current Work USDA-NIFA-AFRI 2015-05673 “Prevalence, Persistence and Control Strategies of Listeria ssp. and Salmonella in Retail Grocery Produce Storage and Sales Environments” 25

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