pnw 36th food safety sanitation 11/8/2016 workshoppostharvest.ucdavis.edu/files/252906.pdf · pnw...
TRANSCRIPT
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
36th Annual Food Safety & Sanitation WorkshopsNovember 8-9, 2016
Part 1. Listeria monocytogenes: Brief Background and Testing MethodsPart 2. L. mono Testing: Lessons Learned from packing house EMPs
Trevor [email protected]://ucfoodsafety.ucdavis.eduhttp://postharvest.ucdavis.edu
FSMA Preventive Controls Rule Environmental Monitoring for Foods with No Terminal Kill Step
Greater Emphasis on EMPs Listeria monocytogenes recognized as a pathogen of concern
Listeria: The group
The Listeria group now contains seventeen speciesaquatica, booriae, cornellensis, fleischmannii, floridensis, grandensis, grayi, innocua, ivanovii, marthii, monocytogenes, newyorkensis, riparia, rocourtiae, seeligeri, weihenstephanensis, welshimeri
• L. monocytogenes is pathogenic to humans and animals
• L. ivanovii infects animals and very rarely causes disease in humans
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
Listeria monocytogenes shortened to “L‐mono”
12 recognized sub‐types of L. mono • 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, and 7 • 1/2a, 1/2b, and 4b responsible for > 95% human illness • type 4b most common in human outbreaks
• Becomes important in Source Tracking at farm, cooler, processor, retail
Swaminathan, B., P. Gerner‐Smidt. The epidemiology of human listeriosis. Microb Infect 2007; 9 (2007): 1236‐1243
McLauchlin, J. 1990. Eur. J. Clin. Microbiol. Infect. Dis. 9:210‐213; Schwartz, B., et al. J. Infect. Dis. 159:680‐685
Environmental Routes of Transmission of Listeria sp. and L. mono
Manure
Food products
Humans
Animal feed/soil/water/environment/decaying vegetation
Food animals
Animal derived food products
Food Processing Plants
Water
Food outletsRetail outlets
Why all the recalls due to Listeria? Way more testingBetter techniques Targeted surveillanceZero tolerance on product It has always been around◦ Listeria monocytogenes common in watershed and waterways near CAFO’s
◦ Likely to be in watershed wherever domestic animal production or recreational animals are penned
PNW 36th Food Safety & Sanitation Workshop
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Listeria Concerns are Back for Raw Agricultural Commodities
Published 2000
United Fresh Listeria Guidance
Released 2013
Zone Concept for Environmental Monitoring
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
Using WGS to Prioritize Master Sanitation Schedule & Customize Environmental Monitoring Programs
� Dump and Transport� Precooling operations� Quality treatment� Pre‐grade and sort� Pre‐pack conditioning or storage� Packing � Pre‐shipping cooling
11,2,3
2 2 2
3,4
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Zone 1 & 2 surfaces may not be ‘internal’ with RAC
Optimal Growth Rate of L.mono100‐fold growth in 5 days @ 39qF
The longer Listeria is associated with a surface….the harder it is to kill
Greatest effect of sanitizer Least effect
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
Listeria finds places to grow on equipment and can persist for decades
L.m. in etched stainless steel H. O¨ lmez, S.D. Temur / LWT ‐ Food Sci. and Technology 43 (2010) 964–970
L.m. biofilm on lettuce
Environmental Sample Collection to Decision and Action‐point
Collection Magical Sample Fairy delivers samples to a
lab for testingResults reported 2‐4
days later
If this seems familiar, you are missing the Devil in the Details…
15
EMP detection, tracking, trending, and corrective actions depend on enrichment proficiency and sensitivity of the detection system
PNW 36th Food Safety & Sanitation Workshop
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If you truly want to find it: You need to be using a sensitive detection method
Time
Lag
Log
Stationary
Death
Num
ber o
f cells
102
106
104
Technology LOD (CFU/mL)
ELFA 105‐106
PCR (DNA) 104‐105
TMA (RNA) 102‐103
A detection method with a low LOD can overcome performance challenges created by less than optimal conditions in other steps of the environmental testing process
Testing method
18
Every enrichment has limitations
Low level of cellsStress condition
Competition Grow to
detectable levels
• Tryptic Soy Broth (TSB)• Buffered Listeria
Enrichment broth (BLEB)
• Brain Heart Infusion (BHI)
• Listeria Enrichment Broth (LEB)• Fraser• Half‐Fraser (FH)• Actero‐ LEB
THE NEED THE BARRIERS
Non‐selective Selective
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
Modified Listeria behavior in enrichment broth
Consequences of sub‐lethal injury on POD
• Sensitive to selective components to which they normally show resistance• Sensitive to enrichment conditions of the sample• Limited production of immuno‐target protein for some detection systems• Some cells of the stressed bacterial population will not initiate growth• Due to repair time, stressed cells tend show longer lag phase • Risk of not reaching the bacterial cell POD necessary for its detection• Therefore; potential false negative results with the detection system
RAC Facility Testing
n= 667
LEB6 Samplings
Multiple facilities
Paired samples
Positive LSP
HF2
3
20
21
Culture positive87%
Negative culture 13%
Half Fraser
Culture positive66%
Negative culture 34%
LEB
31%
55%
BLEB‐B IMS Direct from HF
21.6%44%
BLEB‐B IMS Direct from LEBSuslow et al. 2015 unpublished
PNW 36th Food Safety & Sanitation Workshop
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• Efficiently targets competing flora without affecting the growth of Listeria
• Contains specific ingredients for the recovery of sub-lethally injured Listeria
• Single step enrichment process• Compatible with any pathogen testing system• Low LOD
Optimized media for fast recovery of stressed Listeria
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Growth kinetics of sublethally heat-injured L. monocytogenes at 29ºC
5 CFU/well
_________ Actero™ Listeria media ________Single-step enrichment media
Where does Actero™ Listeria act?
Growth kinetics of healthy L. monocytogenes at 29ºC
• Recovery from environmental swab using Actero‐Lis Broth (AEB) onto CHROM‐LIS agar
• LEB to CHROM‐LIS had only two readily visualized colonies
Colony isolation is greatly facilitated with or without IMS on CHROMagar Listeria using Actero Listeria Broth
Suslow et al. 2015 – unpublished
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
� Expect disagreements between molecular and cultural results for all test platforms �The reverse can also be true
� Culture confirmation could require extensive post‐enrichment sample processing� An initial cultural failure is not always a molecular false‐positive
� Therefore, currently…it is most reasonable to accept the molecular outcome and activate your lot acceptance or corrective action/sanitation plan
Overview of Key Findings ‐ 1
25
Baseline EMP surveys for spatial mapping and source‐tracking
� Overview of objectives �Matching detection to operational units and environment� Contrasting PFGE and WGS similarities and diversity�Common Lm sites and source‐tracking example
5 –year Goal
�Develop data‐driven motivation for all packing operations to refashion themselves as food handlers
�Support validation of practical preventive controls within scale‐appropriate economic constraints
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
Martin Wiedmann / Cornell 5-01
“…the importance of analyzing isolates from both the 1st and 2ndenrichment can be seen. In only 48.9% of positive samples, both the 1st and 2nd enrichments were positive while in 51.1% of positive samples, either the 1st or the 2nd enrichment only was positive.”
2014
Primary Post‐enrichment Screen
Presumptive Lm colonies
qRTPCRvirulence confirmation and serotyping
PFGE Facility & Temporal Diversity Mapping
WGS source‐tracking
T. Suslow et.al. , unpublished 2014‐16
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
� Listeria prevalent in within Zone 2 and 3� areas routinely wet � Typical chlorine, ClO2 , or PAA addition to water doesn’t prevent biofilm buildup
� L. mono persistent following typical suface chlorine sprays� More aggressive sanitation needed
� L. mono persistent over several months � Detectable in dry off‐season conditions � Rebounds once wet operations re‐start
� Some facilities consistently no‐detection
Where does WGS fit?� Linking isolates to spatial and traffic‐flow mapping�More definitive source‐tracking� Better resolve transient and resident Lm� Data‐based evidence for a needed Correction or improved Best Management Practice
2014 3
2014 6
2014 6
2015 8
2014 4
Five isolates from PFGE pulsotype 3 showed significant SNP difference
� Pulsotype 3 considered persistent in Facility 6 based on PFGE � WGS results suggest additional reintroduction from a common source� Diversification in facilities, traffic circulation, and/or cycling among
facilities? � Appeared sporadically in other facilities over 3 seasons
WGS SNP similarity 90%
88%89%
88%
PFGE vs WGS: Listeria monoctytogenes AscI‐ApaI PFGE 98% similarity
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
AscI‐ApaI PFGE 98% similarity
2014 4
2014 6
2014 6
2015 5
2015 6
96%
Five isolates from PFGE pulsotype 22 had far fewer SNP differences
� Isolated from 3 facilities over two years. � Subtype 22 seems to be introduced to
different facilities from an external source with limited diversification
WGS similarity
PFGE vs WGS Listeria monoctytogenes
91%
2014 4
2014 6
2015 8
2014 392%
92%95%
Four isolates from PFGE pulsotype 6 showed SNP difference
� Subtype 6 considered predominantappearing sporadically in 4 facilities over two years
WGS similarity
35
� Understanding isolate diversity is critical to sound source‐tracking and corrective action plan
� Effective root‐cause assessment may depend on using molecular tools � Focus too often on ancillary or secondary positives� Miss key ‘persisters’ by giving equal weight to all sites
� The significance of Zone 2‐3 positives to Zone 1 and product contamination needs resolution
Overview of Key Findings 2
Baseline EMP surveys for spatial mapping and source‐tracking
� Overview of objectives �Matching detection to operational units and environment� Contrasting PFGE and WGS similarities and diversity� Conversion of molecular detection to culture confirmation� Prevalence of single‐site Listeria spp. and L. monocytogenes detection
� Common Lm sites and source‐tracking example
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
How is industry using the information? In the absence of a primary Environmental Swab Assessment… assume L. mono will be present in vulnerable non‐FCS areas
Set corrective measures guidance for non‐FCS positives ◦Prioritize zone Clean vs. Sanitize schedules◦Selection and rotation of sanitizer formulations
You can create a ‘hostile’ environment
90‐day Cleaning and Sanitation Facility Improvement Map
Daily Swab 1 2 3 4 5 6 8 9 10 11 12 14 15 16 17 18 20 21 22 23 24 26 27 28 29 30 32 33 34 35 36 38 39 40 41 42 44 45 46 47 48 50 51 52 53 54 56 57 58 59 60 62 63 64 65 66 68 69 70 71 72 74 75 76 77 78 80 81 82 83 84 86 87 88 89 90
Position
Incoming 1
Incoming 2
Conveyor 1
Roller 1
Conveyor 2
Roller 2
BrushBed 1
Brushed Bed 2
Under BB1
Under BB2
Drop 1
Drop 2
Waxer Brushes 1
Waxer Brushers 2
Polishing Brush 1
Polishing Brush 2
Singulator 1
Singulator 2
Cup Sizer 1
Cup Sizer 2
Grade 1 Belts
Grade 2 Belts
Grade 3 Belts
Drop Rollers 1
Drop Rollers 2
Drop Rollers 3
Auto‐fill Chute 1
Auto‐fill Chute 2
Auto‐Fill Chute 3
Six‐day TPC swab cycle 100’s 1,000’s 10,000’s
Not one factor• Training• Re‐training to data• Replace ‘uncleanables’• Eliminate practices • Alter chemistries
90‐days
PNW 36th Food Safety & Sanitation Workshop
11/8/2016
Key Takeaways � Raw Agricultural Commodity Packing is not the same as Processing�Regardless, implementing a rigorous EMP is strongly recommended or required under FSMA Preventive Controls� Genomic sub‐typing is a critical tool for source‐tracking and prioritization of corrective measures �WGS will likely uncover linkage among detected isolates across wide geospatial distances� This improved knowledge will drive positive change
Trevor SuslowExtension Research Specialist Department of Plant SciencesUniversity of CA, [email protected]://ucfoodsafety.ucdavis.edu
Acknowledgements to Lab Technical Staff and Students• Adrian Sbodio• Janneth Pinzon• Jeremy Roland• Chelsea Kaminski‐Davidson• Polly Wei• Lee Ann Richmond • Host of undergraduates
� Listeria monocytogenes: Lessons Learned � from packing house EMPs
Questions?
42“Oh, if only it were so simple.”