1. Scientific Perspective on Listeria monocytogenes in Ready-to-Eat Food
Régis Pouillot, DVM, PhD
FDA / CFSAN / OAO /
Division of Risk and Decision Analysis /
Risk Analysis Branch
Food Advisory Committee, Dec 7-8, 2015

2. Outline
Epidemiology
Listeria monocytogenes in RTE food (and elsewhere)
Listeria dose-response models
New Data

3. Epidemiology

4. Few cases, severe Pathogen
Estimated annual number of illnesses in the U.S.
(domestically acquired, foodborne)
Estimated annual number of associated deaths
Listeria monocytogenes
1,600
255 (16% of cases)
Salmonella, non typhoidal
1.0 million
378 (.03% of cases)
Norovirus
5.5 million
149 (.002% of cases)
Scallan et al., Emerg Infect Dis 17(1): 7-12.

5. Most cases are sporadic
Outbreak vs. Sporadic Cases
: 1,084 cases reported to CDC via FoodNet
56 (5.2%) only associated to outbreaks
Little idea about the implicated foods for sporadic cases
Some case characteristics (age, gender, illness severity) comparable to outbreaks
Comparable implicated foods?
Few contaminated products will not lead to an outbreak
Interagency Food Safety Analytics Collaboration webinar, Jan 10th, 2014

6. Outbreaks: shift in the implicated foods since 1998
Year
Cases
Food
1998
108
Frankfurters
2005 6
Unknown
2011 2
Undetermined 4 3
Grilled chicken
Chive cheese and ackawi cheese
1999 13
Deli meat
147
Whole cantaloupe 12
Mexican-style cheese 5
2006 15
Aged, blue-veined cheese 11
Pâté
Taco or nacho salad
2012 22
Ricotta cheese
Cheese
2013
Soft ripened cheese
2000
2007
Milk
2014 8 30
2008
Tuna salad
2001 28 20
Sprouts
2002 54 35
Caramel apple
2003
2010
Hog head cheese
2015 10
Ice cream
Sushi rolls
Pre-cut celery
CDC (2013). MMWR Morb Mortal Wkly Rep 62(22):
Cartwright et al., Emerg Infect Dis 19(1): 1-9.
CDC website

7. Listeria Outbreaks and Incidence, 1983-2014
(per million pop)
No. outbreaks
Decrease: 24% during and then reach a plateau
Era
Outbreaks per year
Median cases per
outbreak
Pre-PulseNet
0.3 69
Early PulseNet
2.3 11
Listeria Initiative
2.9
5.5
WGS 8
4.5
Data are preliminary and subject to change
Slide: CDC via M. Wiedmann

8. Overall: Stable number of cases since 2003
Laboratory confirmed cases in FoodNet
CDC MMWR 2014 / 63(15);

9. Conclusions: Epidemiology
Few cases, severe, mostly sporadic
Sharp decrease in the number of cases, then stable since 2003
Evolving knowledge, linked to evolving tools
More, smaller recognized outbreaks
Evolving food implicated in outbreaks
Sporadic cases?

10. Listeria in Ready-to-Eat food (and elsewhere)

11. L. monocytogenes: ubiquitous bacteria
Widely distributed in the environment
Soil, water, vegetation
15% Lm in preharvest env. (NY state)
Manufacturing environment
persistent strains that have found a harborage site within the facility where it may reside for years…
transient strains…
Retail environment
idem
e.g. 241 retail establishments tested
60% positive (at least one positive sample)
6.3% food-contact surface samples were positive
17% of non food-contact surface samples were positive
Milk crates: 34%! Floor drains: 28%!
Environment
Commodities
Processing
Retail
Food
Strawn et al, 2013 Appl Environ Microbiol. 79(24):
Hoelzer et al., J Food Prot 74(7):
Simmons et al. J Food Prot 77(11):

12. In Food: FSIS inspected facilities
Percentage of RTE meat and poultry products testing positive for L. monocytogenes in FSIS- inspected facilities, compared with incidence of listeriosis per 100,000, from CDC FoodNet surveillance
Interagency Risk Assessment: Listeria monocytogenes in Retail Delicatessens Technical Report (2013)
Source data: FSIS and CDC

13. In Food: survey data (0.27%) (4.31%) (0.76%) (4.70%) (0.049%) (1.25%)
FDA/ARS Survey* (2013)
(0.27%)
(4.31%)
(0.76%)
(4.70%)
(0.049%)
(1.25%)
(1.04%)
(2.36%)
2013 prevalence is significantly lower, p<0.0001
* Preliminary results from phase I
Source: Food Advisory Committee Meeting Materials, September 29-30, 2014

14. No-growth food? Known limits Better knowledge of interactions
pH less than or equal to 4.4
Water activity less than or equal to 0.92
Better knowledge of interactions
Robust predictive microbiology models (most studied bacteria)
Remaining issues
Heterogeneity
within product, lot-to-lot
Validation of challenge tests?
see Powell, 2009, IJFM
Augustin and Carlier, 2000, IJFM 56: 53-70
Mejlholm et al, 2010, IJFM 141:

15. Conclusions: L. monocytogenes in RTE food
Ubiquitous bacteria widely distributed in the environment, manufacturing environment, retail environment
Frequently found on non food contact surfaces
Decrease in the prevalence in food over the last decade(s)
FSIS inspected facilities
Other food
(decrease in the number of cases?)
Growth-No growth boundary in practice?

16. Listeria dose-response

17. Lots of bacteria ca. 1.8% RTE food contaminated (Gombas et al., 2003)
Smoked seafood: prevalence: 4.3%, up to CFU/g
Lots of bacteria
Listeria monocytogenes Levels in Food (per serving)
Number of Servings
(per year in the United States)
(per person per year)
3.3  1011
1300
1 to 1000
4,900,000,000 19
103 to 106
620,000,000
2.4
106 to 109
130,000,000
0.5
> 109
73,000,000
0.3
(FDA/FSIS, 2003)
Few cases
2,500 annual cases
(Mead et al., 1999)
1,600 annual cases
(Scallan et al., 2011)

18. Listeria dose-response
Probability of invasive listeriosis following the ingestion of Listeria is then
On average: very low
But function of
Individual Susceptibility ++++
Strain Virulence ++
But no Listeria monocytogenes is safe
Food vehicle ?
Dose

19. Individual Susceptibility
“Healthy” (less 65 year old, no known underlying condition)
1.0
65-74 year old
8.0
more 74 year old 20
Pregnant
116
Chronic Lymphocytic Leukemia
1,138
HIV/AIDS 55
Diabetes 1 44
Diabetes 2
5.4
Heart disease
Non Hispanic women
FoodNet Data,
(per 100,000)
Pouillot et al Clin Infect Dis. 54 Suppl 5:S
Goulet et al.2012 Clin Infect Dis. 54:

20. Virulence heterogeneity
Listeria monocytogenes is one of the most studied intra-cellular pathogen
Entry of L. monocytogenes into certain human epithelial cells is receptor mediated,
Depending on specific interactions between internalins on the bacterial surface and their respective host cell receptors
Point mutations in the inlA gene can lead to virulence attenuation
Nevertheless, all Listeria monocytogenes should be considered as pathogen

21. Dose-Response models Models based on animal data
Model based on outbreak data
Models based on epi data
Hoelzer et al, 2011, Risk anal, 33 (9)

22. How far will we go? ‘Average’ Virulent Strain A Less Virulent Strain B1
0.9
0.8
0.7
0.6
How far will we go?
Pr(Illness|dose)
0.5
0.4
0.3
0.2
0.1 13
log10(Dose)
‘Average’
Virulent Strain A
Less Virulent Strain B
Strain A, Susceptible Individual
Strain B, Non Susceptible Individual

23. FDA 2015 dose-resposne model and butter outbreak in hospitalized patients
Primate DR
DR for this specific outbreak as estimated from data
FDA 2015 DR for transplant organ patients,
highly virulent strain,
moderately virulent strain (this study)
Established DR for Susceptible population, FAO/WHO 2004
Pouillot et al., Risk Analysis, 35(1):90-108, 2015

24. Conclusions: Dose-Response
In order to explain the relatively high frequency of product contamination and the relative rareness of the disease, one must assume that, “on average”, low dose equals low risk
FAO/WHO, 2004
Refined models better characterize the variability in the dose-response: “on average”, low dose equals low risk, but in susceptible subpopulations, a relatively low dose may lead to a measurable risk for some individuals
Pouillot et al, 2015
Animal models and outbreak data model would predict too many cases if applied as-is
The variability in individual susceptibility and strain virulence maybe more important than the dose

25. Listeria monocytogenes risk assessments

26. FDA/FSIS 2003
Quantitative Assessment of Relative Risk to Public Health From Foodborne Listeria monocytogenes Among Selected Categories of Ready-to-Eat Foods
Dose Response considers strain variability and host susceptibility. Scaled to epidemiological data. Characterized uncertainty
Risk: products that support growth

27. FSIS Risk Assessment for L. monocytogenes in Deli Meats (2003)
Plant-to table probabilistic risk assessment built on FDA/FSIS 2003 risk assessment
Evaluated the number of illness prevented and lives saved annually according to various processing interventions
Led to Interim final rule
Alternative 1: Use of post-lethality treatment AND antimicrobial agent/process
Alternative 2: Use of post-lethality treatment OR antimicrobial agent/process
Alternative 3: Use of sanitation procedures only
Source: FSIS

28. FAO/WHO 2004: Risk assessment of L. monocytogenes in ready-to-eat food
Exponential dose-response models
One for susceptible population,
One for non-susceptible population
“The vast majority of cases of listeriosis are associated with the consumption of foods that do not meet current standards for L. monocytogenes in foods, whether that standard is zero tolerance or 100 CFU/g”
FAO/WHO (2004). Risk assessment of Listeria monocytogenes in ready to eat foods - Technical report.

29. FDA/FSIS Interagency Risk Assessment: L
FDA/FSIS Interagency Risk Assessment: L. monocytogenes in Retail Delicatessens (2013)
Sampling surveys and RA attribute most listeriosis cases from deli-meat to retail-sliced deli meat
This RA evaluated the impact of cross-contamination, sanitation, growth in retail deli
Complex discrete event model
FAO/WHO dose response
Key findings
Would explain some sporadic cases
Control Growth, notably through the use of growth inhibitors
Contaminated products that do not support growth can lead to a risk of listeriosis from cross-contamination of product that do support growth
Interagency Risk assessment: Listeria monocytogenes in retail delicatessens (2013)

30. New data

31. Usual data to inform Listeria monocytogenes dose-response models are difficult to obtain
Volunteers
Animal Models
Validity of the animal model?
Hoelzer et al, 2012 Veterinary Research 43(1): 18
Outbreak Data
Long incubation period -> Difficult to find leftovers
Case report
Case control studies
Epidemiological Data

32. FDA Data Collection Efforts
Celery Outbreak, TX, 2010
Limited prevalence and contamination level data
Data on underlying health issues
Growth studies
Cantaloupe Outbreak, Multistate, 2011
Caramel Apple, Multistate, 2015
Ice Cream, Multistate, 2015
Extensive prevalence and contamination data
Opportunity to assess exposure and underlying health issues

33. The case of the caramel apples
Apple does not support growth (pH 3.2),
Caramel does not support growth (low aw)
Caramel coated apple with a stick support growth
Up to 7 log10 in few days at ambient temperature
35 illness including
11 associated with a pregnancy
3 meningitis among otherwise healthy children aged 5-15
(target population!)
CDC website
Bouvier, Science Translational Medicine  7 (311) pp. 311ec184 (2015)
K. A. Glass et al., mBio 6, e (2015).

34. The case of ice-cream 10 cases … … over 5 years!
4 cases, observed in a single hospital, linked to one product of factory A (through milk shakes)
5 cases linked to a second factory (factory B)
… over 5 years!
FDA obtained products from factory A
Product that does not support growth (frozen)
Observed levels of bacteria are relevant to consumed levels

35. Enumeration data “Product A”, from factory A
2,290 samples of Product A tested
all but 13 samples were positive (99.4% positive)
Range: <0.03 MPN/g to > 208 MPN/gm
Highly consistent low contamination levels
15% below 1 MPN/gm
58% below 5 MPN/gm
77% below 10 MPN/gm
92% below 20 MPN/gm
98% below 50 MPN/gm
99.8% below 100 MPN/gm
4 samples > 100 MPN/gm*
*one >208 MPN/g (direct plating: 357 cfu/gm), two = 208 MPN/gm (direct plating: 142 cfu/gm and Non Available),
and one 139 MPN/gm (direct plating: 177cfu/gm)
Chen et al, submitted; Chen et al, IAFP 2015

36. Enumeration of L. monocytogenes in Different Lots of Product A
Average: 8 MPN/g
Chen et al, submitted; Chen et al, IAFP 2015

37. Exposure data (preliminary)
Our estimates from product distribution records
4,000,000 to 33,000,000 contaminated servings sold to the population
62,000 to 471,000 contaminated servings sold to pregnant women: no case identified
16,000 to 56,000 contaminated servings sold to highly susceptible population  4 identified cases
Corresponding dose-response model comparable to previous dose-response models developed from outbreaks
Other outbreaks: limited diffusion of products contaminated at relatively high level
Here: large diffusion of products contaminated at low level
(Pouillot et al, in preparation)

38. Ice cream Outbreak – Tentative Conclusions
We’ll probably never get better data
A no-growth product, with a very low average level of contamination (8 cfu/g) did cause an “outbreak”
It is likely that most patients were exposed to ice cream with < 100 cfu/g
A high dose can’t be excluded
Growth in milk shake > few logs improbable but
“inoculation” of the shaker at each serving. Biofilm? Growth?
The large distribution of a no-growth product, with a low average level of contamination (8 cfu/g) didn’t cause a massive outbreak
The underlying health of the patient, cell-mediated immune status and medications may be more important than the dose
Sufficient for risk management?
Black Swan ? or White Swan?
Few products that support growth (e.g. cheese) -> Outbreak
Very large diffusion of a contaminated product that does not support growth (ice-cream) -> Outbreak

39. Conclusions: Listeria monocytogenes…
The most studied foodborne bacteria
A lot of questions remain unanswered
Risk management in an uncertain world