Scientific Perspective on Listeria monocytogenes in

Ready-to-Eat Food

Régis Pouillot, DVM, PhDFDA / 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

Scallan et al., Emerg Infect Dis 17(1): 7-12.

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)

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Most cases are sporadic

• Outbreak vs. Sporadic Cases• 2004-2011: 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

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Outbreaks: shift in the implicated foods since 1998Year Cases Food Year Cases Food Year Cases Food1998 108 Frankfurters 2005 6 Unknown 2011 2 Undetermined

4 Frankfurters 3 Grilled chicken 2 Chive cheese and ackawi cheese

1999 6 Unknown 13 Deli meat 147 Whole cantaloupe4 Frankfurters 12 Mexican-style cheese 2 Mexican-style cheese

5 Deli meat 2006 2 Unknown 15 Aged, blue-veined cheese11 Pâté 2 Taco or nacho salad 2012 22 Ricotta cheese2 Deli meat 3 Cheese

2013 6 Soft ripened cheese2000 13 Mexican-style cheese 2007 5 Milk

2014 8 Mexican-style cheese

30 Deli meat 2008 5 Tuna salad5 Mexican-style cheese

2001 28 Deli meat 20 Sprouts 5 Sprouts2002 54 Deli meat 8 Mexican-style cheese 35 Caramel apple2003 3 Unknown

2010 8 Hog head cheese2015

10 Ice cream

12 Mexican-style cheese 2 Sushi rolls 30 Cheese

4 Undetermined

10 Pre-cut celery

6 Mexican-style cheese

CDC (2013). MMWR Morb Mortal Wkly Rep 62(22): 448-452.Cartwright et al., Emerg Infect Dis 19(1): 1-9.CDC website

Listeria Outbreaks and Incidence, 1983-2014

1983

1984

1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

2013

2014

012345678

0123456789Total

Outbr...

Pre-PulseNet

0.369

Early PulseNet

2.311

Listeria Initiative

2.95.5

No. outbreaks Incidence (per million pop)

EraOutbreaks per

yearMedian cases per

outbreak

WGS8

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);328-332

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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?

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LISTERIA IN READY-TO-EAT FOOD (AND ELSEWHERE)

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

Processing

Retail

Food

Strawn et al, 2013 Appl Environ Microbiol. 79(24):7618-27Hoelzer et al., J Food Prot 74(7): 1083-1095

Simmons et al. J Food Prot 77(11): 1929-1939

Commodities

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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

Year

1990 1995 2000 2005 2010

L. monocytogenes

ALLR

TE

Testing (%

Positives)

0

1

2

3

4

5

List

erio

sis

Inci

denc

e Pe

r 10

0,00

0

0.0

0.1

0.2

0.3

0.4

0.5

0.6Testing PrevalenceListeriosis Incidence

In Food: survey data

* Preliminary results from phase I

FDA/ARS Survey* (2013)

(0.27%) (4.31%)

(0.76%) (4.70%)

(0.049%) (1.25%)

(1.04%) (2.36%)

Source: Food Advisory Committee Meeting Materials, September 29-30, 2014 13

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No-growth food?• Known limits

– 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 136 10-17

Augustin and Carlier, 2000, IJFM 56: 53-70

Mejlholm et al, 2010, IJFM 141: 137-150

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Conclusions: L. monocytogenesin 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?

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Listeria dose-response

Listeria monocytogenes Levels in Food (per serving)

Number of Servings(per year in the United States)

Number of Servings (per person per year)

0 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

2,500 annual cases(Mead et al., 1999)1,600 annual cases(Scallan et al., 2011)

(FDA/FSIS, 2003)

Lots of bacteria

Few cases

ca. 1.8% RTE food contaminated (Gombas et al., 2003)Smoked seafood: prevalence: 4.3%, up to 105-106 CFU/g

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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

0 20 40 60 80

0.0

0.5

1.0

1.5

2.0

2.5

Age

Inci

denc

e ra

te

Individual Susceptibility

Non Hispanic womenFoodNet Data, 2004-2009

“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 5.4

Pouillot et al. 2014 Clin Infect Dis. 54 Suppl 5:S405-10.Goulet et al.2012 Clin Infect Dis. 54:652-60.

(per

100

,000

)

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

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Dose-Response models

Hoelzer et al, 2011, Risk anal, 33 (9) 1568-81

Models basedon animal data

Model based on outbreak data

Models based on epi data

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

13

log10(Dose)

Pr(Il

lnes

s|do

se)

‘Average’

Virulent Strain A Less Virulent Strain B

Strain A, Susceptible Individual Strain B, Non Susceptible Individual

How far will we go?

22

23

0 5 10 15 20

-8-6

-4-2

0

log10Dose

log 1

0P

rob

FDA 2015 dose-resposne model andbutter outbreak in hospitalized patients

DR for this specific outbreak as estimated from data

FDA 2015 DR for transplant organ patients, highly virulent strain, moderately virulent strain (this study)

PrimateDR

Established DR for Susceptible population, FAO/WHO 2004

Pouillot et al., Risk Analysis, 35(1):90-108, 2015

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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

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LISTERIA MONOCYTOGENES RISK ASSESSMENTS

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• 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

Decreased Risk per Annum

Dec

reas

ed R

isk

per S

ervi

ng

Clusters A and B Clusters C and D Cluster E Very High Risk

(Clusters 1-A, 1-B) Deli Meats Frankfurters (not reheated)

High Risk (Clusters 1-C, 1-D)

Pâté and Meat Spreads Unpasteurized Fluid Milk Smoked Seafood

Moderate Risk (Cluster 1-E)

No food categories Cluster 1

High Risk (Clusters 2-A, 2-B)

High Fat and Other Dairy

Products Pasteurized Fluid Milk Soft Unripened Cheese

Moderate Risk (Clusters 2-C, 2-D)

Cooked RTE Crustaceans

Moderate Risk (Cluster 2-E)

No food categories Cluster 2

Moderate Risk

(Clusters 3-A, 3-B)

No food categories

Moderate Risk (Clusters 3-C, 3-D)

Deli-type Salads Dry/Semi-dry Fermented Sausages Frankfurters (reheated) Fresh Soft Cheese Fruits Semi-soft Cheese Soft Ripened Cheese Vegetables

Low Risk (Cluster 3-E)

Preserved Fish Raw Seafood

Cluster 3

Moderate Risk

(Clusters 4-A, 4-B)

No food categories

Low Risk (Clusters 4-C, 4-D)

No food categories

Very Low Risk (Cluster 4-E)

Cultured Milk Products

Hard Cheese Ice Cream and Other Frozen Dairy Products

Processed Cheese

Cluster 4

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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

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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.

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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)

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NEW DATA

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

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 – Limited prevalence and contamination level data– Growth studies

• Caramel Apple, Multistate, 2015– Growth studies

• Ice Cream, Multistate, 2015– Extensive prevalence and contamination data– Opportunity to assess exposure and underlying health issues

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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 websiteBouvier, Science Translational Medicine 7 (311) pp. 311ec184 (2015)K. A. Glass et al., mBio 6, e01232-15 (2015).

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The case of ice-cream• 10 cases …

– 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

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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

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Enumeration of L. monocytogenes in Different Lots of Product A

Average: 8 MPN/g

Chen et al, submitted; Chen et al, IAFP 2015

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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)

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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

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Conclusions: Listeria monocytogenes…

• The most studied foodborne bacteria

• A lot of questions remain unanswered

• Risk management in an uncertain world