From Experimental Infections in Animals to Quantifying Subtypes in Foods: Data Collection for L. monocytogenes Dose-Response

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1 From Experimental Infections in Animals to Quantifying Subtypes in Foods: Data Collection for L. monocytogenes Dose-Response Yuhuan Chen, Ph.D. FDA Center for Food Safety and Applied Nutrition

2 Overview Recent advancements made in diverse research fields Highlights from selected research Challenges and future considerations Summary Quantifiable differences in dose response due to variability in molecular determinants of L. monocytogenes subtypes

3 Recent Advancements in Knowledge from Diverse Research Physiopathogenicity of L. monocytogenes (Lm) infections Elucidation of the role of internalins Characterization of Lm subtypes Distribution of Lm subtypes in ready-to-eat foods Variability in prevalence and levels Dose responses in humans in relation to subtypes Systemic infections in animals Outbreak vs. virulence attenuated strains

4 L. monocytogenes Infections Key Steps Pathophysiology Invasion Replication Spread Key molecular determinants Internalins (e.g., inla, inlb) Listeriolysin (e.g., hly) Actin (e.g., acta) Host receptors differences Humans Animals (Listeria DR Working Group and David Weingaertner, 2011)

5 Physiological Differences among Animal Models Compared to Humans Bonazzi, Lecuit and Cossart, 2009

6 PMSC type 4 PMSC type 5 PMSC type 6 PMSC type 12 PMSC type 1 PMSC type 2 PMSC type 3 Characterization of L. monocytogenes (Lm) Subtypes Full-length InlA Wildtype, No PMSC in inla Truncated InlA A mutation in inla Presence of PMSC Attenuated mammalian virulence Seven types of PMSC mutation among isolates in U.S. (Van Stelten et al. 2010) Full-length inla and locations of premature stop codon, PMSC (adapted from Van Stelten and Nightingale, 2008)

7 Variability in Prevalence of Subtypes: Full-length vs. inla PMSC Lm strains carrying inla PMSC commonly found in foods but rarely associated with human cases (Van Stelten et al. 2010) Human isolates 5.1% (26 of 507) Food isolates 45% (226 of 502) Variability of Lm strains among food and environmental isolates (Ward et al., 2010) Outbreak-associated strains found in food/environment, 7.6% Lm strains with inla PMSC found in food/environment, 48.5% Full-length inla found at a higher frequency in clinical isolates than in food isolates (Jacquet et al., 2004) Human isolates 96% Food isolates 65%

8 A Case Study Are there differences in the concentrations of Lm subtypes in foods? How do such differences translate into dose response differences in humans?

9 Stepping Back: A (Short) History

10 The NFPA Survey Covered - Total Ready-to-Eat Food Samples - Collected in CA and MD from Jan to Dec. 2001

11 Data Obtained for Eight RTE Categories No. Positives (No. Samples Tested) Products MD CA MD+CA Fresh Soft Cheese 4 (1450) 1 (1481) 5 (2931) Bagged Salads 8 (1465) 14 (1501) 22 (2966) BVSMR Cheese 7 (1473) 30 (1497) 37 (2970) Seafood Salads 88 (1225) 27 (1221) 115 (2446) Smoked Seafood 43 (1281) 71 (1363) 114 (2644) Luncheon Meats 54 (4599) 28 (4600) 82 (9199) Deli Salads 103 (4295) 99 (4256) 202 (8549)

12 Data from the Food Survey Prevalence and concentrations Overall 577 positive samples (1.82% ) Enumeration of positive samples MPN enumeration and direct plating (MPN/g and CFU/g) 502 L. monocytogenes isolates Isolates from eight RTE food categories

13 Concentration Distribution of Positive Samples (No. isolates in each range) Concentration interval Lm Lm All Isolates (log CFU/g) Subgroup A Subgroup B [-2] - [-1] 398 [-1] Lm Subtype k Average dose 8.0x10 4 Average dose in CFU/ serving, determined from the frequency data and serving size distribution (serving size data from FDA/FSIS 2003 Lm risk assessment)

14 Molecular Subtyping of L. monocytogenes Isolates Subtyping targeted a virulence factor inla, encoding internalin A (InlA) InlA facilitates invasion (L. monocytogenes crossing intestinal barrier) Subtyping 502 food isolates and 507 human isolates from multiple States Including 42 isolates from listeriosis cases in MD and CA FoodNet sites, , provided by CDC inla allelic type data published by Van Stelten, Simpson, Ward and Nightingale (2010)

15 Concentration Distributions Partitioned by inla Subtypes (No. isolates in each range) Concentration interval Wildtype inla PMSCs in inla All Lm isolates a PMSC4 in inla [-2] - [-1] [-1] Average dose b 1.8x x x x10 6 a Data for no subgrouping for comparison. b CFU/ serving, determined from the frequency data and serving size distribution (serving size data from FDA/FSIS 2003 Lm risk assessment)

16 Database Used for Dose Response Analysis Food survey data Frequency of contamination by subtypes Concentration for each subtype (CFU/g) Clinical data Relative frequency of subtype over observed cases Epidemiology data Number of cases per year Consumption data Number of servings Serving size

17 Subtype-Specific DR Model P r k / I P / µ k I k P k µ k P / k P k P I I : average dose ingested : fraction of illness cases attributed to subtype k : fraction of food exposure attributed to subtype k : probability of illness from exposure to Lm regardless of subtype

18 Dose-Response in Relation to L. monocytogenes Subtypes based on Epi and Food Data 0-1 Probability (log) of illness (invasive lsteriosis) Wildtype: log r = (-8.1) [-10.4] Subtypes with PMSC in inla: log r = (-10.7) [-13.8] Dose (log CFU) (DR curve with circles: all L. monocytogenes isolates regardless of subtype, log r = (-9.7) [-12.8])

19 Comparison of Virulence Potential between Subgroups/Subtypes based on Epi and Food Data Molecular subgroups/subtypes a r-value (log) Confidence interval (95% CI) Wildtype (no PMSC) vs. PMSCs in inla [3.24] b [ ] Wildtype (no PMSC) vs. PMSC4 in inla INF [4.06] Not quantifiable [ ] Lineage I vs. Lineage II 2.42 [2.65] [ ] Ribotype DUP-1062A vs. DUP-1042B 3.53 [4.19] [ ] a Results from published studies from Gray et al. (2004), Nightingale et al. (2008); Van Stelten et al. (2010), and Chen et al. (2006&2011) b Values outside (inside) brackets: based on human illness and clinical isolate data from MD&CA (from multiple U.S. states), keeping other inputs the same

20 Dose-Response in Relation to Subtypes based on Guinea Pigs Data Probability of spleen infection Dose (log CFU) Solid line female guinea pigs outbreak strain Long dashed line male guinea pigs outbreak strain Short dashed line male guinea pigs attenuated strain (food isolate with PMSC in inla) Van Stelten et al., AEM April 2011; data for female guinea pigs provided by Mary A. Smith

21 Questions Remain: Low Dose Extrapolation for the Same Subtype A Probability of infection for isolate with inla PMSC Guinea pigs dose response: Log-logistic (purple dashed line) Exponential (blue dashed line) Log-logistic (solid orange line) Beta-Poisson (solid green line) Dose (log CFU) (Van Stelten et al., 2011) (Chen et al., 2011) Which DR model is plausible? Further research is needed.

22 Summary Recent advancements in data collection greatly enhance our understanding of the multiple facets of L. monocytogenes (Lm) dose response. Molecular determinants of pathogenicity (role of InlA) Differences in human and animal host receptors Variability in Lm subtypes prevalence/levels in RTE foods Lm subtype virulence may vary by 4 logs (>10,000-fold) Based on data relevant to humans and subtyping mechanistically related to defined genetic markers, provided assumptions made Challenges remain Relating data from diverse research fields to listeriosis in humans Reducing uncertainties, e.g., extrapolating from high to low dose, application to specific subpopulations such as the elderly

23 Acknowledgements Participating Experts at the Joint IRAC/JIFSAN L. monocytogenes Dose Response Workshop Interagency Listeria Dose Response Working Group Sherri Dennis, FDA Disclaimer: The findings and conclusions in this presentation do not necessarily reflect the official views of FDA.