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N. D. Andritsos *, S. D. Paramithiotis , E. H. Drosinos , M. A. Mataragas

Bayesian inference for quantifying Listeria monocytogenes presence in minced pork meat as well as sensitivity and specificity of culture methods. Summary

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N. D. Andritsos *, S. D. Paramithiotis , E. H. Drosinos , M. A. Mataragas

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  1. Bayesian inference for quantifying Listeriamonocytogenes presence in minced pork meat as well as sensitivity and specificity of culture methods Summary The aim of the present study was the detection of the food-borne pathogen Listeria monocytogenes in minced pork meat samples, so as to estimate the prevalence (p) and concentration (c) of the bacterium, as well as to evaluate the sensitivity (Se) and specificity (Sp) of three culture media (i.e. PALCAM, ALOA and RAPID’L.mono) most commonly used for the detection/enumeration of listeriae in foods, by the application of Bayesian inference. Samples (n=100) taken from butcher’s shops and supermarkets were tested for L. monocytogenes presence by plating in parallel onto the three differential diagnostic media. Species confirmation was doubled checked by performing biochemical (tests) and molecular (PCR) identification. Finally, 22 samples were identified positive to L. monocytogenes (p=22%). Se was estimated at 72.7%, 68.2% and 77.3%, whereas Sp at 100%, 94.9% and 88.5% for PALCAM, ALOA and RAPID’L.mono, respectively. Based on the results obtained by plating onto the culture media, two Bayesian models were set up. The first model utilized the parallel use of all three culture media, while the latter used only the results obtained from ALOA and RAPID’L.mono. Both these models were in accordance and estimated satisfactorily (error<20%) the values of the tested parameters (p, Se, Sp), so the capability of the second model to predict the p of the pathogen was further assessed. For that reason, an independent validation experiment was performed through the analysis of extra samples (n=10). The results showed a good correlation between the observed and the predicted p value (error=3.27%). As far as the c (CFU/kg of total samples) of the pathogen is concerned, another Bayesian model was set up with the help of the Excel and estimated it with 95% confidence interval (CI) at 10.9 (CI: 5.9-17.9 log CFU/kg), 13.9 (CI: 7.8-22.8 log CFU/kg) and 17.2 (CI: 10.7-26.2 CFU/kg) for PALCAM, ALOA and RAPID’L.mono, respectively. Conclusively, the parallel use of at least two culture media adds value for L. monocytogenes detection in meat samples, because of the ability to predict the p of the pathogen without the need of further confirmation testing, as this is implied by Bayesian inference. Introduction/Objectives Mince is an excellent substrate for microbial growth. The main objective of this work was to estimate the prevalence and concentration of L. monocytogenes in minced pork meat, as well as to evaluate the sensitivity and specificity of the three culture media used (i.e. PALCAM, ALOA and RAPID’L.mono), by the application of Bayesian inference. Table 2: Predicted values of detection parameters given by Bayesian modeling for quantifying L. monocytogenes presence in minced pork meat, after subculturing on ALOA and RAPID’L.mono Methods Sampling 100 samples of fresh minced pork meat (500 g/sample) obtained from local markets (butcher’s shops and supermarkets) in Athens (Greece). • Detection/enumeration of L. monocytogenes • Parallel use of 3 differential diagnostic culture media: • PALCAM (Biolife, Milano, Italy) non-chromogenic medium • ALOA (Biolife) • RAPID’L.mono (Bio-Rad, Paris, France) • Detection according to ISO 11290-1:1996 • Enumeration according to ISO 11290-2:1998 a Value refering to the combination of confirmation results for every single culture medium used B CI: 95% Confidence Interval Discussion/Conclusions Microbiological data could be biased due to false-positive and false-negative results produced by a culture method during routine analysis. Therefore, the use of three culture methods in parallel adds value for L. monocytogenes detection. Chromogenic media (e.g. ALOA and RAPID’L.mono) are preferentially better compared to non-chromogenic ones (e.g. PALCAM), as their ability to differentiate individual species (e.g. L. monocytogenes) can be utilized by Bayesian inference without the need of further specie confirmation. RAPID’L.mono was more sensitive than ALOA in detecting L. monocytogenes in mince, but showed a significantly reduced specificity value due to many false-positive results produced. Differences between the observed and predicted values of p, Se and Sp given by Bayesian modeling for quantifying L. monocytogenespresence in minced pork meat, after subculturing on ALOA and RAPID’L.mono, were below enough of the acceptable limit (<20%) (Ross, 1996). Besides, a good correlation was established between the observed and the predicted p value (error=3.27%) of an independent validation experiment performed. Conclusively, the parallel use of at least two culture media adds value for L. monocytogenesdetection in meat, because of the ability to predict the p of the pathogen without the need of further confirmation testing. chromogenic media N. D. Andritsos*,S. D. Paramithiotis, E. H. Drosinos, M. A. Mataragas Laboratory of Food Quality Control & Hygiene, Department of Food Science & Technology, Agricultural University of Athens, IeraOdos 75, GR-118 55 Athens, Greece *Poster Author: • Confirmation of L. monocytogenes • Species confirmation was doubled checked by performing: • Biochemical tests: motility, Gram stain, catalase, oxidase, haemolysis, fermentation of rhamnose, xylose, mannitol & methyl-α-D-mannopyranoside • Polymerase Chain Reaction (PCR) according to D’ Agostino et al. (2004) Bayesian inference Bayesian models for p, Se and Sp estimation were build using the WinBUGS software. Furthermore, to estimate c, from the positive samples, a Bayesian model was set up in the Excel and was simulated with the @Risk 4.5 software to construct the posterior distribution. Results Table 1: Observed values of detection parameters for quantifying L. monocytogenes presence in minced pork meat a The value of the tested parameter for each culture medium used results after species confirmation has taken place B Value refering to the combination of confirmation results for every single culture medium used Acknowledgement This work was supported by the A. S. Onassis Public Benefit Foundation, through granting a PhD scholarship on Food Quality Management & Hygiene Assurance Systems (Scholarship Code No.: G-ZC 003-2/2008-2009) to the author N. D. Andritsos. • References • D’ Agostino M., Wagner M., Vazquez-Boland J. A. et al., 2004. J. Food Prot., 67, 1646-1655. • ISO, 1996. International Standard, EN-ISO 11290-1. Geneva, International Organization for Standardization. • ISO, 1998. International Standard, EN-ISO 11290-2. Geneva, International Organization for Standardization. • Ross T, 1996. J. Appl. Bacteriol., 81, 501-508. Figure 1: Concentration of L. monocytogenes in minced pork meat depending on the culture medium used for the detection of the pathogen

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