Quantitative microbiological risk assessment as a tool to obtain useful information for risk managers - specific application to Listeria monocytogenes and ready-to-eat meat products

The presence of Listeria monocytogenes in a sliced cooked, cured ham-like meat product was quantitatively assessed. Sliced cooked, cured meat products are considered as high risk products. These ready-to-eat, RTE, products (no special preparation, e.g. thermal treatment, before eating is required), support growth of pathogens (high initial pH = 6.2–6.4 and water activity = 0.98–0.99) and has a relatively long period of storage at chilled temperatures with a shelf life equal to 60 days based on manufacturer's instructions. Therefore, in case of post-process contamination, even with low number of cells, the microorganism is able to reach unacceptable levels at the time of consumption. The aim of this study was to conduct a Quantitative Microbiological Risk Assessment (QMRA) on the risk of L. monocytogenes presence in RTE meat products. This may help risk managers to make decisions and apply control measures with ultimate objective the food safety assurance. Examples are given to illustrate the development of practical risk management strategies based on the results obtained from the QMRA model specifically developed for this pathogen/food product combinatio

Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in fresh cut vegetables

Innovative diagnostic methods were developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and evaluate their efficacy and viability for routine analysis. To this purpose, ready-to-eat fresh fruits and vegetables were collected throughout the production chain. A multidisciplinary approach, including a newly developed ELISA method compared to ISO procedures, was applied to detect the pathogenic bacteria after harvesting, processing and shelf-life. Results obtained exhibited the technical efficiency of the developed methods showing similar sensitivity, specificity, negative predictive values and negative likelihood ratios

Estimation of listeria monocytogenes and escherichia coli O157:H7 prevalence and levels in naturally contaminated rocket and cucumber samples by deterministic and stochastic approaches

The aims of the present study were to determine the prevalence and levels of Listeria monocytogenes and Escherichia coli O157:H7 in rocket and cucumber samples by deterministic (estimation of a single value) and stochastic (estimation of a range of values) approaches. In parallel, the chromogenic media commonly used for the recovery of these microorganisms were evaluated and compared, and the efficiency of an enzyme-linked immunosorbent assay (ELISA)-based protocol was validated. L. monocytogenes and E. coli O157:H7 were detected and enumerated using agar Listeria according to Ottaviani and Agosti plus RAPID'L.mono medium and Fluorocult plus sorbitol MacConkey medium with cefixime and tellurite in parallel, respectively. Identity was confirmed with biochemical and molecular tests and the ELISA. Performance indices of the media and the prevalence of both pathogens were estimated using Bayesian inference. In rocket, prevalence of both L. monocytogenes and E. coli O157:H7 was estimated at 7% (7 of 100 samples). In cucumber, prevalence was 6% (6 of 100 samples) and 3% (3 of 100 samples) for L. monocytogenes and E. coli O157:H7, respectively. The levels derived from the presence-absence data using Bayesian modeling were estimated at 0.12 CFU/25 g (0.06 to 0.20) and 0.09 CFU/25 g (0.04 to 0.170) for L. monocytogenes in rocket and cucumber samples, respectively. The corresponding values for E. coli O157:H7 were 0.59 CFU/25 g (0.43 to 0.78) and 1.78 CFU/25 g (1.38 to 2.24), respectively. The sensitivity and specificity of the culture media differed for rocket and cucumber samples. The ELISA technique had a high level of cross-reactivity. Parallel testing with at least two culture media was required to achieve a reliable result for L. monocytogenes or E. coli O157:H7 prevalence in rocket and cucumber samples

Validation of innovative methods for human pathogen bacteria detection in fresh cut vegetables

In the framework of the QUAFETY FP7 \u2013 EU project innovative diagnostic methods have been developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and evaluate their efficacy and viability for routine analysis. For this purpose, ready-to-eat fresh fruits and vegetables, have been collected throughout the production chain. More accurately, a total of 48 samples of rocket, mixed salad and piel de sapo melon have been provided by Italian, Portuguese and Greek SMEs. A multidisciplinary approach, including newly developed ELISA and MPN-qPCR methods as well as ISO procedures have been used to detect the pathogenic bacteria after harvesting, processing, packaging and shelf-life. Results obtained exhibited the technical efficiency of the developed methods. More accurately, both methods had similar sensitivity, specificity, negative predictive values and negative likelihood ratios. False positive results obtained by the ELISA method resulted in the reduction of positive predictive values. Regarding their efficacy and viability for routine analysis it is mostly dependent upon available equipment and technical expertise

In Vitro Gene Transcription of Listeria monocytogenes after Exposure to Human Gastric and Duodenal Aspirates

The aim of the present study was to assess, for the first time to our knowledge, Listeria monocytogenes CFU changes, as well as to determine the transcription of key virulence genes, namely, sigB, prfA, hly, plcA, plcB, inlA, inlB, inlC, inlJ, inlP, and lmo2672 after in vitro exposure to human gastric and duodenal aspirates. Furthermore, investigations of the potential correlation between CFU changes and gene regulation with factors influencing gastric (proton pump inhibitor intake and presence of gastric atrophy) and duodenal pH were the secondary study aims. Gastric and duodenal fluids that were collected from 25 individuals undergoing upper gastrointestinal endoscopy were inoculated with L. monocytogenes serotype 4b strain LQC 15257 at 9 log CFU•mL-1 and incubated at 378C for 100 min and 2 h, respectively, with the time corresponding to the actual exposure time to gastric and duodenal fluids in the human gastrointestinal tract. Sampling was performed upon gastric fluid inoculation, after incubation of the inoculated gastric fluids, upon pathogen resuspension in duodenal fluids and after incubation of the inoculated duodenal fluids. L. monocytogenes CFU changes were assessed by colony counting, as well as reverse transcription quantitative PCR by using inlB as a target. Gene transcription was assessed by reverse transcription quantitative PCR. In 56% of the cases, reduction of the pathogen CFU occurred immediately after exposure to gastric aspirate. Upregulation of hly and inlC was observed in 52 and 58% of the cases, respectively. On the contrary, no upregulation or downregulation was noticed regarding sigB, prfA, plcA, plcB, inlA, inlB, inlJ, inlP, and lmo2672. In addition, sigB and plcA transcription was positively and negatively associated, respectively, with an increase of the pH value, and inlA transcription was negatively associated with the presence of gastric atrophy. Finally, a positive correlation between the transcriptomic responses of plcB, inlA, inlB, inlC, inlJ, inlP, and lmo2672 was detected. This study revealed that the CFU of the pathogen was negatively affected after exposure to human gastroduodenal aspirates, as well as significant correlations between the characteristics of the aspirates with the virulence potential of the pathogen. © 2020 International Association for Food Protection. All rights reserved

Microbial ecosystem of traditional dry fermented sausages in Mediterranean countries and Slovakia.

This study highlighted the wide diversity of the processes for the manufacturing of traditional dry sausages in the Mediterranean countries (Spain, Italy, France, Greece and Portugal) and East-Central Europe (Slovakia). In particular, the temperature range of the “fermentation” step was broad, from low temperature 2°C to high 26°C together with variable length (1-8 days). Also a wide range both in temperature (2-25°C) and length (5-90 days) was noticed for the ripening step. Statistical analyses revealed that Gram-positive catalase-positive cocci (GCC+), yeasts/moulds and Enterobacteriaceae discriminated the ripened sausages according to the geographical origins while Lactic Acid Bacteria (LAB) constituted the dominant bacteria of the ripened products in all the countries. LAB counts, which increased during the fermentation step and aw which decreases during ripening, discriminated the three manufacturing steps (batter, fermented, ripened). In general, traditional dry fermented sausages did not present health risks in general, although the presence of some pathogens and spoilage microbiota in some sausages highlighted the importance of maintaining sound hygienic procedures

Tools for the performance assessment and improvement of food safety management systems ; review

Food business operators are challenged to combine requirements from different stakeholders (e.g. government, retailers) into a company specific Food Safety Management System (FSMS). Tools to diagnose the performance of an implemented FSMS (diagnostic tools), tools to help a selection process (selection tools), and tools to improve the FSMS performance (improvement tools) are presented. These tools have been validated in European food processing industries (meat and dairy chain). They are organised towards an on-line freely available FSMS support application which can be used for the internal auditing process. These tools should empower food business operators to improve the performance of FSMS and to produce safer food products

Strategies to reduce biogenic amine accumulation in traditional sausage manufacturing

Different strategies in the reduction of biogenic amine accumulation during the manufacture of five European traditional fermented sausages were studied concerning sausage formulation, the increase of sugar in the Italian salame abruzzese reduced the accumulation of cadaverine up to 43%. However, the addition of sugar in the French saucisson did not show a significant amine reduction. The inoculation of a decarboxylase-negative autochthonous starter culture reduced the biogenic amine accumulation in a different manner depending on the species and strain(s). The highest reduction was achieved by Lactobacillus sakei used in the Greek aeros thasou, resulting in a total putrescine reduction and a significant decrease in tyramine (62%) and histamine (71%). In Portuguese chouriços cadaverine reduction was only of 45% when a single strain of Staphylococcus equorum was inoculated, whereas a single strain of L. sakei or a mixture of S. equorum yielded a 75% and 89% of reduction, respectively. In Spanish fuet, a combination of L. sakei CTC6626 plus S. xylosus CTC6013 had only a very slight effect on tyramine reduction (19%) in Spanish fuet, whereas L. sakei CTC494 plus S. xylosus CTC6013 was capable to reduce tyraminogenesis by nearly 50%, suggesting that L. sakei CTC494 was the strain responsible for the additional tyramine reduction

Traditional dry fermented sausages produced in small-scale processing units in Mediterranean countries and Slovakia. 1. Microbial ecosystems of processing environments

Microbial ecosystems were surveyed in 314 environmental samples from 54 Southern and Eastern European small-scale processing units (PUs) manufacturing traditional dry fermented sausages. The residual microflora contaminating the surfaces and the equipment were analysed after cleaning and disinfection procedures. All the PU environments were colonised at various levels by spoilage and technological microflora with excessive contamination levels in some of the PUs. Sporadic contamination by pathogenic microflora was recorded. Salmonella and Listeria monocytogenes were detected in 4.8% and 6.7% of the samples, respectively, and Staphylococcus aureus was enumerated in 6.1% of the samples. Several critical points were identified, such as the machines for S. aureus and the tables and the knives for L. monocytogenes; this knowledge is crucial for the improvement of hygiene control systems in small and traditional meat processing industries. The variability of the residual contamination emphasized the different cleaning, disinfecting and manufacturing practices routinely followed by these small-scale processing units