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The production of healthy, nutritious, tasty, and safe foods requires efficient strategies to control foodborne pathogens along the food chain. One of these pathogens is the notorious foodborne Listeria monocytogenes. L. monocytogenes is a robust, ubiquitously present human pathogen, and the cause of life-threatening listeriosis in the very young, elderly, pregnant, and immunocompromised persons, the so-called YOPI population. The incidence of L. monocytogenes infections is low, but the severity of listeriosis and the high mortality rate rank it among the top three causes of death by foodborne disease.There is an inherent heterogeneity in microbial populations, and this heterogeneity gives L. monocytogenes the capacity to cope with stresses during transmission from the environment to the human host. Stochastic differences in stress response between individual cells of a population, lead to the differential survival of cells after lethal stresses such as heat or low pH, and ultimately result in tailing of the inactivation curve. The heterogeneity in a population can be either transient, where certain cells temporarily have different properties, or stable, where individual cells have undergone genetic changes that make them better able to resist (lethal) stress. Cells with genetic changes are called stable variants, and can be isolated from the tail of the inactivation curve.    Almost all research that has been done with L. monocytogenes has focussed on the diversity that is already present in a population. Therefore, in chapter 2, we investigated the rate at which new diversity is generated by mutations. Using a high-throughput protocol, we have experimentally determined the mutation rate of 20 L. monocytogenes strains, and found a mutator strain with an insertion in the DNA mismatch repair gene mutS, that resulted in a 100-1000-fold increase in mutation rate. To our knowledge, this is the first mutator strain of L. monocytogenes isolated from fo

Since the publication of the 2004 risk assessment, outbreaks of illness and resultant deaths due to L. monocytogenes continue to occur across the globe. Continued effort is needed to summarize and critically evaluate the most recent information on L. monocytogenes in RTE foods. New data to improve and further inform the 2004 Risk Assessment is available for nearly every factor considered previously, including new quantitative data on L. monocytogenes contamination of foods.To facilitate this work, an FAO/WHO expert meeting was held by virtual means from 20 October to 6 November 2020 to review and discuss the available data and background documents, and to assess the need to modify and update risk assessment models/tools. This report focuses on the deliberations and conclusions of the expert meeting.

Spices and dried aromatic herbs impart flavour when added to food, and they may include many parts of the plant, including berries, flowers, leaves, roots and seeds. A number of different pathogens have been found in spices on the market, especially Salmonella spp., B. cereus and C. perfringens. There have also been several disease outbreaks associated with spices and dried aromatic herbs. An increased concern and attention to the safety of spices and dried aromatic herbs prompted, the Codex Committee on Food Hygiene (CCFH) to request FAO and WHO to undertake a risk assessment on microbiological hazards in these food commodities.An expert meeting of the FAO/WHO Joint Expert Meeting on Microbiological Risk Assessment (JEMRA) considered the global evidence on the burden of illness, prevalence and concentration of selected microbial hazards with respect to various spices and dried aromatic herbs, and interventions aimed at controlling them in these commodities. The experts developed the approach to rank the health risks related to the commodity-pathogen combinations, and assessed the performance of the existing Codex sampling plan for Salmonella against several contamination scenarios.

It is well-established that Extended-spectrum beta-lactamase-producing (ESBL-) Escherichia coli challenge reliable detection of campylobacters during enrichment in Bolton broth (BB) following ISO 10272-1:2017. The overgrowth of Campylobacter by ESBL-E. coli in the enrichment medium BB can lead to false-negative detection outcomes, but the cause for the growth suppression is yet unknown. A plausible reason could be the competition-induced lack of certain growth substrates. Therefore, this study aimed to investigate whether campylobacters and ESBL-E. coli compete for the same medium components and whether this is the cause for the observed growth repression. The availability of possible growth substrates in BB was determined and changes in their extracellular concentration were measured over time during mono-culture enrichment of C. jejuni, C. coli or ESBL-E. coli as well as in co-culture enrichments of campylobacters and ESBL-E. coli. Comparative analysis showed lactate and fumarate utilization by C. jejuni and C. coli exclusively, whereas ESBL-E. coli rapidly consumed asparagine, glutamine/arginine, lysine, threonine, tryptophan, pyruvate, glycerol, cellobiose, and glucose. Both campylobacters and ESBL-E. coli utilized aspartate, serine, formate, a-ketoglutarate and malate. Trends in compound utilization were similar for C. jejuni and C. coli and trends in compound utilization were rather comparable during enrichment of reference and freeze-stressed campylobacters. Since final cell densities of C. jejuni and C. coli in co-cultures were not enhanced by the addition of surplus L-serine and final cell densities were similar in fresh and spent medium, growth suppression seems not to be caused by a lack of substrates or production of inhibitory compounds. We hypothesized that oxygen availability was limiting growth in co-cultures. Higher oxygen availability increased the competitive fitness of C. jejuni 81-176 in co-culture with ESBL-E. coli in duplicate experiments, as

Fresh leafy greens like lettuce are often consumed raw and can be contaminated by foodborne pathogens. Washing fresh-cut lettuce can help alleviate foodborne outbreaks and diffuse cases, but the washing process must be controlled to avoid pathogenic cross-contamination. The thesis aimed to evaluate the effectiveness of chemical disinfection of the water during fresh-cut lettuce washing to reduce pathogenic cross-contamination. First, the effectiveness of chlorine, chlorine dioxide (ClO2), ozone, and peracetic acid (PAA) during produce processing was studied along with legislation and disinfection by-product production. Next, the effectiveness of sodium hypochlorite, ClO2, and a silver-copper solution to reduce Salmonella enterica serovar Typhimurium and pathogenic Escherichia coli were assessed on the laboratory scale. Results showed that the water’s organic load, wash water temperature, and pathogenic characteristics influenced the effectiveness. Then, the effectiveness of ClO2 (5 mg/L and 3 mg/L) on reducing supplemented nonpathogenic E. coli during fresh-cut lettuce washing at the industrial scale was studied. Also, the effectiveness of a PAA solution (75 mg/L) on reducing supplemented nonpathogenic E. coli during fresh-cut lettuce washing at the laboratory and industrial scales was studied. The results showed that laboratory experiments disinfecting water with ClO2 and a PAA solution had similar findings, which were also confirmed at the industrial scale. Finally, a multi-criteria decision analysis was applied to a case study to determine the best control strategy during fresh-cut lettuce processing when disinfection was applied directly in the wash tank. Results showed that PAA was the overall preferred control strategy. This proposed disinfection strategy appears to be the right balance between safety and effective fresh-cut produce disinfection

Undissociated lactic acid has been shown to play a major role in complete growth inhibition of Listeria monocytogenes in Gouda cheese. In addition, low water activity conditions may contribute to growth inhibition. In the current study, it was assessed whether the major factors that inhibit growth of L. monocytogenes in Gouda cheese are the factors that determine growth in other types of ready-to-eat cheese as well. Various types of cheeses were selected, some of which had been associated with listeriosis, while others had not. Based on the composition of the different cheese types, the concentrations of undissociated lactic acid were calculated for each type. The ability to support growth of L. monocytogenes was predicted using the Gamma model, based on literature data on total lactic acid content, moisture content, fat content, pH, Aw, and temperature, and optimal growth rates in milk at 30–37 °C. In addition, the actual specific growth rates of L. monocytogenes in the various cheeses were calculated based on available experimental growth data. In 9 out of the 10 RTE cheeses reviewed, the undissociated lactic acid concentrations and aw determined growth/no growth of L. monocytogenes. No growth was correctly predicted for feta, Cheddar and Gouda, and growth was correctly predicted for ricotta, queso fresco, Camembert, high-moisture mozzarella, cottage and blue cheese. Growth of L. monocytogenes was not observed in practice upon inoculation of Emmental, whereas growth in this cheese type was predicted when including the above mentioned factors in the models. Other factors, presumably acetic and propionic acid, are thought to be important to inhibit growth of the pathogen in Emmental. The results from our study show that for cheeses in which lactic acid is a main acid, our model based on undissociated lactic acid, temperature, pH and aw gives a good prediction of potential outgrowth of L. monocytogenes. Implications for L. monocytogenes legislation are discussed per

Several chemical disinfection strategies may be used to control cross-contamination of pathogens during fresh-cut produce washing. Deciding which strategy to select not only requires the use of technical information on the relevant criteria but can also make use of stakeholder perception. This study aimed to describe the application of a Multi-Criteria Decision Analysis (MCDA) to support decision-makers when determining a control strategy that best meets the views of various stakeholders and their possible conflicting interests. In this case study, five control strategies that can be used to treat the wash water, which comes in direct contact with the produce in the wash tank during processing, were examined. These strategies aimed to control pathogenic cross-contamination during fresh-cut lettuce washing at processors. These strategies included the use of free chlorine, chlorine dioxide, ozone, peracetic acid, or no wash water disinfectant. The performance analysis was based on five main criteria: Effectiveness, Technological aspects, Consumer acceptance, Economics, and Health. Scores for these criteria were evaluated using scientific literature, while the weights of the criteria were assessed using stakeholders. Results for the five control strategies were determined per stakeholder group, which included fresh-cut processors, producers and suppliers of disinfectants and equipment, scientists, and government representatives, as well as per individual. Stakeholders were shown to have different views on the relative importance of the criteria; however, the criterion “Health” was consistently considered most important. The ranking of the control strategies was similar for each stakeholder group and over stakeholders. Overall, the results showed that peracetic acid is the preferred control strategy. Based on the results of these analyses, the MCDA approach may assist in the complex decision to select a control strategy to control potential cross-contamination directly

Thermophilic sporeformers are a group of bacteria that proliferate at high temperatures (mostly above 50°C as optimum growth temperatures) and can form spores as a mean to survive in unfavourable environmental conditions. They are of primary concern for food processing plants that apply high temperatures. Even though they have been reported as not pathogenic, they could pose a threat to the quality of food products when they grow because their growth produces off flavour and changes the texture of the products. For this reason, they are sometimes used as a hygiene indicator in food processing environments. Customers of intermediate products also often set strict specifications for thermophilic sporeformers. Those specifications are difficult to achieve, thus present an important challenge to the dairy industry worldwide. To meet the strict specifications, a systematic approach to understanding the source of the contamination and the transmission routes of thermophilic bacilli in dairy-concentrate processing environments is needed.In this thesis, the composition of the natural flora of thermophilic sporeformers present along an entire dairy-concentrate processing line is studied (Chapter 2), and the environmental and physiological factors contributing to their rapid growth in dairy-concentrate processing environments is explored (Chapter 2, Chapter 3).Furthermore, it has been reported that biofilms contribute to the persistence of thermophilic sporeformers in dairy-concentrate processing environments. For this reason, specific genes that are involved in biofilm formation and development in thermophilic sporeformers, were also subjects of study in this thesis (Chapter 4). This has produced a better understanding of the mechanism of biofilm formation in thermophilic sporeformers. Lastly, we applied comparative analysis to explore the genomic and physiological characteristics of a group of dairy and non-dairy associated thermophilic bacilli in relation to the contaminat

Fresh leafy greens like lettuce can be consumed raw and are susceptible to foodborne pathogens if they become contaminated. Recently, the number of reported pathogenic foodborne outbreaks related to leafy greens has increased. Therefore, it is important to try to alleviate the human health burden associated with these outbreaks. Processing of fresh-cut lettuce, including washing, is a step in the supply chain that needs to be well controlled to avoid cross-contamination. Current measures to control the quality of lettuce during washing include the use of chemicals like chlorine; however, questions regarding the safety of chlorine have prompted research for alternative solutions with peracetic acid (PAA). This study evaluates the effectiveness of a PAA (c.a. 75 mg/L) solution on the reduction of a commensal E. coli strain during the washing of fresh-cut lettuce. Experiments were performed at the laboratory scale and validated at the industrial scale. We observed that the use of PAA was not adversely affected by the organic load in the water. The contact time and dose of the PAA showed to be relevant factors, as observed by the approximately 5-log reduction of E. coli in the water. Results showed that once introduced during washing, E. coli remained attached to the lettuce, thus supporting the need to control for pathogenic bacteria earlier in the supply chain (e.g., during primary production) as well as during washing. Moreover, our results showed that the use of PAA during washing did not have an apparent effect on the levels of fluorescent pseudomonads (FP) and total heterotrophic bacteria (THB) in lettuce. Overall, our results at the laboratory and industrial scales confirmed that during the processing of fresh-cut produce, where the accumulation of soil, debris, and other plant exudates can negatively affect washing, the use of a PAA (c.a. 75 mg/L) solution was an effective and safe wash water disinfectant that can potentially be used at the industrial scale

Campylobacter jejuni and Campylobacter coli continue to be the leading cause of zoonotic gastroenteritis in the European Union, making reliable detection in food important. Low storage temperatures and atmospheric oxygen concentrations during food production can cause sub-lethal damage or transient non-culturability which is why ISO 10272-1:2017 includes an enrichment step to repair cell damage and increase cell concentrations, thereby supporting detection of campylobacters from foods. The aim of this study was to assess the variability in lag-duration of C. jejuni and C. coli during enrichment after different food-relevant stress treatments and evaluate its impact on growth kinetics and reliability of detection outcomes. Therefore, 13 C. jejuni and 10 C. coli strains were subjected to cold stress during refrigerated and frozen storage. Refrigerated storage did not significantly reduce culturability, but frozen storage reduced cell concentrations by 1.6 ± 0.1 log10cfu/ml for both species. Subsequently, cells were enriched following ISO 10272-1:2017-A and cell concentrations were determined over time and lag-duration and growth rate were determined by fitting the Baranyi-model. Without prior stress treatment, mean lag-duration for C. jejuni and C. coli was 2.5 ± 0.2 h and 2.2 ± 0.3 h, respectively. Refrigerated storage increased lag-duration for C. jejuni to 4.6 ± 0.4 h and for C. coli to 5.0 ± 0.4 h and frozen storage increased lag-duration to 5.0 ± 0.3 h and 6.1 ± 0.4 h for C. jejuni and C. coli, respectively. Comparison of strain- and biological variability showed that differences in recovery after cold stress can be attributed mainly to strain variability since strain variability after refrigeration and freeze stress increased respectively 3-fold and 4-fold while biological variability remained constant. A subset of strains was also subjected to oxidative stress that reduced cell concentrations by 0.7 ± 0.2 log10 cfu/ml and comparison of recovery patterns after o

Foodborne illnesses are a significant cause of morbidity and mortality worldwide and are therefore important to manage for the protection of public health. In the course of the last half century, when it comes to prioritizing resources for their management, there has been a gradual shift of focus from the hazards that cause the illnesses (microorganisms, chemical and physical agents) to the actual risk that these hazards pose to the population (probability and severity of adverse health effects in relation to hazards in food) and their impact on public health. To accommodate these different schools of thought, new health metrics could be operationalized (DALY, Disability Adjusted Life Year) but also new benchmarks were created for the control of foodborne hazards in food supply chains (ALOP, Appropriate Level of Protection; FSO, Food Safety Objective). These risk-based metrics have been proposed by international organizations as a means of establishing a link between governmental public health policy and the management of hazards in the food supply chain. The aim of this thesis was to develop four different case studies on the operationalization of these risk-based metrics (DALY, ALOP, FSO). Cases were selected so as to deal with different microbial hazards, food products and population groups as well as addressing a number of different real life settings faced by competent authorities in various countries. The first case study used a range of publicly available data to rank all foodborne risks in Greece on the basis of DALYs, in order to set national priorities for food safety management. The use of the results of this study for setting ALOPs is part of the general discussion of the thesis. The second case study dealt with setting of ALOP and FSO targets for Listeria monocytogenes in deli meats in the Netherlands. Here the focus was on a severe, low incidence disease, mainly relevant for susceptible subpopulations, usually associated with medium to high doses of th

In this PhD thesis, the potential of outgrowth of L. monocytogenes was assessed in Dutch-type Gouda cheese. It was demonstrated that L. monocytogenes, which can cause listeriosis, is not able to grow in or on Dutch-type Gouda. The factors present in Gouda cheese that can lead to full inhibition of growth of L. monocytogenes were identified and safety criteria aiming for complete inhibition of growth of L. monocytogenes in Dutch-type Gouda cheese are suggested. Dutch-type Gouda cheese is a semi-hard cheese made from bovine milk that is pasteurized when produced at an industrial scale. It is a ready-to-eat food with a pH > 5.0 and water activity aw > 0.94. In absence of scientific evidence that this product does not support growth, Dutch-type Gouda is classified by the European legislation as a ready-to-eat food product able to support growth of L. monocytogenes. In two challenge studies described in this thesis, it was demonstrated that Dutch-type Gouda cheese does not support growth of L. monocytogenes when inoculated in and on the product. During the cheese making process, entrapment but no growth of L. monocytogenes in the curd was observed. During subsequent ripening of the cheeses, no growth was observed, and upon prolonged ripening periods (>2 months) inactivation was found. In the second challenge study, a limited transfer of L. monocytogenes from brine to the outer layers of cheeses was observed, and during brining and ripening viable numbers of L. monocytogenes did not increase. The variation in aw inside Gouda cheese was assessed by determining the profiles of water and NaCl and the resulting aw in nature-ripened and foil-ripened Gouda cheese during brining and ripening. An empirical model was derived for Gouda cheese in which aw is expressed as a function of the NaCl-in-moisture content. Dutch-type cheeses contain organic acids that are known to have potential inhibitory effects on L. monocytogenes. The MICs of organic acids for 6 different L. monocytogene

Global consumption of vegetables and the associated reported foodborne illnesses have been increasing in tandem. This study was commissioned to analyze the microbial risk from “farm to fork” along the fresh vegetable supply chain in Rwanda to explore microbial safety options that can contribute to an integrated system to detect, control and prevent foodborne infections. Specific study objectives were developed, the first being to estimate the burden of foodborne infectious illnesses using the available data to obtain insight into the general plight of food safety issues and to also develop a framework for future investigations. Second, to investigate the microbial safety status, handling practices and risk exposure factors along the vegetable supply chain. To understand the overall impact of foodborne illnesses on human health in Rwanda the burden of food related illnesses in year 2013 was estimated using the DALY (disability adjusted life year) metric, as encouraged by the World Health Organization. DALY is a measure that combines years of life lost due to premature death and healthy years lost due to disability during sickness. Study findings indicate that for the year 2013, watery diarrhea occurred all year round as by the surveillance system data, resulting to an estimated 672 (95% credible interval [CrI] 424 ‒ 932) DALY per million inhabitants, bloody diarrhea was seasonal coinciding with the rainy months and caused an estimated 213 (95% CrI 50 ‒ 475) DALY per million, typhoid and cholera manifested as outbreaks with an estimated 73 (95% CrI 57 ‒ 91) and 1 (95% CrI 0 ‒ 2) DALY per million respectively. These data show that the health burden is high and we suspect that a large proportion is caused by consumption of contaminated food. Also investigated in this study, was the prevalence of foodborne pathogens in farm vegetables and agricultural water (used for irrigation and on-farm washing of vegetables) in Rwanda. In agricultural water from rivers, lakes, lagoon

In this study, factors relevant to nature-ripened Gouda cheese were evaluated for their potential to inhibit growth of Listeria monocytogenes. Factors included water activity, pH, undissociated acetic and lactic acid, diacetyl, free fatty acids, lactoferrin, nitrate, nitrite and nisin. In addition, the effect of temperature was evaluated. For each factor, the actual concentrations and values relevant to Gouda cheese were obtained and the inhibitory effect of these individual factors on growth of L. monocytogenes was assessed. This evaluation revealed that undissociated lactic acid is the most important factor for growth inhibition of L. monocytogenes in Gouda cheese and that, additionally, low water activity as present in the cheese rind and after prolonged ripening times can also cause full growth inhibition. Gouda cheeses have a typical total lactic acid content of 1.47% w/w. In a 2-week old Gouda cheese, with a pH value of 5.25 and a moisture content of 42% w/w, the concentration of undissociated lactic acid in the water phase is 10.9 mM. Growth of L. monocytogenes is not supported when the undissociated lactic acid concentration is > 6.35 mM. Concentrations of undissociated lactic acid in the water phase of Gouda cheese will be higher than this value when the total lactic acid content is > 0.86% w/w at a pH < 5.25 (relevant to young Gouda cheese), or > 1.26% w/w at a pH < 5.50 for mature Gouda cheese (moisture content of 35% w/w). This study underlines the importance of undissociated lactic acid as growth inhibitor for L. monocytogenes in Gouda cheese.

Microbial population heterogeneity allows for a differential microbial response to environmental stresses and can lead to the selection of stress resistant variants. In this study, we have used two different stress resistant variants of Listeria monocytogenes LO28 with mutations in the rpsU gene encoding ribosomal protein S21, to elucidate features that can contribute to fitness, stress-tolerance and host interaction using a comparative gene profiling and phenotyping approach. Transcriptome analysis showed differential expression of 319 genes of which 116 genes were upregulated and 114 genes were downregulated in both rpsU variants. Upregulated genes included a major contribution of SigB-controlled genes such as intracellular acid resistance-associated glutamate decarboxylase (GAD) (gad3), genes involved in compatible solute uptake (opuC), glycerol metabolism (glpF, glpK, glpD), and virulence (inlA, inlB). Downregulated genes in the two variants involved mainly genes involved in flagella synthesis and motility. Phenotyping results of rpsU variants matched the gene profiling data including enhanced freezing resistance conceivably linked to compatible solute accumulation, higher glycerol utilisation rates, and better adhesion to Caco 2 cells presumably linked to higher expression of internalins. Also, bright field and electron microscopy analysis confirmed reduced flagellation of the variants. The activation of SigB-mediated stress defence offers an explanation for the multiple-stress resistant phenotype in rpsU variants.

Everyday reports of food scandals and recalls are published. One day it’s scary bacteria in meat, and another day it’s dangerous pesticides in fruits. According to some, meat needs to be cooked well to prevent food-borne illness while others warn not to heat food to prevent the formation of poisonous substances. Many consumers worry about the conflicting and confusing messages about food hazards This nutrition and health course will teach you about the hazards associated with food and give you the tools to assess and quantify the dangers they can present. You will learn the effects of food processing, the shared responsibility in the food chain and the prevention of food poisoning/intoxication. After this course, you will be able to differentiate what is a food myth vs. an actual risk. Finally, you will gain a contemporary view of how different risks can be weighed in a scientific way.