Ecology of pathogens in enrichments

Transcription

1 Ecology of pathogens in enrichments foodborne pathogens, enrichment, competition, modelling Heidy den Besten ( heidy.denbesten@wur.nl) Wilma Hazeleger (wilma.hazeleger@wur.nl) Tjakko Abee (tjakko.abee@wur.nl) BSc and MSc 4-6 months Microbiological testing of foods is an essential activity to check for the presence of pathogens or to verify that control measures are effectively reducing the prevalence of pathogens to acceptable levels. Detection efficacy is however hampered by the fact that pathogens in food are often damaged due to low storage temperature or food preservation and may only represent a small fraction of the total microflora in food. Analytical testing methods therefore incorporate a timeconsuming enrichment procedure to recover and selectively amplify damaged target pathogens to much higher concentrations allowing subsequent detection. It is generally recognized that the selective enrichment procedure is still based on trial-and-error practices, and can be qualified as a black box that often suffers from a false negative outcome. Insight in the ecology of enrichment cultures is essential to better understand this black box that forms the basis of successful detection of foodborne pathogens. Two projects are focussing on enrichment ecology: 1. Enrichment ecology of Listeria monocytogenes The enrichment procedure for Listeria monocytogenes suffers from under-representation of specific (pathogenic) serotypes, thereby reducing its reliability to detect all serotypes of this pathogenic species. This project aims to investigate and model the growth kinetics of various L. monocytogenes serotypes with(out) competitors during enrichment to test the hypothesis whether the enrichment favours detection of only a selection of serotypes. 2. Enrichment ecology of Campylobacter The enrichment procedure for Campylobacter is hindered by antibiotic resistant microorganisms, such as ESBLs that outcompete Campylobacter during enrichment. These antibiotic resistant microorganisms are naturally present in especially poultry, and prevent reliable detection of Campylobacter in these foods. This project aims to investigate and model the growth kinetics of Campylobacter and competitors in various modified enrichment broths to possibly improve the selectivity. concentration of cells competitor pathogen enrichment time

2 Design a model system for soil soil, literature research, design model system, Listeria Heidy den Besten (heidy.denbesten@wur.nl) Wilma Hazeleger Tjakko Abee BSc and MSc 4-6 months Some pathogens that cause foodborne diseases are able to survive from soil-to-food. One example is Listeria monocytogenes that has been isolated from soil indicating that soil can serve as reservoir for this pathogen. Survival of the microorganism in this niche is a critical step if transmission from soil to food substrates such as plants is considered. To date, no model system is available to study the survival of pathogens in soil. The structure of soil and soil characteristics like low water content and high availability of minerals, will influence the ability of pathogens to survive. This projects aims to design a model medium to simulate soil characteristics, which can be used to study microbial behaviour. Analysis of the factors influencing survival of Listeria monocytogenes in the soil environment, like adhesion to clay particles, is important in understanding the environmental selection and subsequent transmission of strains to food and man.

3 How can a pathogen successfully transit to the host? Listeria monocytogenes, serotypes, survival and transmission potential Heidy den Besten (heidy.denbesten@wur.nl) Wilma Hazeleger (wilma.hazeleger@wur.nl) Tjakko Abee (tjakko.abee@wur.nl) BSc and MSc 4-6 months Listeria monocytogenes is ubiquitously found in the environment and this suggests that human exposure to L. monocytogenes must be frequent. Incidence of infection is however low and the severe symptoms of the disease occur mainly in the susceptible population which includes pregnant women, the very young or elderly and the immunocompromised. Although twelve serotypes can cause disease, at least 95% of L. monocytogenes strains isolated from human listeriosis cases (both outbreak and sporadic) belong to only three serotypes: 1/2a, 1/2b, and 4b. To date, it is not clear whether the observed serotype distribution reflects potential differences in their virulence. Another explanation could be that other characteristics of the organism s ecology and physiology enable strains of these serotypes to more efficiently colonize in foods and food processing environments and survive in the gastro-intestinal tract. This project aims to investigate the survival dynamics and transmission potential of L. monocytogenes strains from different origin and with different serotype from the environment to man The microbial stability and safety of minimally processed foods are based on the use of mild preservation factors (for example lowered water activity and storage at low temperature). These mild stress factors can trigger the so-called adaptive stress response in the bacterial cell. When the adaptive stress response is activated, the bacteria can gain increased resistance to lethal stresses. Increasingly, it is known that bacteria which originate from the same population, show diversity in their stress response. In this project, bacterial cells will be cultured and imaged on strips made of Anopore to facilitate monitoring of cells at the early stage of stress exposure. Anopore stips are

4 Which nutrients are essential for growth? Listeria monocytogens, physiology, growth requirements Heidy den Besten Wilma Hazeleger Tjakko Abee BSc and MSc 4-6 months Which nutrients are essential for Listeria monocytogenes to grow? And on which carbon sources can it grow? The answers to these questions will give insight into the physiology of this pathogen. In the natural habitats of Listeria monocytogenes only a limited number of nutrient sources might be available, thereby determining its growth potential. This projects aims to determine the essential nutritional growth requirements and the range of substrates it can use, to understand how Listeria monocytogenes can survive and grow in diverse circumstances.

5 Variability of Salmonella during enrichment Salmonella, spoilage flora, growth rate, competition, enrichment, detection Martine Reij Gerrieke van Middendorp Heidy den Besten BSc and MSc 3-6 months For the detection of pathogens usually an enrichment step is applied, during which the pathogen can recover and multiply. Both pathogens and spoilage flora, however, grow simulaneously during enrichment and competition between pathogens and spoilage flora during enrichment is imminent. After non-selective enrichment, usually a second selective enrichment is applied. This selective step aims to allow the target organism (usually a pathogen) to proliferate while the growth of other microorganisms will be limited by inhibitory compounds and non-optimal growth conditions. During selective enrichment pathogens and other flora compete as well. This study aims at determining the kinetics of Salmonella serovars during pre-enrichment in Buffered Pepton Water (BPW) and the variability thereof. In addition, the effect of antibiotics and relation between the growth of spoilage flora and the development or inactivation of pathogenic flora during pre-enrichment.

6 Rapid identification of enterobacteriaceae in food and food environments Enterobacteriaceae, competition, identification, transfer, tracing Martine Reij Gerrieke van Middendorp BSc and MSc 3-6 months Matrix-assisted laser ionization/desorption time of flight mass spectrometry (MALDI-TOF MS) has been widely used for identification of micro-organisms in the medical field. The identification of the bacteria is obtained by extracting their proteins, analysing the protein patterns and comparing with a database to identify the microorganisms at genus, species and sometimes at subspecies level. The main advantage of MALDI-TOF MS comparing to other identification methods, is that it is a fast technique giving reliable results by analysing many samples in a limited amount of time. For food-related organisms, however, MALDI-TOF MS has so far not been widely used amongst other due to the lack of suitable databases. The aim of this project is to explore the possibilities of using MALDI-TOF MS in the field of food safety microbiology and hygiene. Within this area the following topics are being studied: Building and extending databases for rapid identification of enterobacteriaceae Exploring the potential of Malditof MS to detect low levels of organisms. Application in mixed cultures and food matrices

7 Inactivation of pathogens in spices and herbs model validation, gamma irradiation, aw, temperature Ioanna Stratakou Heidy den Besten Marcel Zwietering BSc and MSc 3-6 months Spices and dried herbs are products of low water activity, which inhibits microbiological growth. However, spices and herbs can be naturally contaminated with high numbers of microorganisms, among them potentially pathogenic bacteria and toxigenic moulds, for example, Salmonella spp., Clostridium perfringens, Bacillus cereus and aflatoxigenic Aspergillus spp.[1, 2]. Treatments like irradiation, microwave, and fumigation aim to both reducing microbial counts and preserving the characteristics of these commodities. The effect of these decontamination treatments has been determined by meta-analysis of published studies. Inactivation rates of pathogens by irradiation, gas treatments and steam in spices and herbs were estimated and the corresponding variability. While some microorganisms can be inactivated at a satisfactory level, others can be very troublesome and survive decontamination. This project aims to design and conduct inactivation experiments in spices and herbs which will provide insight on the different pathogen survival rates and reveal the variability of the applied decontamination treatments. The results will be used for model validation and compared to the data found in literature. 1. Sospedra, I., J.M. Soriano, and J. Mañes, Assessment of the microbiological safety of dried spices and herbs commercialized in spain. Plant Foods for Human Nutrition, (4): p Witkowska, A.M., et al., The microbiological quality of commercial herb and spice preparations used in the formulation of a chicken supreme ready meal and microbial survival following a simulated industrial heating process. Food Control, (3-4): p

8 Survival of pathogens in spices and herbs at storage conditions kinetics, aw, temperature Ioanna Stratakou Heidy den Besten Marcel Zwietering BSc and MSc 3-6 months Spices and dried herbs are products of low water activity, which inhibits microbiological growth. However, spices and herbs can be naturally contaminated with large numbers of microorganisms, among them potentially pathogenic bacteria and toxigenic moulds, for example, Salmonella spp., pathogenic Escherichia coli strains, Clostridium perfringens, Bacillus cereus and aflatoxigenic Aspergillus spp.[1, 2]. These pathogenic organisms can survive for a long time in spices and herbs. In this project, data from scientific literature on pathogen survival in spices and herbs will be collected and analysed. A meta-analysis will be done to evaluate significant factors affecting survival. Also, the survival of selected pathogens will be monitored in different matrices, one herb and one spices, using differences pathogenic strains. These data will be used to model the survival of pathogens in spices and herbs and put it into perspective to the data found in literature. 1. Sospedra, I., J.M. Soriano, and J. Mañes, Assessment of the microbiological safety of dried spices and herbs commercialized in spain. Plant Foods for Human Nutrition, (4): p Witkowska, A.M., et al., The microbiological quality of commercial herb and spice preparations used in the formulation of a chicken supreme ready meal and microbial survival following a simulated industrial heating process. Food Control, (3-4): p

9 Sampling plans for spices and herbs microbiological limits, sample size, low aw food commodities, microbial distributions Ioanna Stratakou Heidy den Besten Marcel Zwietering BSc and MSc 6 months Spices and herbs are produced from diverse plants from mostly tropical and subtropical soils and climates. Depending on the product, different parts of the plant are used to produce the spice or the herb. In case of mustard and nutmeg the seeds are used, for pepper the berries, in oregano and bay the leaves and so on [1]. These products commonly carry large numbers of bacteria and moulds while herbs mainly carry microorganisms of soil origin. Spore-forming organisms capable of causing gastroenteritis and Salmonella spp. can be found in spices. In fresh herbs pathogenic types E. coli have been reported [2]. For a producer to bring a safe product in the market he has to ensure that levels of microorganisms in a product do not pose threat to human health and the way to achieve this is not only to prevent and control contamination, but also to monitor the process. Sampling is a method to monitor a product s safety throughout the food chain and the sampling strategy plays a significant role whether the monitoring is effective. In this project, a batch of spices will be examined for Salmonella spp. and/or with Enterobacteriaceae. This will provide information on the distribution of these organisms in batches of spices which is important to evaluate and efficient sampling strategies. 1. ICMFS, 1980, Microorganisms in Foods 1: Their Significance and Methods of Enumeratio. 2nd ed. (1978); reprinted 1982, 1988 with revisions. Toronto: University of Toronto Press. ISBN: , pp ICMFS, 1986, Microorganisms in Foods 2. Sampling for microbiological analysis: Principles and specific applications. International Commission on Microbiological Specifications for Foods. 2nd ed. (1986). Toronto: University of Toronto Press. ISBN: , pp