Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54
Hayrapetyan, Hasmik - \ 2017
Wageningen University. Promotor(en): T. Abee, co-promotor(en): M.N. Nierop Groot. - Wageningen : Wageningen University - ISBN 9789463431194 - 181
microorganisms - bacillus cereus - food contamination - biofilms - foodborne pathogens - abiotic conditions - sporulation - micro-organismen - bacillus cereus - voedselbesmetting - biofilms - voedselpathogenen - abiotiek - sporulatie
Biofilms are surface-associated communities of microbial cells embedded in a matrix of extracellular polymers. It is generally accepted that the biofilm growth mode represents the most common lifestyle of microorganisms. Next to beneficial biofilms used in biotechnology applications, undesired biofilms can be formed by spoilage and pathogenic microorganisms in food production environments. Bacillus cereus is a foodborne human pathogen able to cause two types of food poisoning, emetic and diarrheal. B. cereus can persist in factory environments in the form of biofilms, which can become a source of food contamination. This thesis adds to the knowledge about (a)biotic factors and conditions that affect B. cereus biofilm formation, including the effect of type of substratum such as polystyrene and stainless steel, with the latter supporting the highest biofilm formation for all tested strains including two reference strains and 20 food isolates. The ability of B. cereus to use a variety of iron sources was subsequently studied in these 22 strains and linked to the genes encoding iron transport systems present in the respective genomes, revealing significant diversity in the capacity to use complex and non-complex iron sources for growth and biofilm formation. For spore forming Bacilli, biofilm formation and sporulation are two intertwined cellular processes and studies in wet and dry (air-exposed) biofilms revealed differences in sporulation rate and efficacy, with biofilm-derived spores showing higher heat resistance than their planktonic counterparts. Additionally, comparative phenotype and transcriptome analysis of B. cereus wild type and a Sigma 54 deletion mutant provided insight into the pleiotropic role of this transcriptional regulator in B. cereus biofilm formation and physiology in general. Taken together, this knowledge improves our understanding of the biofilm lifecycle of this notorious food-borne human pathogen and provides clues which can help to reduce the domestication of this microorganism in production environments.
Improvement of methods for the detection of Gram-negative foodborne pathogens
Margot, H.F.T. - \ 2016
Wageningen University. Promotor(en): Marcel Zwietering; Han Joosten, co-promotor(en): R. Stephan. - Wageningen : Wageningen University - ISBN 9789462578708 - 158
gram negative bacteria - pathogens - foodborne pathogens - detection - real time pcr - salmonella - escherichia coli - mung beans - gramnegatieve bacteriën - pathogenen - voedselpathogenen - detectie - real time pcr - salmonella - escherichia coli - mungbonen
Foodborne diseases are a major source of morbidity and mortality worldwide. In most cases, these diseases are caused by contaminated food products, but transmission can also subsequently occur via person to person contact. The ability to detect the pathogens is an important aspect in the verification of food safety. A major proportion of foodborne disease is caused by Gram-negative bacteria. In this thesis, the detection of Gram-negative foodborne pathogens is addressed by looking at the successive steps from enrichment to detection with Salmonella, Shiga toxin-producing E. coli and Cronobacter spp. as example pathogens. The detection of foodborne pathogens using microbiological culture media aiming at the resuscitation and growth of bacteria is still regarded as the gold standard and included in many reference methods. However, cultural methods are time and labour-intensive. Since an immediate response is required in case of contamination and during outbreaks there is a strong interest in methods that deliver information on the microbiological status of the product as quickly and reliable as possible. Rapid cultural methods and commercially available real-time PCR systems for the detection of Salmonella and STEC were compared with regards to their sensitivity and specificity. It was shown that most of the marketed systems are as reliable as the standard methods. However, false-positive results were obtained with real-time PCR systems for the detection of Salmonella. Rapid cultural methods that were based on procedures without the pre-enrichment step, reduced the time to detection but did show some ambiguous results with difficult matrices such as tea. Of the seven rapid tests for the detection of STEC, one did not detect relevant Stx subtypes.
In order to be detected, pathogens need to multiply to reach a minimum threshold level. However, because they are often sublethally injured due to hostile processing and storage conditions, they first need to be resuscitated. For most pathogens, (Salmonella, STEC and Cronobacter spp.) the first step in the detection is an enrichment including resuscitation in a non-selective medium such as BPW. Modifications to BPW were compared with respect to their ability to promote growth of unstressed and stressed Gram-negative pathogens. The aim was to develop a medium that could be used for the enrichment of pathogens in horizontal methods using only one enrichment step. The resuscitation of stressed Cronobacter cells was improved in BPW supplemented with an additional iron source and sodium pyruvate along with low levels of compounds for the inhibition of Gram-positive bacteria. However, it was observed that BPW containing these supplements allowed for less resuscitation of STEC when compared to regular BPW. Based on these results it was concluded that the application of a one-broth enrichment in food products with a high number of competing bacteria is not recommended due to the overgrowth of the target bacteria. Limitations of the current method for the detection of STEC from sprouted seeds were noticed. Therefore, the growth of stressed STEC cells from different serotypes was assessed in media used for the enrichment of Enterobacteriaceae. In addition, the growth of STEC was examined in the enrichment of sprouts using different media and incubation temperatures. It was shown that the high level of competitors was inhibiting the detection of the target pathogen and that the similarity of target and competing bacteria prevents the design of a selective enrichment procedure. In order to get a better insight in the enrichment ecology, the microbiome of mungo bean sprouts was analysed using Illumina HiSeq sequencing prior to and during the enrichment in BPW and EE-broth at different temperatures. The majority of the sprout flora was composed of bacteria belonging to the phylum Proteobacteria. Enrichment in BPW increased the proportion of Firmicutes whereas the incubation in EE-broth enriched Proteobacteria. The results point out that with the application of a selective medium like EE-broth, growth of the competitive microflora that complicates the detection of STEC is promoted. It was shown that EE-broth also resulted in good growth of STEC however, the problematic situation of low maximum population densities of the target strain in the matrix is still present. The probability of detection is not only influenced by the natural flora of a food product, but also by the physiological state of the pathogen. The influence of stress on the lag time of single cells and the resulting probability of detection were determined for Cronobacter spp. in powdered infant formula. Lag time was calculated from optical density measurement data and different scenarios were modelled. Lag time was longest after acid stress and lag time increase coincided with increased lag time variability. The probability of detection, however, depended both on the sampling plan and on the duration of the lag phase.
This thesis provides a critical evaluation of rapid methods and valuable new insights on enrichment procedures, the role of competitors in bacterial enrichment procedures and the limitations of selective agents. This information will be of great help to further improve microbiological methods and thereby contribute to more effective management of food safety.
Salmonella spp. in the feed chain in the Netherlands : monitoring results of five years (2008 to 2012)
Yassin, H. ; Adamse, P. ; Fels, H.J. van der - \ 2015
Wageningen : RIKILT Wageningen UR (RIKILT report 2015.005) - 131
salmonella - voedselpathogenen - veevoederindustrie - voer - voederveiligheid - voedselonderzoek - monitoring - salmonella - foodborne pathogens - feed industry - feeds - feed safety - food research - monitoring
Salmonella spp. is an important food-borne pathogen in humans. In the Netherlands, monitoring Salmonella spp. in the feed and food chain has become an important issue since 1997. Monitoring results from different sectors, such as broiler meat and eggs, are analysed annually to determine the prevalence of Salmonella spp. The objective of this study was to analyse Salmonella spp. prevalence in feed materials in the Netherlands during the years 2008-2012. Data from the Dutch feed industry, stored in the GMP+ monitoring database, were provided by the Dutch Product Board Animal Feed for use in the current study. These data included results of, on average, 10080 compound feed and 9109 feed material samples per year. This high total number of samples reflects the intensive monitoring program in the Netherlands.
Preventing Campylobacter at the Source: Why Is It So Difficult?
Wagenaar, J.A. ; French, N.P. ; Havelaar, A.H. - \ 2013
Clinical infectious diseases 57 (2013)11. - ISSN 1058-4838 - p. 1600 - 1606.
united-states - new-zealand - disease burden - broiler meat - vertical transmission - foodborne pathogens - source attribution - risk-assessment - poultry - spp.
Campylobacteriosis in humans, caused by Campylobacter jejuni and Campylobacter coli, is the most common recognized bacterial zoonosis in the European Union and the United States. The acute phase is characterized by gastrointestinal symptoms. The long-term sequelae (Guillain-Barre syndrome, reactive arthritis, and postinfectious irritable bowel syndrome) contribute considerably to the disease burden. Attribution studies identified poultry as the reservoir responsible for up to 80% of the human Campylobacter infections. In the European Union, an estimated 30% of the human infections are associated with consumption and preparation of poultry meat. Until now, interventions in the poultry meat production chain have not been effectively introduced except for targeted interventions in Iceland and New Zealand. Intervention measures (eg, biosecurity) have limited effect or are hampered by economic aspects or consumer acceptance. In the future, a multilevel approach should be followed, aiming at reducing the level of contamination of consumer products rather than complete absence of Campylobacter.
Risk assessment strategies as a tool in the application of the Appropriate Level of Protection (ALOP) and Food Safety Objective (FSO) by risk managers
Gkogka, E. ; Reij, M.W. ; Gorris, L.G.M. ; Zwietering, M.H. - \ 2013
International Journal of Food Microbiology 167 (2013)1. - ISSN 0168-1605 - p. 8 - 28.
salmonella-typhimurium dt104 - bacterial cross-contamination - broiler supply chain - minced pork meat - listeria-monocytogenes - thermal inactivation - foodborne pathogens - escherichia-coli - assessment model - bacillus-cereus
In the course of the last decade, the Appropriate Level of Protection (ALOP), the Food Safety Objective (FSO) and their associated metrics have been proposed by the World Trade Organization and Codex Alimentarius as a means for competent authorities to ultimately translate governmental public health policy regarding food safety into risk-based targets for the food industry. The industry needs to meet these targets through the effective choice of control measures that are part of its operational food safety management system. The aim of this study was to put the practical application of ALOP and FSO to the test in the case of Salmonella in chicken meat in the Netherlands. Two different risk assessment approaches were applied to derive potential ALOP and FSO values, a ‘top-down’ approach based on epidemiological data and a ‘bottom-up’ approach based on food supply chain data. To this end, two stochastic models specific to the Dutch situation were built. Comparisons between 23 countries in Europe were also made using the top-down model. The mean estimated current Level Of Protection values were similar for the two approaches applied, with the bottom-up model yielding 87 cases per 100,000 inhabitants per year (95% CI: 0.03, 904) and the top-down model 71 (95% CI: 9.9, 155). The estimated FSO values on the other hand were considerably different with the mean ‘top down’ FSO being - 4.6 log CFU/g (95% CI: - 5.4, - 4.1) and the mean ‘bottom-up’ FSO - 6.0 log CFU/g (95% CI: - 8.1, - 2.9) reflecting major differences in the output distributions of this parameter obtained with the two approaches. Significant differences were observed between current LOP values for different EU countries, although it was not clear whether this was due to actual differences in the factors influencing the risk of salmonellosis or due to the quality of the available data. Keywords Risk assessment; Stochastic modelling; Salmonellosis; Foodborne disease; Public health targets
Assessment of Food Safety Management Systems in the global fresh produce chain
Kirezieva, K.K. ; Jacxsens, L. ; Uyttendaele, M. ; Boekel, T. van; Luning, P.A. - \ 2013
Food Research International 52 (2013)1. - ISSN 0963-9969 - p. 230 - 242.
techno-managerial approach - irrigation water - public-health - microbiological quality - foodborne pathogens - enteric pathogens - escherichia-coli - cut lettuce - outbreaks - contamination
Foodborne outbreaks appear to increase with more incidences linked to fresh produce and derived food products. This indicates inadequacies in Food Safety Management Systems (FSMSs), which are currently implemented in companies along the fresh produce chain. However, the information related to these inadequacies is restricted and little is known about the status of the FSMS. This paper describes the development of a tool for assessment of FSMS implemented in the fresh produce chain. The tool consists of indicators and grids to assess activities that are important for fresh produce, and the system output in terms of microbiological and chemical food safety (that is, pesticide residues and emerging mycotoxins). Three sets of indicators, one for each stage of the production chain (primary production, processing and trade), have been validated by experts and tested in companies. The tool enables an integral and comprehensive assessment of FSMS across the entire supply chain. Users of the tool can identify improvement opportunities and learn how to develop towards more advanced levels of activities. For research purposes differences in FSMS can be identified and linked to type of commodity, production system, country, etc.
Surface behaviour of S. Thyphimurium, S. Derby, S. Brandenburg and S. Infantis
Castelijn, G.A.A. ; Parabirsing, J.A. ; Zwietering, M.H. ; Moezelaar, R. ; Abee, T. - \ 2013
Veterinary Microbiology 161 (2013)3-4. - ISSN 0378-1135 - p. 305 - 314.
enterica serovar typhimurium - food contact surfaces - biofilm-formation - listeria-monocytogenes - salmonella spp. - foodborne pathogens - cross-contamination - escherichia-coli - stainless-steel - feed factories
Cross-contamination due to Salmonella on the surface of processing equipment greatly contributes to contamination of pork products. Therefore, a clear understanding of surface and survival behaviour of relevant Salmonella serovars in pork processing environments is needed to develop better strategies for Salmonella control. Within this study the biofilm forming behaviour of S. Typhimurium, S. Derby, S. Brandenburg and S. Infantis isolates was analysed using the crystal violet assay. This assay, commonly used to analyse total biofilm formation, revealed variation in biofilm forming capacity between and within serovars. This has not been shown before for S. Derby, S. Brandenburg and S. Infantis. From each serovar, isolates with different biofilm forming capacity were selected to analyse biofilm formation on stainless steel. This revealed no significant differences between biofilm formation on polystyrene compared to stainless steel. Furthermore a relation was observed between biofilm forming capacity of an isolate and survival on stainless steel surfaces. On such surfaces, biofilms showed greater and longer survival than planktonic cells, and they were less susceptible to peracetic acid disinfection treatments. However, the latter effect was marginal and only observed in the presence of organic material, which drastically decreased the activity of peracetic acid. With the obtained results a hierarchical cluster was also performed to identify differences and similarities between the four different serovars. This indicated that the surface behaviour of S. Typhimurium was more comparable to S. Infantis than to S. Derby or S. Brandenburg
Survival of Listeria monocytogenes on a conveyor belt material with or without antimicrobial additives
Chaitiemwong, N. ; Hazeleger, W.C. ; Beumer, R.R. - \ 2010
International Journal of Food Microbiology 142 (2010)1-2. - ISSN 0168-1605 - p. 260 - 263.
food-processing environments - stainless-steel surfaces - foodborne pathogens - cross-contamination - escherichia-coli - silver ions - stress - products - growth - rubber
Survival of Listeria monocytogenes on a conveyor belt material with or without antimicrobial additives, in the absence or presence of food debris from meat, fish and vegetables and at temperatures of 10, 25 and 37 °C was investigated. The pathogen survived best at 10 °C, and better at 25 °C than at 37 °C on both conveyor belt materials. The reduction in the numbers of the pathogen on belt material with antimicrobial additives in the first 6 h at 10 °C was 0.6 log unit, which was significantly higher (P <0.05) than the reduction of 0.2 log unit on belt material without additives. Reductions were significantly less (P <0.05) in the presence of food residue. At 37 °C and 20% relative humidity, large decreases in the numbers of the pathogen on both conveyor belt materials during the first 6 h were observed. Under these conditions, there was no obvious effect of the antimicrobial substances. However, at 25 °C and 10 °C and high humidity (60–75% rh), a rapid decrease in bacterial numbers on the belt material with antimicrobial substances was observed. Apparently the reduction in numbers of L. monocytogenes on belt material with antimicrobial additives was greater than on belt material without additives only when the surfaces were wet. Moreover, the presence of food debris neutralized the effect of the antimicrobials. The results suggest that the antimicrobial additives in conveyor belt material could help to reduce numbers of microorganisms on belts at low temperatures when food residues are absent and belts are not rapidly dried
Simulation modelling and risk assessment as tools to identify the impact of climate change on microbiological food safety – The case study of fresh produce supply chain
Jacxsens, L. ; Luning, P.A. ; Vorst, J.G.A.J. van der; Devlieghere, F. ; Leemans, R. ; Uyttendaele, M. - \ 2010
Food Research International 43 (2010)7. - ISSN 0963-9969 - p. 1925 - 1935.
minimally processed vegetables - escherichia-coli o157-h7 - agricultural land-use - time rt-pcr - ambient-temperature - iceberg lettuce - cryptosporidium oocysts - foodborne pathogens - contaminated water - future scenarios
The current quality assurance and control tools and methods to prevent and/or to control microbiological risks associated with fresh produce are challenged due to the following pressures upon the food supply chain, i.e. changing consumption patterns, globalization and climate change. It demonstrates the need for scientific research and development of new and/or improved tools, techniques and practices to adapt the current risk management systems. In this paper, a conceptual research approach is presented to analyse the complexity of the climate change and globalization challenge on the fresh produce supply chain taken as a case study. The factors which affect the vulnerability of the fresh produce chain demand a multidisciplinary research approach. The proposed knowledge-based modelling system is believed to be a most appropriate way to identify problems and to offer solutions to monitor and prevent microbiological food safety risks during all phases of food production and supply. To explore the potential impact of climate change and globalization, baseline information can be obtained by surveillance and performance measurement of implemented food safety management systems. Simulation of climate change scenarios and the logistic chain of fresh produce, along with mathematical models to optimize packaging technology to maintain quality and safety of fresh produce are tools to provide insights in the complex dynamic ecosystem. They are the basis for elaboration of risk assessment studies to scientifically support management options and decisions to new microbiological threats related to globalization and climate change in the fresh produce supply chain. This research concept as such will contribute to develop strategies in order to guarantee the (microbiological) food safety of fresh produce on the long term
Comprehensive analysis and differentiated assessment of food safety control systems: a diagnostic instrument
Luning, P.A. ; Bango, L. ; Kussaga, J. ; Rovira, J. ; Marcelis, W.J. - \ 2008
Trends in Food Science and Technology 19 (2008)10. - ISSN 0924-2244 - p. 522 - 534.
critical control point - techno-managerial approach - hazard analysis - haccp system - listeria-monocytogenes - quality management - catering establishments - processing equipment - foodborne pathogens - conceptual-model
In this article, an instrument is presented to diagnose microbial safety control activities in a food safety management system. The need of such a tool is derived from the importance of microbial safety control and the need for improvement of existing control systems. Careful diagnosis of these systems provides the basis for their improvement. The diagnostic instrument provides a comprehensive checklist of crucial control activities, addressing major technology-dependent and managerial activities in design and operation of preventive measures, intervention processes, and monitoring systems. Secondly, it provides detailed grids describing three levels of execution for each safety control activity to enable a differentiated assessment of ones food safety control system situation. The basic assumption underlying the diagnostic instrument is that activities on a higher level are more predictable and better able to achieve a desired safety outcome, due to more insight in underlying mechanisms and more accurate information. Finally, we discuss that using the instrument may contribute in finding effective types and levels of control activities within given contextual dependencies.
Quantification of Campylobacter jejuni cross-contamination via hands, cutlery, and cutting board during preparation of a chicken fruit salad
Verhoeff-Bakkenes, L. ; Beumer, R.R. ; Jonge, R. de; Leusden, F.M. van; Jong, A.E.I. de - \ 2008
Journal of Food Protection 71 (2008)5. - ISSN 0362-028X - p. 1018 - 1022.
food-handling practices - foodborne pathogens - domestic kitchen - united-states - risk-factors - survival - infection - safety - salmonella - surfaces
Using artificially contaminated chicken, the quantitative overall effect of Campylobacter jejuni cross-contamination, either via cutlery, cutting board, or hands, on the microbiological quality of a chicken salad was tested to identify the most critical transfer route. The end contamination level of salads prepared according to different scenarios, with or without cross-contamination, was compared. It was shown that the mean transfer rate calculated for all salads prepared allowing cross-contamination was 0.12% of the initial number of C. jejuni on the chicken fillet (8.8 ± 0.2 log CFU). The difference in calculated transfer rates for the tested cross-contamination routes was not significantly different (P > 0.05). The prevention of cross-contamination by replacing cutlery and cutting board after handling raw chicken and the prevention of hand contact resulted in considerably reduced end contamination levels (
Cross-contamination in the kitchen: effect of hygiene measures
Jong, A.E.I. de; Verhoeff-Bakkenes, L. ; Nauta, M.J. ; Jonge, R. de - \ 2008
Journal of Applied Microbiology 105 (2008)2. - ISSN 1364-5072 - p. 615 - 624.
food-handling practices - foodborne pathogens - campylobacter-jejuni - domestic kitchen - united-states - stainless-steel - survival - chicken - safety - risk
Aims: To determine the effect of hygiene measures on cross-contamination of Campylobacter jejuni at home and to select a safe tracer organism for C. jejuni. Methods and Results: Comparative tests were conducted with nonpathogenic Escherichia coli and Lactobacillus casei and L. casei was chosen as the safe tracer organism. Salads containing chicken breast fillet contaminated with a known number of C. jejuni and L. casei were prepared according to different cross-contamination scenarios and contamination levels of salads were determined. Cross-contamination could be strongly reduced when cleaning cutting board and cutlery with hot water (68°C), but generally was not prevented using consumer-style cleaning methods for hands and cutting board. Conclusions: Dish-washing does not sufficiently prevent cross-contamination, thus different cutting boards for raw meat and other ingredients should be used and meat¿hand contact should be avoided or hands should be thoroughly cleaned with soap. Lactobacillus casei can be used as a safe tracer organism for C. jejuni in consumer observational studies. Significance and Impact of the Study: Cross-contamination plays an important role in the transmission of food-borne illness, especially for C. jejuni. This study delivers suitable data to quantitatively assess the risk of campylobacteriosis caused by cross-contamination and it shows the effect of different preventive hygiene measures.
A quantitative analysis of cross-contamination of Salmonella and Campylobacter spp. via domestic kitchen surfaces
Kusumaningrum, H.D. ; Asselt, E.D. van; Beumer, R.R. ; Zwietering, M.H. - \ 2004
Journal of Food Protection 67 (2004)9. - ISSN 0362-028X - p. 1892 - 1903.
risk-assessment - food safety - foodborne pathogens - exposure assessment - chicken products - retail chicken - prevalence - poultry - model - quantification
Epidemiological data indicate that cross-contamination during food preparation in the home contributes noticeably to the occurrence of foodborne diseases. To help prevent such occurrences, the inclusion of a cross-contamination model in exposure assessments would aid in the development and evaluation of interventions used to control the spread of pathogenic bacteria. A quantitative analysis was carried out to estimate the probability of contamination and the levels of Salmonella and Campylobacter spp. on salads as a result of cross-contamination from contaminated chicken carcasses via kitchen surfaces. Data on the prevalence and numbers of these bacteria on retail chicken carcasses and the use of unwashed surfaces to prepare foods were collected from scientific literature. The rates of bacterial transfer were collected from laboratory experiments and literature. A deterministic approach and Monte Carlo simulations that incorporated input parameter distributions were used to estimate the contamination of the product. The results have shown that the probability of Campylobacter spp. contamination on salads is higher than that of Salmonella spp., since both the prevalence and levels of Campylobacter spp. on chicken carcasses are higher than those of Salmonella spp. It is realistic to expect that a fraction of the human exposure to Campylobacter spp., in particular, originates from cross-contamination in private kitchens during food handling. The number of human campylobacteriosis cases could be reduced either by reducing the degree of Campylobacter spp. contamination on chicken carcasses or by improving the hygiene in private kitchens. To eliminate the cross-contamination route, it is important to use separate surfaces or to properly wash the surfaces during the preparation of raw and cooked foods or ready-to-eat foods.