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Detection, Occurrence, Grow Th and Inactivation of Cronobacter Spp

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Detection, Occurrence, Grow Th and Inactivation of Cronobacter Spp kaft_def.qxd 20/03/2010 11:36 Page 1 Detection, occurrence, growth of andinactivation Detection, occurrence, growth and inactivation of Cronobacter spp. (Enterobacter sakazakii) Cronobacter spp. M.C. Kandhai 2010 Kandhai M.C. spp. Chantal Kandhai Propositions 1. In the last decennium, detection methods for Cronobacter spp. have been improved, which has led to an increase in the isolation of Cronobacter spp. from a wide variety of food products and environments. This fact in itself is not a reason to consider Cronobacter spp. as an emerging pathogen. 2. The use of sterile liquid formulae is an effective control measure to reduce the probability of infections in low and very low birth weight infants due to Cronobacter spp.. 3. As, generally, pathogens are relevant in foods only at such low numbers that multiplication is required to allow their detection, the enrichment step must be regarded as the most important step in the entire detection procedure. Yet, relatively little research effort is dedicated to optimizing this procedure. 4. Improvements in food safety management can only successfully be achieved in a food enterprise when there is a true food safety culture in the food enterprise. 5. Children are an extraordinary gift and enhance their mother's career by improving her multitasking skills. 6. Patience is the most important virtue in joint scientific writing. Propositions belonging to the thesis, entitled: “Detection, occurrence, growth, and inactivation of Cronobacter spp. (Enterobacter sakazakii)” M.C. Kandhai Wageningen, 23 April 2010. Detection, occurrence, growth and inactivation of Cronobacter spp. (Enterobacter sakazakii) M.C. Kandhai Thesis committee Thesis supervisors Prof. dr. L.G.M. Gorris Chairholder of the European Chair in Food Safety Microbiology Wageningen University Prof. dr. ir. M.H. Zwietering Professor of Food Microbiology Wageningen University Thesis co-supervisor Dr. ir. M.W. Reij Researcher and Lecturer at Laboratory of Food Microbiology Wageningen University Other members Prof.dr.ir. M.A.J.S. van Boekel, Wageningen University Prof. S. Fanning, UCD Center for Food Safety, Ireland Prof. dr. ir. A.H. Havelaar, University of Utrecht Dr. ir. A.E. Heuvelink, Food and Consumer Product Safety Authority, Zutphen This research was conducted under the auspices of the Graduate School of Voeding, Levensmiddelentechnologie, Agrobiotechnologie en Gezondheid (VLAG). Detection, occurrence, growth and inactivation of Cronobacter spp. (Enterobacter sakazakii) M.C. Kandhai Thesis Submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Doctorate Board to be defended in public on Friday 23 April 2010 at 4 PM in the Aula. M.C. Kandhai Detection, occurrence, growth and inactivation of Cronobacter spp. (Enterobacter sakazakii), 240 pages. Thesis, Wageningen University, Wageningen, NL (2010) With references, with summaries in Dutch and English ISBN 978-90-8585-556-9 To Fons, Aditya, Sarada & Ishaan “Life is constantly changing. Whatever time you have is yours, for tomorrow might never come” Abstract The genus Cronobacter consists of Gram-negative, motile, non-spore forming, facultative anaerobic bacteria, and was originally defined as one species “Enterobacter sakazakii” within the genus Enterobacter in 1980. Cronobacter spp. have been documented as a rare cause of outbreaks and sporadic cases of neonatal meningitis, necrotizing enterocolitis, and sepsis in infants with a high mortality. Among these infants, those at greatest risk are infants less than 2 months of age, particularly pre-term infants, low birth weight (LBW) infants (< 2500 g), and immuno-compromised infants. At the onset of the work for this thesis, Cronobacter spp. had been isolated from milk- based powdered formulae which have a direct link to the sub-population at greatest risk. However, there was a need to more closely investigate whether and where Cronobacter spp. occurs in environments in which these powdered are manufactured and packed but also to investigate other sources which could lead to exposure of vulnerable sub-populations. The main objective of this study was to develop isolation and detection methods that would allow quick and reliable investigation into the occurrence of the micro-organism in potential sources. Furthermore, more insight into the growth behavior of Cronobacter spp. in reconstituted infant formula was necessary in order to provide data to be used in Microbiological Risk Assessment (MRA) dedicated to this particular food product. A selective enrichment method was developed for the rapid and reliable enrichment and detection of Cronobacter spp. in environmental samples. The detection method which was developed is based on two features of Cronobacter spp. combined: their yellow pigmented colonies when grown on tryptone soy agar and their constitutive -glucosidase, which can be detected in a 4-h colorimetric assay. The initially developed method and refinements thereof were applied for routine screening for the presence of Cronobacter spp. in environmental samples and a variety of food products manufactured or marketed in The Netherlands. The detection method described in this thesis has been the basis for a series of media for Cronobacter spp. that have recently been commercialized. Quantitative data on product contamination at manufacture, during preparation, and also growth after reconstitution are required in order to assess the risk associated with Cronobacter spp. exposure. Next to that, tools are needed to asses the micro-organisms growth potential as well as its inactivation (thus, its survival) due to specific control measures applied. In this thesis, predictive growth models were developed that capture key growth parameters. Minimum– and maximum temperatures estimated with the Secondary Rosso equation were 3.6 ºC and 47.6 ºC, respectively. The estimated lag time of the micro- organisms was found to vary from 83.3 ± 18.7 h at 10 ºC to 1.73 ± 0.43 h at 37 ºC and could be described with the hyperbolic model and reciprocal square root relation. The models for growth rates and lag times as a function of temperatures obtained during this study allow estimating the potential growth of Cronobacter spp. in reconstituted infant formula stored at any temperature below 47 C. As growth rates of Cronobacter at refrigeration temperatures are relatively small, caregivers are advised to store reconstituted infant at low temperature as a control measure to prevent microbial growth. It is evident that storage of reconstituted formula in a refrigerator may require a significant amount of the time before the formula reach the targeted refrigeration temperature. Therefore, a mathematical model was built to predict the temperature profile and the resulting growth of Cronobacter spp. during cooling, i.e. under dynamic temperature conditions. Predictions showed that proliferation of Cronobacter spp. during cooling strongly depends on the size of the container used for storage and that it may be prevented by limiting the volume to be cooled to portion-size only or by reconstituting at temperatures of 25 °C or lower. The survival of two Cronobacter strains in dry powdered infant formula (PIF) was tested and compared to the survival of six other bacterial strains after inoculation and storage at several temperatures between 7 and 42 ºC. The effect of temperature on survival in PIF, was described using both the Weibull distribution model and the log-linear model. Differences were found in the rate of survival that can be due to difference in the resistance to inactivation in dry environments between Cronobacter species, which could be relevant to consider when establishing quantitative risk assessments on consumer risks related to PIF. The research described in this thesis contributes to the existing knowledge on the natural habitat of Cronobacter spp. and its occurrence and behavior in PIF. The models developed for quantifying the growth of Cronobacter spp. in reconstituted formulae under various conditions can be applied in risk assessments set-up to estimate the probability of vulnerable sub-populations becoming ill after consuming infant formulae. International and national governmental bodies may use these predictive models in risk assessments and to establish guidelines for health care professionals to provide effective hygiene training to parents and professional caregivers to ensure that PIF is prepared handled and stored appropriately. Contents Abstract 6 Chapter 1 Introduction 11 Chapter 2 A new protocol for the detection of Cronobacter spp. applied to environmental samples 43 Chapter 3 A simple and rapid cultural method for enrichment of Cronobacter spp. in environmental samples 55 Chapter 4 Occurrence of Cronobacter spp. in food production environments and households 69 Chapter 5 A study into the prevalence of Cronobacter spp. in The Netherlands 77 Chapter 6 The effect of pre-culturing conditions on the lag time and specific growth rate of Cronobacter spp. in reconstituted infant formula 109 Chapter 7 Growth of Cronobacter spp. under dynamic temperature conditions occurring during cooling of reconstituted powdered infant formula 133 Chapter 8 Survival of Cronobacter spp. in comparison to other bacteria in dry infant formulae 165 Chapter 9 Discussion, and concluding remarks 193 Samenvatting 227 Acknowledgement 231 List of publications 233 Curriculum vitae 237 Overview of
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