Strain- and serotype-dependent affinity of Shiga toxin-producing Escherichia coli for bovine milk fat globules.

Shiga toxin-producing Escherichia coli (STEC) are widely detected in raw milk products intended for human consumption. Although STEC are a worldwide public health problem, the pathogenicity of STEC in cheese remains unclear. In fact, bacterial association with compounds in raw milk cheeses could reduce their pathogenicity. A previous study showed the association of 2 STEC strains with raw milk cream in a natural creaming assay. Different concentrations of each strain were required to saturate the cream. In this study, we hypothesized that all STEC strains could be associated with milk fat globules (MFG) in raw milk and that the bacterial load required for saturation of the cream is serotype dependent. We evaluated the affinity of STEC strains belonging to the O157:H7, O26:H11, and O103:H2 serotypes for bovine raw milk cream and analyzed saturation of the cream layer by natural creaming assay. We used 12 STEC strains and 3 strains belonging to another pathotype to assess the effects of serotypes on this phenomenon. We performed sucrose density gradient centrifugation assays with 2 STEC model strains to confirm the results obtained by natural creaming. The localization of STEC within MFG-enriched creams was observed by confocal and electron microscopy. We recovered approximately 10 times more STEC from the cream layer after natural creaming than from raw bovine milk. The concentration of STEC required to saturate the cream layer (the saturation concentration) was estimated for each strain by nonlinear regression, highlighting a strain and serotype effect. Moreover, the concentration of STEC in the cream was milk fat level dependent. However, even in nonsaturating conditions, a high level of STEC was still present in the aqueous phase, after fat separation. Thus, natural creaming should not be used as the sole preventive measure to remove STEC from naturally contaminated raw milk. The results of our study suggest that cream saturation is a complex mechanism, most likely involving specific interactions between STEC and raw MFG.

Modeling of Clostridium perfringens vegetative cell inactivation in beef-in-sauce products: a meta-analysis using mixed linear models.

The aim of the present study was to predict Clostridium perfringens vegetative cell inactivation during the final reheating step of two beef-in-sauce products prepared and distributed in a French hospital for exposure in risk assessment. In order to account for variability according to experts and international organization recommendations, published data were used to estimate the thermal inactivation parameters of a probabilistic model. Mixed effects models were proposed to describe variability on D(ref) the decimal reduction time at temperature T(ref). Many models differing by their description of variability on D(ref) were tested. Based on goodness-of-fit and parsimony of the model, the one including three random effects was chosen. That model describes random effects of vegetative cell culture conditions, strains and other uncontrolled experimental factors. In order to check the ability of the model to predict inactivation under dynamic thermal conditions, model validation was carried out on published non isothermal data. This model was then used to predict C. perfringens vegetative cell inactivation using temperature profiles inside beef-in-sauce products registered in a French hospital and to explore control measures easier to apply than French regulations.

The importance of incorporating age and sex when backcalculating length in bullhead Cottus gobio.

The present study backcalculated body length for a data set of a bullhead Cottus gobio population located at different sampling sites in a river network. Model comparison between various growth models, which included successively new parameters, showed the effect and importance of taking sex, age and the location in the river network into account. The data sets obtained by backcalculation were fitted by the von Bertalanffy growth function, which revealed the effect of the backcalculation formula on the estimation of the von Bertalanffy growth parameters. Fitting results and parameter estimates showed again the importance of incorporating age and sex when backcalculating body length in the C. gobio population studied.

Bayesian modeling of Clostridium perfringens growth in beef-in-sauce products.

Models on Clostridium perfringens growth which have been published to date have all been deterministic. A probabilistic model describing growth under non-isothermal conditions was thus proposed for predicting C. perfringens growth in beef-in-sauce products cooked and distributed in a French hospital. Model parameters were estimated from different types of data from various studies. A Bayesian approach was proposed to model the overall uncertainty regarding parameters and potential variability on the 'work to be done' (h(0)) during the germination, outgrowth and lag phase. Three models which differed according to their description of this parameter h(0) were tested. The model with inter-curve variability on h(0) was found to be the best one, on the basis of goodness-of-fit assessment and validation with literature data on results obtained under non-isothermal conditions. This model was used in two-dimensional Monte Carlo simulations to predict C. perfringens growth throughout the preparation of beef-in-sauce products, using temperature profiles recorded in a hospital kitchen. The median predicted growth was 7.8×10(-2) log(10) cfu·g(-1) (95% credibility interval [2.4×10(-2), 0.8]) despite the fact that for more than 50% of the registered temperature profiles cooling steps were longer than those required by French regulations.

Quantitative risk assessment for Escherichia coli O157:H7 in frozen ground beef patties consumed by young children in French households.

A quantitative risk assessment for Escherichia coli O157:H7 in frozen ground beef patties consumed by children under 10 years of age in French households was conducted by a national study group describing an outbreak which occurred in France in 2005. Our exposure assessment model incorporates results from French surveys on consumption frequency of ground beef patties, serving size and consumption preference, microbial destruction experiments and microbial counts on patties sampled from the industrial batch which were responsible for the outbreak. Two different exposure models were proposed, respectively for children under the age of 5 and for children between 5 and 10 years. For each of these two age groups, a single-hit dose-response model was proposed to describe the probability of hemolytic and uremic syndrome (HUS) as a function of the ingested dose. For each group, the single parameter of this model was estimated by Bayesian inference, using the results of the exposure assessment and the epidemiological data collected during the outbreak. Results show that children under 5 years of age are roughly 5 times more susceptible to the pathogen than children over 5 years. Exposure and dose-response models were used in a scenario analysis in order to validate the use of the model and to propose appropriate guidelines in order to prevent new outbreaks. The impact of the cooking preference was evaluated, showing that only a well-done cooking notably reduces the HUS risk, without annulling it. For each age group, a relation between the mean individual HUS risk per serving and the contamination level in a ground beef batch was proposed, as a tool to help French risk managers.

Persistence of Shiga toxin-producing Escherichia coli O26 in various manure-amended soil types.

AIMS: To evaluate the behaviour of Shiga toxin-producing Escherichia coli (STEC) O26 strains inoculated in manure-amended soils under in vitro conditions. METHODS AND RESULTS: Four green fluorescent protein (GFP)-labelled STEC O26 strains were inoculated in duplicate (at 10(6) CFU g(-1)) in three different manure-amended soil types, including two loam soils (A and B) and one clay loam soil (C), and two incubation temperatures (4 and 20 degrees C) were tested. STEC counts and soil physical parameters were periodically monitored. STEC O26 cells were able to persist during extended periods in soil even in the presence of low moisture levels, i.e. less than 0 x 08 g H2O g(-1) dry soil. At 4 and 20 degrees C, STEC could be detected in soil A for 288 and 196 days, respectively, and in soils B and C for at least 365 days postinoculation at both temperatures. The ambient temperature (i.e. 20 degrees C) was significantly associated with the highest STEC count decline in all soils tested. CONCLUSIONS: The temperature and soil properties appear to be contributory factors affecting the long-term survival of STEC O26 in manure-amended soils. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information regarding the ecology of STEC O26 in manure-amended soils and may have implications for land and waste management.

Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry.

AIMS: The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. METHODS AND RESULTS: Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 10(6) CFU ml(-1) in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8.5 to 9.5 units during the last 50 days. CONCLUSION: STEC O26 strains were able to survive in cow slurry for an extended period. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26.

Growth of Shiga-toxin producing Escherichia coli (STEC) and bovine feces background microflora in various enrichment protocols.

Cattle are an important reservoir for STEC and eating food contaminated with fecal material is a frequent source of human STEC infection. It is thus essential to reliably determine the prevalence of STEC contamination in cattle. Currently, different enrichment protocols are used before the detection of Shiga-Toxin producing Escherichia coli (STEC) in fecal samples. However, there have not been any studies performed that have compared the effectiveness of these various enrichment protocols for the growth of non-O157 STEC in fecal samples. The objective of this present study was to characterize the effects of different enrichment factors on the simultaneous growth of the feces background microflora (BM) and two non-O157 STEC strains. The different factors studied were the basal medium (TSB and EC), the effect of novobiocin in the broth (N+ or N-) and the incubation temperature (37 or 40 degrees C). The BM and STEC growth data were simultaneously fitted by using a competitive growth model. The STEC final levels obtained after 24h were higher for the protocols with novobiocin and/or EC compared to the others. However, novobiocin inhibited the growth of one STEC strain. We observed that the addition of novobiocin into broths is not advisable for optimal growth conditions. Moreover, given high BM and low STEC levels often observed in feces, predictions made with the growth model highlighted that false negative results could more likely appear with protocols using TSB without novobiocin than with protocols using EC. In conclusion, the use of EC broth in enrichment protocols seems to be more appropriate for detecting non-O157 STEC from bovine fecal samples. This can help avoid false negative results that cause an underestimation of the STEC prevalence in cattle.

Supplementation of enrichment broths by novobiocin for detecting Shiga toxin-producing Escherichia coli from food: a controversial use.

AIMS: To investigate the assumption that usage of novobiocin (20 mg l(-1)) in Shiga toxin-producing Escherichia coli (STEC) enrichment broths could achieve false-negative results. METHODS AND RESULTS: First, the minimum inhibitory concentration (MIC) of 74 E. coli O157:H7 and 55 non-O157:H7 STEC strains to novobiocin was determined. Second, to visualize the potential impact of novobiocin on the STEC growth during the enrichment step, the growth experiments were carried out in trypticase soy broth (TSB) with and without 20 mg l(-1) of novobiocin. The MIC values varied from 32 to > 64 mg l(-1) for the 74 E. coli O157:H7 strains, and from 16 to > 64 mg l(-1) for the 55 non-O157:H7 STEC strains. The E. coli O157:H7 strains were significantly (P < 0.001) more resistant to novobiocin than the non-O157:H7 STEC strains. The present study shows that the addition of novobiocin into enrichment broths inhibits the growth of some non-O157:H7 STEC strains, and slows down the growth of some STEC strains. CONCLUSIONS: Enrichment broths supplemented by novobiocin could lead to false-negative results for detecting STEC from food. SIGNIFICANCE AND IMPACT OF THE STUDY: We strongly suggest that novobiocin should not be systematically added into enrichment broths for detecting STEC from food.

Growth and survival of non-O157:H7 Shiga-toxin-producing Escherichia coli in cow manure.

AIMS: The main objective of this study was to evaluate the behaviour of non-O157:H7 Shiga-toxin-producing Escherichia coli (STEC) strains in cow manure. METHODS AND RESULTS: A mixture of eight green-fluorescent-protein-labelled STEC strains was inoculated around 10(6)-10(7) CFU g(-1) into four manure heaps. Two heaps were regularly turned and the two others remained unturned. STEC counts and physical parameters (temperature, pH, moisture content and oxido-reduction potential) were monitored for 1000 manure samples. The highest mean pH values were obtained near the surface at the base of all manure heaps. At the surface, the moisture content decreased from 76.5% to 42% in turned heaps. Temperatures reached 65 degrees C near the main body of all manure heaps, and only 35 degrees C near the superficial parts located at the base of them. These two sites (the centre and the base) were associated with D values for the STEC counts of 0.48 and 2.39 days, respectively. We were able to detect STEC strains during 42 days in turned manure heaps and during at least 90 days in unturned ones. CONCLUSIONS: These results emphasize the long-term survival of non-O157:H7 STEC in cow manure. SIGNIFICANCE AND IMPACT OF THE STUDY: Good management practices (e.g. turning) should be respected in order to minimize the risk of environmental contamination by STEC.

Serological and molecular ecology of Listeria monocytogenes isolates collected from 13 French pork meat salting-curing plants and their products.

The purpose of this study was dual: 1. to evaluate the serotype distribution of 1028 Listeria monocytogenes isolates collected in 13 French salting factories and their products and 2. to identify sources of L. monocytogenes contamination in these factories and trace the routes of spread by PFGE (Pulsed-Field Gel Electrophoresis) typing. Serotypes 1/2a, 1/2b, 1/2c, 4b and 4e occurred. Pulsotype diversity was high among strains collected in plants and products. Furthermore, strains showing similar pulsotypes occurred on the same surfaces after an interval of at least two weeks and in unrelated factories. Forty five strains were genetically closely related to a 4b serotype L. monocytogenes strain isolated from a human clinical case of listeriosis. Our results highlighted the fact that L. monocytogenes is introduced into meat processing plants through raw meat. To overcome such contamination, suppliers of raw material should adhere to specific microbiological control measures. In addition, more attention should be focused on the appropriateness and compliance with procedures of cleaning and disinfection.

Modeling and predicting the simultaneous growth of Escherichia coli O157:H7 and ground beef background microflora for various enrichment protocols.

The simultaneous growth of Escherichia coli O157:H7 (O157) and the ground beef background microflora (BM) was described in order to characterize the effects of enrichment factors on the growth of these organisms. The different enrichment factors studied were basal medium (Trypticase soy broth and E. coli broth), the presence of novobiocin in the broth, and the incubation temperature (37 degrees C or 40 degrees C). BM and O157 kinetics were simultaneously fitted by using a competitive growth model. The simple competition between the two microfloras implied that O157 growth stopped as soon as the maximal bacterial density in the BM was reached. The present study shows that the enrichment protocol factors had little impact on the simultaneous growth of BM and O157. The selective factors (i.e., bile salts and novobiocin) and the higher incubation temperature (40 degrees C) did not inhibit BM growth, and incubation at 40 degrees C only slightly improved O157 growth. The results also emphasize that when the level of O157 contamination in ground beef is low, the 6-h enrichment step recommended in the immunomagnetic separation protocol (ISO EN 16654) is not sufficient to detect O157 by screening methods. In this case, prior enrichment for approximately 10 h appears to be the optimal duration for enrichment. However, more experiments must be carried out with ground beef packaged in different ways in order to confirm the results obtained in the present study for non-vacuum- and non-modified-atmosphere-packed ground beef.

Isolation of E. coli O157:H7 and non-O157 STEC in different matrices: review of the most commonly used enrichment protocols.

AIMS: To review and characterize the enrichment protocols used for detecting all Shiga-Toxin producing Escherichia coli (STEC) from different matrices. METHODS AND RESULTS: Firstly, the frequency distribution of the factors characterizing the enrichment protocols is described; secondly, a multiple correspondence analysis is performed to display profiles of association of these factors, and thirdly, published results concerning the relative performances of the protocols are summarized. Trypticase Soy Broth (TSB) is reported as the most frequently used enrichment broth. More often, one antibiotic is added in enrichment broths and these broths are incubated for a duration of 16-24 h at 35-37 degrees C. It also appears that the incubation temperature does not seem to be related to the type of serogroup looked for and that antibiotics are used regardless of the matrix analysed. Finally, results relating to the enrichment protocol efficacy are rare and differ from one study to another. CONCLUSIONS: Statistical studies must be conducted so as to assess the efficacy of the main enrichment protocols investigated in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reviews the most commonly used enrichment protocols and highlights the lack of results as to their relative efficacy.

Effect of temperature, water-phase salt and phenolic contents on Listeria monocytogenes growth rates on cold-smoked salmon and evaluation of secondary models.

Salting and smoking are ancient processes for fish preservation. The effects of salt and phenolic smoke compounds on the growth rate of L. monocytogenes in cold-smoked salmon were investigated through physico-chemical analyses, challenge tests on surface of cold-smoked salmon at 4 degrees C and 8 degrees C, and a survey of the literature. Estimated growth rates were compared to predictions of existing secondary models, taking into account the effects of temperature, water phase salt content, phenolic content, and additional factors (e.g. pH, lactate, dissolved CO2). The secondary model proposed by Devlieghere et al. [Devlieghere, F., Geeraerd, A.H., Versyck, K.J., Vandewaetere, B., van Impe, J., Debevere, J., 2001. Growth of Listeria monocytogenes in modified atmosphere packed cooked meat products: a predictive model. Food Microbiology 18, 53-66.] and modified by Giménez and Dalgaard [Giménez, B., Dalgaard, P., 2004. Modelling and predicting the simultaneous growth of Listeria monocytogenes and spoilage micro-organisms in cold-smoked salmon. Journal of Applied Microbiology 96, 96-109.] appears appropriate. However, further research is needed to understand all effects affecting growth of L. monocytogenes in cold-smoked salmon and to obtain fully validated predictive models for use in quantitative risk assessment.

Use of Bayesian modelling in risk assessment: application to growth of Listeria monocytogenes and food flora in cold-smoked salmon.

An attempt to use a Bayesian approach to model variability and uncertainty separately in microbial growth in a risk assessment is presented. It was conducted within the framework of a French project aiming at assessing the exposure to Listeria monocytogenes in cold-smoked salmon. The chosen model describes the effect of time and temperature on bacterial growth. A Bayesian approach close to the one proposed by Pouillot et al. [Int. J. Food Microbiol. 81 (2003) 87] is used to estimate the variability and uncertainty of growth parameters from both literature data and data experimentally acquired during the project. Variability between strains and between products is taken into account. The growth of the food flora of cold-smoked salmon is also modelled by the same method. The results obtained for both models are used to predict the simultaneous growth of L. monocytogenes and food flora in cold-smoked salmon with a competitive model, expressing variability and uncertainty through a second-order Monte Carlo simulation.

[From prevalence to predictive values: considerations on the antimicrobial susceptibility testing dealing with change of susceptibility rates]. / De la prévalence aux valeurs prédictives: l'antibiogramme face à l'évolution de la résistance aux antibiotiques.

The aim of the study was to quantify the influence of the variation of the susceptibility prevalence (frequencies of susceptible, intermediate and resistant isolates) on the predictive value of antimicrobial susceptibility testing reports performed with disk diffusion method, under several scenarios of distinct susceptibility prevalence. Prevalence variation effect was assessed through a modeling approach that enabled to take into account the technical variability and to control prevalence through scenarios. Results show how the prevalence impacts on the disk diffusion performance with level of discrepancies varying with prevalence. The phenomenon may be quantitatively noteworthy for some antimicrobial agents considering the prevalence recorded with time or location, leading to lowered performances. For instance, with amoxicillin+clavulanic acid, predictive values of susceptible and resistant reports varied respectively from 70 to 96 and from 33 to 97 %. For a 18-mm diameter, the probability the isolate is truly susceptible varied from 16 to 73 % according to the prevalence tested. Characteristic of the prevalence effect are described and consequences on zone diameter breakpoint policy raised. They include to (i) reevaluate disk diffusion breakpoint consistency when the prevalence impact is noteworthy and (ii) estimate the consequences of an international harmonized breakpoint policy on prediction quality and appropriate patient management.

Prevalence of Listeria monocytogenes in 13 dried sausage processing plants and their products.

The aims of the present study were: (i) to investigate the occurrence of Listeria monocytogenes in dried sausage processing plants on surfaces before and during processing, (ii) to study the contamination in meat and sausages at different stages of maturation, (iii) to assess the distribution of L. monocytogenes in the different plants and products studied. Thirteen dried sausage processing plants were sampled at two different times of the working day. The studies were repeated twice to evaluate the persistence of the pathogen. A total of 1029 samples were collected. Among swabbed samples, 15% were positive before the beginning of the working day and 47.3% during working day. Results showed that effectiveness of cleaning and disinfecting operations could be linked with the complexity of processing lines and machines used. The presence of L. monocytogenes in mixed meat amounted to 71.6% of the collected samples. A decrease of the contamination rate in dry sausage was noted, particularly during the drying stage. Nevertheless 3 sausages studied presented a low contamination rate (<3 cfu/g) when ready for consumption. A total of 996 strains of L. monocytogenes were characterised by biochemical tests and serotyping. A majority of isolates were 1/2a (49.5%), 1/2c (19.5%) and 1/2b (13%) strains. A high heterogeneity of serotypes was observed in all plants, raw meat and in sausages during maturation.

Fate of Listeria monocytogenes in experimentally contaminated French sausages.

Listeria monocytogenes has been recognized as one of the most important foodborne pathogens dealt with by the food. The bacterium has been found in every part along the pork processing industry from the slaughterhouse to the cutting room and the delicatessen factories. During the fermentation and drying of sausages, L. monocytogenes tends to decrease substantially. However, despite the various hurdles in the dry sausage manufacturing process, L. monocytogenes is able to survive and is detected in the final products. The present study has evaluated growth and survival of eight different L. monocytogenes strains (originating from sausage, sausage industry environment and from clinical cases of listeriosis) in experimentally inoculated French sausages with 10(4) cfu g(-1). This study points out the fact that the decrease of L. monocytogenes contamination rate during the manufacturing process of sausages is strain dependent (p < 0.001) and mainly due to the drying and maturation step than to the fermentation itself. Whatever the strains studied, almost no decrease of the contamination rate was noted during the fermentation step. However hurdle-adapted strains (those isolated from sausages or sausage industry environment) were more difficult to cure from sausages (decrease by 1.5 log10) than non-adapted strains (decrease by 3 log10) at the end of the drying period (day 35), when sausages were ready for consumption. These sausages became safe only at the best before date. As a consequence, L. monocytogenes and more particularly those "adapted" strains might represent a very important issue for hygienists since these strains originating from sausages or production environment themselves are likely to contaminate sausages during manufacturing and remain in the final products. However, the high inoculum levels used in the study (10(4) cfu g(-1)) are not representative of the natural contamination of L. monocytogenes commonly encountered in the raw material for sausages. If such contamination happened to be inferior to 100 cfu g(-1), then the manufacturing process used in this study would be able to produce "safe" sausages according to the European regulation requiring the absence of L. monocytogenes in 25 g of food with a tolerance of below 100 cfu g(-1) at the best before date.

Modelling the effect of a temperature shift on the lag phase duration of Listeria monocytogenes.

The aim of this work is to study and model the effect of a temperature shift on h(0), the product of the growth rate by the lag phase duration (mulambda). Our work is based on the data of Whiting and Bagi [Int. J. Food Microbiol. 73 (2002) 291], who studied the influence of both the pre-incubation temperature (T(prior)) and the growth temperature (T(growth)) on lambda values of Listeria monocytogenes. We introduce a new model to describe the evolution of the parameter h(0) as a function of T(prior) and T(growth), and compare it to Whiting and Bagi's published polynomial model that describes the influence of T(prior) and T(growth) on lambda independently of mu. For exponential as well as stationary phase cells, h(0) increases almost linearly with the magnitude of the temperature shift. A simple linear model of h(0) turns out to be more suitable to predict lambda values than a polynomial model of lambda.