Do farm audits improve milk quality?

Milk quality is assessed using bulk milk analysis and by farm audits in the Netherlands. However, the extent of the effect that dairy farm audits have on milk quality is unknown. Data from over 13,000 audits performed on 12,855 dairy farms from February 2006 to April 2008 were merged with laboratory test results of 325,150 bulk milk samples collected 6 mo before and after the audit. A linear mixed model with the method of restricted maximum likelihood was conducted to study whether the total bacterial counts (TBC) of bulk milk were lower during the periods before and after the dairy farm audit. Results showed that TBC values were 2 to 6% lower (i.e., 0.010 to 0.026 log cfu/mL) for a period from 1.5 to at least 6 mo after an audit. Additionally, several variables were significantly associated with bulk milk TBC values: seasonality, total number of attention points (given if some checklist points were not appropriate), audit type, audit result, and the categories milking equipment maintenance, and utility room-tank maintenance. The TBC values increased with a higher level of attention points. Furthermore, the farms rejected based on the audit results had the highest average TBC values and the approved farms had the lowest values. If dairy farms had an overall negative audit assessment and consequently needed a re-audit in the following year, the TBC values of bulk milk were more likely to be higher. Auditing may provide dairy farmers the opportunity to receive advice about factors that influence bulk milk TBC values, for a period of at least 6 mo following the audit.

Risk assessment strategies as a tool in the application of the Appropriate Level of Protection (ALOP) and Food Safety Objective (FSO) by risk managers.

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.

Modelling homogeneous and heterogeneous microbial contaminations in a powdered food product.

The actual physical distribution of microorganisms within a batch of food influences quantification of microorganisms in the batch, resulting from sampling and enumeration by microbiological tests. Quantification may be most accurate for batches in which microorganisms are distributed homogeneously. However, when the distribution is non-homogeneous, quantification may result in an under-, or overestimation. In the case of pathogens being non-homogeneously distributed, this heterogeneity will impact on public health. Enumeration data are commonly modelled by the Lognormal distribution. Although the Lognormal distribution can model heterogeneity, it does not allow for complete absence of microorganisms. Studies that validate the appropriateness of using Lognormal or other statistical distributions are scarce. This study systematically investigated laboratory and industrial scale batches of powdered infant formula, modelled the enumeration data using a range of statistical distributions, and assessed the appropriateness of individual models. For laboratory scale experiments, batches of milk powder were contaminated by distributing similar numbers of cells of Cronobacter sakazakii either homogeneously throughout a batch of milk powder or by distributing the cells in a localised part of the batch. Each batch was then systematically sampled and the distribution determined by enumerating the samples. By also enumerating the remainder of the batch, a balance could be made of the total number of microorganisms added and of the number retrieved from a batch. Discrete, as well as continuous statistical distributions, were fitted to enumeration data and the parameters estimated by Maximum Likelihood. The data were fitted both as censored and uncensored data. Enumeration data obtained for an industrial batch of powdered infant formula were investigated in this way as well. It was found that Normal, Poisson and Zero-Inflated Poisson distributions fitted the data sets very poorly. In case of homogeneous contamination, there was not a notable difference between the ability of Negative Binomial, Poisson-Lognormal, Weibull, Gamma, and Lognormal distributions to model the data. Overall, either the Negative Binomial distribution or the Poisson-Lognormal distribution fitted the data best in the 10 batches studied, especially when part of a data set contained zeros and/or the numbers were low. The Negative Binomial fitted the laboratory batches best and the Poisson-Lognormal fitted the industrial batch best.

Actual distribution of Cronobacter spp. in industrial batches of powdered infant formula and consequences for performance of sampling strategies.

The actual spatial distribution of microorganisms within a batch of food influences the results of sampling for microbiological testing when this distribution is non-homogeneous. In the case of pathogens being non-homogeneously distributed, it markedly influences public health risk. This study investigated the spatial distribution of Cronobacter spp. in powdered infant formula (PIF) on industrial batch-scale for both a recalled batch as well a reference batch. Additionally, local spatial occurrence of clusters of Cronobacter cells was assessed, as well as the performance of typical sampling strategies to determine the presence of the microorganisms. The concentration of Cronobacter spp. was assessed in the course of the filling time of each batch, by taking samples of 333 g using the most probable number (MPN) enrichment technique. The occurrence of clusters of Cronobacter spp. cells was investigated by plate counting. From the recalled batch, 415 MPN samples were drawn. The expected heterogeneous distribution of Cronobacter spp. could be quantified from these samples, which showed no detectable level (detection limit of -2.52 log CFU/g) in 58% of samples, whilst in the remainder concentrations were found to be between -2.52 and 2.75 log CFU/g. The estimated average concentration in the recalled batch was -2.78 log CFU/g and a standard deviation of 1.10 log CFU/g. The estimated average concentration in the reference batch was -4.41 log CFU/g, with 99% of the 93 samples being below the detection limit. In the recalled batch, clusters of cells occurred sporadically in 8 out of 2290 samples of 1g taken. The two largest clusters contained 123 (2.09 log CFU/g) and 560 (2.75 log CFU/g) cells. Various sampling strategies were evaluated for the recalled batch. Taking more and smaller samples and keeping the total sampling weight constant, considerably improved the performance of the sampling plans to detect such a type of contaminated batch. Compared to random sampling, stratified random sampling improved the probability to detect the heterogeneous contamination.

Factors influencing the accuracy of the plating method used to enumerate low numbers of viable micro-organisms in food.

This study aims to assess several factors that influence the accuracy of the plate count technique to estimate low numbers of micro-organisms in liquid and solid food. Concentrations around 10CFU/mL or 100CFU/g in the original sample, which can still be enumerated with the plate count technique, are considered as low numbers. The impact of low plate counts, technical errors, heterogeneity of contamination and singular versus duplicate plating were studied. Batches of liquid and powdered milk were artificially contaminated with various amounts of Cronobacter sakazakii strain ATCC 29544 to create batches with accurately known levels of contamination. After thoroughly mixing, these batches were extensively sampled and plated in duplicate. The coefficient of variation (CV) was calculated for samples from both batches of liquid and powdered product as a measure of the dispersion within the samples. The impact of technical errors and low plate counts were determined theoretically, experimentally, as well as with Monte Carlo simulations. CV-values for samples of liquid milk batches were found to be similar to their theoretical CV-values established by assuming Poisson distribution of the plate counts. However, CV-values of samples of powdered milk batches were approximately five times higher than their theoretical CV-values. In particular, powdered milk samples with low numbers of Cronobacter spp. showed much more dispersion than expected which was likely due to heterogeneity. The impact of technical errors was found to be less prominent than that of low plate counts or of heterogeneity. Considering the impact of low plate counts on accuracy, it would be advisable to keep to a lower limit for plate counts of 25 colonies/plate rather than to the currently advocated 10 colonies/plate. For a powdered product with a heterogeneous contamination, it is more accurate to use 10 plates for 10 individual samples than to use the same 10 plates for 5 samples plated in duplicate.

Inactivation rates of Cronobacter spp. and selected other bacterial strains in powdered infant formulae stored at different temperatures.

The aim of this study was to determine the survival of two strains of Cronobacter (Enterobacter sakazakii) and six other bacterial strains inoculated into dry powdered infant formula (PIF) stored for 22 weeks at several temperatures between 7 and 42 degrees C. The experimental setup involved a relatively high initial concentration of bacteria, around 10(4) CFU/g of powder, and enumeration of survivors with a minimum detection level of 100 CFU/g. For all strains tested, it was found that the number of bacterial cells decreased faster with increasing temperature. Cronobacter spp. cells generally survived better at high temperatures (37 and 42 degrees C) than the other bacteria, while such a difference in survival was not apparent at other temperatures. To describe the effect of temperature on survival, both the Weibull distribution model and the log-linear model were tested. At 22 degrees C, decline rates of 0.011 and 0.008 log units per day were found for Cronobacter sakazakii ATCC 29544 and Cronobacter strain MC10, respectively. Assuming a linear relationship between log-transformed D-values and temperature, z-values estimated for C. sakazakii ATCC 29544 and Cronobacter MC10 were 13.3 and 23.5 degrees C, respectively. Such differences found in resistance among Cronobacter spp. would be relevant to consider when establishing quantitative risk assessments on consumer risks related to PIF.

Recall costs balanced against spoilage control in Dutch custard.

The relation between the moment at which a recall of Dutch custard is initiated and the direct costs of this recall was investigated. A simulation model of the custard supply chain was developed to compare scenarios with and without a quarantine of 48 h at the storage of the production plant. The model consists of 3 parts: 1) the distribution of a 24,000-L batch of custard over the supply chain over time is simulated; 2) the time to detect spoilage bacteria with a recontamination test procedure is simulated; and 3) the direct recall costs of custard over the different parts of the supply chain are calculated. Direct recall costs increase from about 25,000 euros/batch to 36,171 euros/batch from 57 to 135 h in the situation without quarantine and from 25,000 euros/batch to 36,648 euros/batch from 123 h to 163 h for the situation with quarantine. Then costs decrease because more and more custard is at the consumer level and only 0.13% of the consumers will ask for a refund. With low true contamination probabilities quarantine is not profitable, but at later detection moments with high probabilities it is. We conclude that a simulation model is a helpful tool to evaluate the efficiency of risk management strategies like end product testing and a quarantine situation.

Growth of Cronobacter spp. under dynamic temperature conditions occurring during cooling of reconstituted powdered infant formula.

Reconstituted infant formulae are excellent growth media for Cronobacter spp. (formerly Enterobacter sakazakii) and other microorganisms that may be present in such products. Immediate consumption or rapid cooling and storage at a low temperature are therefore recommended as control measures to prevent microbial growth. Placing a container filled with reconstituted liquid formula in the refrigerator, however, does not mean that the temperature of the liquid is directly the same as the set-point of the refrigerator. This study describes the temperature profiles and methods to predict lag time and possible growth of Cronobacter spp. during the cooling process in three types of containers. The overall heat transfer coefficients (alpha) were determined and were shown to have a very large variability in both household refrigerators and an air-ventilated refrigerator equipped with a fan. A mathematical model was built to predict the growth of Cronobacter spp. under dynamic temperature conditions using three models for the lag time. The various estimations for the lag time had a remarkably strong impact on the predicted growth. The assumption of a constant k-value (k = lag time x specific growth rate = lambda x micro = 2.88) fitted the experimental data best. Predictions taking into account the large variability in heat transfer showed that proliferation of Cronobacter spp. during cooling may be prevented by limiting the volume to be cooled to portion size only, or by reconstituting at temperatures of 25 degrees C or lower. The model may also be used to predict growth in other situations where dynamic temperature conditions exist.

Perspective on the risk to infants in the Netherlands associated with Cronobacter spp. occurring in powdered infant formula.

Cronobacter spp. has been responsible for severe infections in infants. Relative risks associated with this organism in powdered infant formula (PIF) have been described in several studies. To set priorities and decide on risk management options, it is important for risk managers to have a quantitative perspective on the absolute level of risk of this pathogen within the totality of the burden of illnesses in the population. This study set-out to establish such a perspective for The Netherlands. It addresses the impact of heterogeneity in the distribution of the micro-organism in PIF on risk levels. Based on the assumptions in this study, 60% of formula-fed infants are estimated not to be exposed to Cronobacter spp. during their neonatal period. The mean exposure was calculated to be about 1cfu per infant over the total neonatal period. Even after thorough mixing, artificially contaminated powder shows counts which are more variable than expected from a normal, homogeneous distribution. Therefore, mean exposure levels may not represent a good basis for calculating risks. The burden of disease of Cronobacter infections to the Dutch population was estimated to be 19-24 Disability Adjusted Life Years (DALYs) per year, of which 95% are due to meningitis. As compared to other illnesses Cronobacter infections represent 0.5-2.4% of the total estimated burden of foodborne infections and intoxications. The organism is estimated to be responsible for 0.5-0.7% of the meningitis burden to the entire population of The Netherlands.

Effects of preculturing conditions on lag time and specific growth rate of Enterobacter sakazakii in reconstituted powdered infant formula.

Enterobacter sakazakii can be present, although in low levels, in dry powdered infant formulae, and it has been linked to cases of meningitis in neonates, especially those born prematurely. In order to prevent illness, product contamination at manufacture and during preparation, as well as growth after reconstitution, must be minimized by appropriate control measures. In this publication, several determinants of the growth of E. sakazakii in reconstituted infant formula are reported. The following key growth parameters were determined: lag time, specific growth rate, and maximum population density. Cells were harvested at different phases of growth and spiked into powdered infant formula. After reconstitution in sterile water, E. sakazakii was able to grow at temperatures between 8 and 47 degrees C. The estimated optimal growth temperature was 39.4 degrees C, whereas the optimal specific growth rate was 2.31 h(-1). The effect of temperature on the specific growth rate was described with two secondary growth models. The resulting minimum and maximum temperatures estimated with the secondary Rosso equation were 3.6 degrees C and 47.6 degrees C, respectively. The estimated lag time varied from 83.3 +/- 18.7 h at 10 degrees C to 1.73 +/- 0.43 h at 37 degrees C and could be described with the hyperbolic model and reciprocal square root relation. Cells harvested at different phases of growth did not exhibit significant differences in either specific growth rate or lag time. Strains did not have different lag times, and lag times were short given that the cells had spent several (3 to 10) days in dry powdered infant formula. The growth rates and lag times at various temperatures obtained in this study may help in calculations of the period for which reconstituted infant formula can be stored at a specific temperature without detrimental impact on health.

A new protocol for the detection of Enterobacter sakazakii applied to environmental samples.

Enterobacter sakazakii is a motile, peritrichous, gram-negative rod that was previously known as a yellow pigmented Enterobacter cloacae. It is documented as a rare cause of outbreaks and sporadic cases of life-threatening neonatal meningitis, necrotizing enterocolitis, and sepsis. E. sakazakii has been isolated from milk powder-based formulas, and there is thus a need to investigate whether and where E. sakazakii occurs in these manufacturing environments. For this purpose, a simple detection method was developed based on two features of E. sakazakii: its yellow pigmented colonies when grown on tryptone soy agar and its constitutive alpha-glucosidase, which is detected in a 4-h colorimetric assay. Using this screening method, E. sakazakii strains were isolated from three individual factories from 18 of 152 environmental samples, such as scrapings from dust, vacuum cleaner bags, and spilled product near equipment. The method is useful for routine screening of environmental samples for the presence of E. sakazakii.

Recontamination as a source of pathogens in processed foods.

Food products that have been submitted to an adequate heat-treatment during processing are free of vegetative pathogens and, depending on the treatments, of sporeformers and are generally regarded as safe. Processed products such as pâté, ice cream, infant formulae and others have nevertheless been responsible for food-borne illnesses. Thorough epidemiological investigations of several of these outbreaks have demonstrated that the presence of vegetative pathogens such as Salmonella spp. or Listeria monocytogenes in the consumed products was frequently due to post-process recontamination. The majority of studies on pathogens in foods are devoted to investigations on their presence in raw materials or on their growth and behaviour in the finished products. Reference to recontamination is, however, only made in relatively few publications and very little is published on the sources and routes of these pathogens into products after the final lethal processing step. The investigation of an outbreak, including epidemiological studies and typing of strains, is very useful to trace the origin and source of the hazard. Published data demonstrate that the presence of pathogens in the vicinity of unprotected product in processing lines represents a significant risk of recontamination. Microbiological Risk Assessment studies can be conducted as part of governmental activities determining appropriate protection levels for populations. Although recontamination has been identified as a relevant cause of food incidences, it is often not considered in such studies. This paper advocates that an effort should be made to develop our knowledge and information on recontamination further and start using it systematically in the exposure assessment part of Microbiological Risk Assessment studies.

Continuous degradation of trichloroethylene by Xanthobacter sp. strain Py2 during growth on propene.

Propene-grown Xanthobacter sp. strain Py2 cells can degrade trichloroethylene (TCE), but the transformation capacity of such cells was limited and depended on both the TCE concentration and the biomass concentration. Toxic metabolites presumably accumulated extracellularly, because the fermentation of glucose by yeast cells was inhibited by TCE degradation products formed by strain Py2. The affinity of the propene monooxygenase for TCE was low, and this allowed strain Py2 to grow on propene in the presence of TCE. During batch growth with propene and TCE, the TCE was not degraded before most of the propene had been consumed. Continuous degradation of TCE in a chemostat culture of strain Py2 growing with propene was observed with TCE concentrations up to 206 microns in the growth medium without washout of the fermentor occurring. At this TCE concentration the specific degradation rate was 1.5 nmol/min/mg of biomass. The total amount of TCE that could be degraded during simultaneous growth on propene depended on the TCE concentration and ranged from 0.03 to 0.34g of TCE per g of biomass. The biomass yield on propene was not affected by the cometabolic degradation of TCE.

Membrane bioreactor with a porous hydrophobic membrane as a gas-liquid contactor for waste gas treatment.

A novel type of bioreactor for waste gas treatment has been designed. The reactor contains a microporous hydrophobic membrane to create a large interface between the waste gas and the aqueous phase. To test the new reactor, propene was chosen because of its high air/water partition coefficient, which causes a low water concentration and hampers its removal from air. Propene transfer from air to a suspension of propene-utilizing Xanthobacter Py2 cells in the membrane bioreactor proved to be controlled by mass transfer in the liquid phase. The resistance of the membrane was negligible. Simulated propene transfer rates agreed well with the experimental data. A stable biofilm of Xanthobacter Py2 developed on the membrane during prolonged operation. The propene flux into the biofilm was 1 x 10(-6) mol m(-2) s(-1) at a propene concentration of 9.3 x 10(-2) mol m(-3) in the gas phase. (c) 1995 John Wiley & Sons, Inc.

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex.

The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.