Martine W. Reij

Occurrence of Enterobacter sakazakii in food production environments and households

Enterobacter sakazakii occasionally causes illness in premature babies and neonates. Contamination of infant formulae during factory production or bottle preparation is implicated. Advice to health-care professionals focuses on bottle preparation, but the effectiveness of prevention depends on the degree of contamination and contamination sites, which are generally unknown. To keep contamination to a minimum in the finished product depends on knowledge of the occurrence of E sakazakii. We used a refined isolation and detection method to investigate the presence of this micro-organism in various food factories and households. Environmental samples from eight of nine food factories and from five of 16 households contained E sakazakii. The widespread nature of this micro-organism needs to be taken into account when designing preventive control measures.

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.

Development of a Standard Test To Assess the Resistance of Staphylococcus aureus Biofilm Cells to Disinfectants

ABSTRACT A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.

Effects of Preculturing Conditions on Lag Time and Specific Growth Rate of Enterobacter sakazakii in Reconstituted Powdered Infant Formula

ABSTRACT 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°C. The estimated optimal growth temperature was 39.4°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°C and 47.6°C, respectively. The estimated lag time varied from 83.3 ± 18.7 h at 10°C to 1.73 ± 0.43 h at 37°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.

Comparison of two optical-density-based methods and a plate count method for estimation of growth parameters of Bacillus cereus.

ABSTRACT Quantitative microbiological models predicting proliferation of microorganisms relevant for food safety and/or food stability are useful tools to limit the need for generation of biological data through challenge testing and shelf-life testing. The use of these models requires quick and reliable methods for the generation of growth data and estimation of growth parameters. Growth parameter estimation can be achieved using methods based on plate counting and methods based on measuring the optical density. This research compares the plate count method with two optical density methods, namely, the 2-fold dilution (2FD) method and the relative rate to detection (RRD) method. For model organism Bacillus cereus F4810/72, the plate count method and both optical density methods gave comparable estimates for key growth parameters. Values for the maximum specific growth rate (μmax) derived by the 2FD method and by the RRD method were of the same order of magnitude, but some marked differences between the two approaches were apparent. Whereas the 2FD method allowed the derivation of values for lag time (λ) from the data, this was not possible with the RRD method. However, the RRD method gave many more data points per experiment and also gave more data points close to the growth boundary. This research shows that all three proposed methods can be used for parameter estimation but that the choice of method depends on the objectives of the research.

Risk-based Estimate of Effect of Foodborne Diseases on Public Health, Greece

TOC summary: These infections may account for 896 disability-adjusted life years per 1 million inhabitants annually.

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.

Quantification of the Emetic Toxin Cereulide in Food Products by Liquid Chromatography-Mass Spectrometry Using Synthetic Cereulide as a Standard

ABSTRACT Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K+ ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K+ content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.

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.