Variation for virulence was examined amongst 20 field collections of Plasmodiophora brassicae from France. Out of the 10 brassica lines tested, seven reacted differentially to inoculation; of these, two oilseed rape cultivars exhibited previously unreported differential responses. Some of the differential lines used previously to classify pathotypes of P. brassicae were susceptible to all collections, suggesting that pathogen populations in France may be different from those reported elsewhere. Good pathotype discrimination was obtained using a set of three cultivars of Brassica napus. Five pathotypes, P 1 , P 2 , P 3 , P 4 and P 5 , were detected and their occurrence was unrelated to host type. Pathogenic variation amongst 17 single-spore isolates derived from three field collections was studied, and five pathotypes were identified. Four isolates were classified as pathotype P 1 , pathogenic on all three differential hosts, and eight as pathotype P 4 , pathogenic on none of the three differentials. The five other isolates were classified as pathotypes P 3 , P 6 and P 7 , the latter two expressing patterns of reaction not observed for field collections. The fractionation of different individual pathotypes from one original spore suspension confirmed the genetic heterogeneity of field populations of P. brassicae.

This study was designed to investigate the growth potential of Salmonella enteritidis in liquid egg white at 30°C and to examine the mechanism of egg white resistance to Salmonella growth. We observed a low and variable growth in whole egg white: Salmonella cell counts rose by 2 log units during the 4 to 6 days of incubation. Treatments to render the egg white components more homogeneous and to facilitate the circulation of nutrients had no effect on the low and variable growth of Salmonella cells. To investigate whether a lack of nutrients or the presence of inhibitory factors could explain this low growth, the growth of various strains at 30°C in egg white filtrate (egg white without protein) was examined. Growth was fast and comparable with growth observed in optimum medium (tryptic soy broth). The addition of 10% egg white to the filtrate decreased the growth of Salmonella enteritidis to the same level observed in egg white, leading us to conclude that inhibitory factors, probably proteins, inhibit the growth of S. enteritidis. To determine the role of the different egg white proteins and to identify which of these inhibit S. enteritidis growth, the effect of each protein added to the filtrate was evaluated. To test the inhibitory potency of three binding proteins, supplementation with their corresponding ligands was also studied. Our study shows that ovotransferrin, or iron deficiency resulting from iron binding to ovotransferrin, was the major protein or mechanism implicated in the inhibition of the growth of S. enteritidis in egg white.

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The presence of Staphylococcus intermedius in food remains unclear because routine laboratory analysis does not discriminate between S. intermedius and Staphylococcus aureus, a major cause of food poisoning. Both species share many phenotypic characteristics, including coagulase and thermonuclease production. In both species, some strains can produce enterotoxin and therefore can be the cause of food poisoning outbreaks. Although the ID32 Staph System (bioMérieux, SA, Marcy l'Etoile, France), based on a miniaturized phenotypic characterization, gives satisfactory results for discriminating between these two species, some rapid molecular PCR-based methods have been developed to identify S. aureus specifically, but they do not identify S. intermedius. Here, we developed a rapid, accurate, and discriminative multiplex PCR method that targets species-specific sequences in the nuc gene, which encodes thermonuclease in the two species. The test includes an internal positive control that targets a highly conserved region of 16S ribosomal RNA gene (rDNA). A total of 116 strains were used to validate our test. The test gave no signal on the following Staphylococcus species: S. epidermidis, S. chromogenes, S. hyicus, S. warneri, S. xylosus, S. lentus, and S. sciuri. It allowed a 100% successful discrimination between S. aureus (44 strains tested) and S. intermedius (57 strains) isolated from different origins.

Salmonella enterica serovar Enteritidis is the prevalent egg-product-related food-borne pathogen. The egg-contamination capacity of S. Enteritidis includes its exceptional survival capability within the harsh conditions provided by egg white. Egg white proteins, such as lysozyme and ovotransferrin, are well known to play important roles in defence against bacterial invaders. Indeed, several additional minor proteins and peptides have recently been found to play known or potential roles in protection against bacterial contamination. However, although such antibacterial proteins are well studied, little is known about their efficacy under the environmental conditions prevalent in egg white. Thus, the influence of factors such as temperature, alkalinity, nutrient restriction, viscosity and cooperative interactions on the activities of antibacterial proteins in egg white remains unclear. This review critically assesses the available evidence on the antimicrobial components of egg white. In addition, mechanisms employed by S. Enteritidis to resist egg white exposure are also considered along with various genetic studies that have shed light upon egg white resistance systems. We also consider how multiple, antibacterial proteins operate in association with specific environmental factors within egg white to generate a lethal protective cocktail that preserves sterility.

Chicken egg white protects the embryo from bacterial invaders by presenting an assortment of antagonistic activities that combine together to both kill and inhibit growth. The key features of the egg white anti-bacterial system are iron restriction, high pH, antibacterial peptides and proteins, and viscosity. Salmonella enterica serovar Enteritidis is the major pathogen responsible for egg-borne infection in humans, which is partly explained by its exceptional capacity for survival under the harsh conditions encountered within egg white. However, at temperatures up to 42°C, egg white exerts a much stronger bactericidal effect on S. Enteritidis than at lower temperatures, although the mechanism of egg white-induced killing is only partly understood. Here, for the first time, the impact of exposure of S. Enteritidis to egg white under bactericidal conditions (45°C) is explored by global-expression analysis. A large-scale (18.7% of genome) shift in transcription is revealed suggesting major changes in specific aspects of S. Enteritidis physiology: induction of egg white related stress-responses (envelope damage, exposure to heat and alkalinity, and translation shutdown); shift in energy metabolism from respiration to fermentation; and enhanced micronutrient provision (due to iron and biotin restriction). Little evidence of DNA damage or redox stress was obtained. Instead, data are consistent with envelope damage resulting in cell death by lysis. A surprise was the high degree of induction of hexonate/hexuronate utilization genes, despite no evidence indicating the presence of these substrates in egg white.

High-intensity electric fields have been successfully applied to the destruction of Salmonella Enteritidis in diaultrafiltered egg white. The effects of electric field strength (from 20 to 35 kV x cm(-1)), pulse frequency (from 100 to 900 Hz), pulse number (from 2 to 8), temperature (from 4 to 30 degrees C), pH (from 7 to 9), and inoculum size (from 10(3) to 10(7) CFU x ml(-1)) were tested through a multifactorial experimental design. Experimental results indicate that, for Salmonella inactivation, the electric field intensity is the dominant factor with a strongly positive effect, strengthened by its positive interaction with pulse number. Pulse number, temperature, and pH have also significant positive effects but to a lesser extent. In the most efficient conditions, the pulsed electric field (PEF) treatment is capable of 3.5 log10 reduction in viable salmonellae. Simultaneously, the measure of surface hydrophobicity does not indicate any increase after PEF treatment. These results suggest that no protein denaturation occurs, unlike what is observed after comparable heat treatment in terms of Salmonella inactivation (55 degrees C for 15 min).

-The conventional plate-counting technique, while still routinely used in laboratories requiring micro-organism screening or enumeration, remains time-consuming and tiresome for the operator when large numbers of samples are assayed. We developed a miniaturized method based on serial tenfold dilution of samples in 96-well micro-plates followed by multiple pour-plating into 24-well micro-plates with an agar overlay procedure, providing easy colony counting and interpretation. The accuracy, sensitivity and reproducibility of this miniaturized method were tested using the conventional plate-counting technique as reference for viability measurements of Escherichia coli cells submitted to a sub-lethal thermic stress, enumeration of lactic bacteria during milk fermentation, and analysis of the total aerobic flora, coliform and Staphylococcus aureus content of spoiled food. No significant differences were found between the results obtained with our miniaturized method and the conventional one, regardless of the bacteria and media used. Providing a reduction in overall cost and handling time, this reliable miniaturized method should be useful for experiments involving large ranges of cell concentration as well as great numbers of samples and replicates. Résumé -Développement d'une méthode de dénombrement microbien rapide et peu coûteuse basée sur la miniaturisation de la méthode classique de dénombrement après culture sur gélose. La méthode classique de dénombrement des micro-organismes après culture en milieu gélosé est toujours très utilisée dans les laboratoires de microbiologie. Elle est cependant coûteuse en temps et devient fastidieuse pour des études mettant en jeu un grand nombre d'échantillons et/ou nécessitant de nombreuses répétitions. Nous avons développé une méthode miniaturisée basée sur le même principe que la méthode classique mais utilisant des plaques 96 puits pour la phase de dilution décimale des échantillons et des plaques 24 puits pour la phase d'ensemencement des dilutions dans l'agar, ceci permettant le traitement d'un grand nombre d'échantillons en gardant la même facilité de comptage et d'interprétation des colonies. La fiabilité, la sensibilité et la reproductibilité de cette méthode miniaturisée ont été vérifiées en utilisant la technique conventionnelle comme référence pour des dénombrements de cellules d'Escherichia coli soumises à un traitement thermique subléthal, de bactéries lactiques au cours de la fermentation du lait en yaourt et de la flore méso-phile, des coliformes totaux et de Staphylococcus aureus dans des aliments. Aucune différence significative n'a été mise en évidence entre les résultats obtenus par les deux méthodes, quels que soient les bactéries et le milieu de culture. Cette microméthode de dénombrement après culture en milieu gélosé permet donc des réductions de coût et de temps de manipulation avec la même fiabilité que la méthode classique. Elle peut donc être utile pour toutes les expériences impliquant une gamme étendue de concentrations bactériennes ainsi ...