Noël Grosset

Isolation and characterization of a psychrotolerant toxin producer, Bacillus weihenstephanensis, in liquid egg products.

A psychrotolerant bacteria of the Bacillus cereus group was found responsible for the spoilage of whole liquid egg products. By sequencing a 16S rRNA region and performing a PCR amplification of specific 16S rRNA and cspA signatures, a Bacillus weihenstephanensis was identified. Characterization of this strain shows its ability to grow in defined medium as well as in whole liquid egg at refrigerated temperatures. The strain isolated possesses genes encoding for hemolysin BL, nonhemolytic enterotoxin, and B. cereus enterotoxins and produces enterotoxins with cytotoxic activity in whole liquid egg, even at refrigerated temperatures. The isolate exhibits a clear ability to stick and form biofilms on stainless steel, the most common material used in egg breaking factories, as well as on model hydrophilic (glass) and hydrophobic (polytetrafluoroethylene) materials. These findings show the necessity to monitor for Bacillus contamination in egg products that are often used in the composition of particularly susceptible finished products such as cream, dessert, dairy, meat, and seafood.

Genetic diversity of food-isolated Salmonella strains through Pulsed Field Gel Electrophoresis (PFGE) and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR).

All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.

Biodiversity of psychrotrophic bacteria of the Bacillus cereus group collected on farm and in egg product industry

The aim of the present study was (i) to type, by genotypic and phenotypic methods, a collection of psychrotrophic bacteria belonging to the Bacillus cereus group collected in a farm and in 6 egg breaking industries during a period covering a warm and a cold season, and (ii) to characterize the egg product spoilage (growth in liquid whole egg) and the sanitary risk potential (cytotoxic activity on Caco-2 cells and adhesion on stainless steel) of each isolate of the collection. The investigation of specific psychrotrophic and mesophilic signatures together with the study of ability to grow at 6 °C and/or at 43 °C on optimal agar medium allowed highlighting twelve profiles, the major one corresponding to the species Bacillus weihenstephanensis (46.2% of the collection). The diversity of the profiles depended on the season and on the origin of the isolates. In terms of food spoilage, all the isolates were able to grow at the same level in liquid whole egg and in optimal medium, even at low temperature. Under the same conditions, the cytotoxic activity depended on the isolate, the medium and the temperature. At 10 °C, no isolate was cytotoxic at 10 °C in liquid whole egg and only one, belonging to the Bacillus weihenstephansensis species, in the optimal medium. All the isolates were able to adhere on stainless steel at various levels, from 2.6±0.2 log cfu/cm(2) to 4.9±0.1 log cfu/cm(2). A large majority (80.8%) was strongly adhering and could lead to the formation of biofilms in industrial equipments.

Global overview of the risk linked to the Bacillus cereus group in the egg product industry: identification of food safety and food spoilage markers

To evaluate the food safety and spoilage risks associated with psychrotrophic Bacillus cereus group bacteria for the egg product industry and to search for relevant risk markers.

Real time PCR gene profiling and detection of Salmonella using a novel target: The siiA gene.

The objective of this study was to develop and evaluate a SYBR Green real time PCR method for the specific detection of Salmonella spp using a novel target, the siiA gene. Primer specificity testing was done on a panel of 76 Salmonella strains and 32 non-Salmonella strains. The primers directed against the siiA gene amplified all Salmonella strains tested, while non-Salmonella strains were not amplified. The melting temperatures of the 107 bp amplicons were consistently specific as they gave melting peaks around 75.5°C. The precision of the assay, based on intra and inter-run variations, was shown to be widely acceptable. In the second part of this study, 45 Salmonella strains were screened for the presence of 6 virulence-associated genes (sopB, cat2, safC, sefB and SC1248) located in several Salmonella Pathogenicity Islands (SPIs) and the spvC gene from the Salmonella virulence plasmid. The prevalence of these genes ranged from 51% to 100%. Variable virulence gene profiles were obtained even within the same serotype.

Biochemical and micrographic evidence of Escherichia coli membrane damage during incubation in egg white under bactericidal conditions.

Bacterial membranes are often thought to be the main targets of the antimicrobial activity of egg white. In order to test this hypothesis, the state of the membranes of Escherichia coli K-12 cells during either bactericidal (45°C) or bacteriostatic (30°C) incubation in egg white at natural alkaline pH was studied by biochemical methods. Namely, the permeability of the outer membrane was evaluated through its ability to incorporate a hydrophobic fluorescent probe (1-N-phenylnaphthylamine), and the permeability of the cytoplasmic membrane was evaluated through the release of a specific intracellular enzyme (β-galactosidase). The bacteria were observed by atomic force microscopy in order to support the biochemical results. At 45°C, the outer membrane of E. coli K-12 incorporated the hydrophobic probe, suggesting that it was disrupted. In addition, the cytoplasmic β-galactosidase was released at this temperature. The atomic force microscopy analysis revealed the formation of spheroplasts, which provided further evidence of the cell wall disruption and a progressive release of cellular contents. At 30°C, biochemical and micrographic experiments confirmed that membrane integrity was preserved. These techniques provide a useful approach for studying the mechanisms of bacterial cell death in egg white.

Identifying risk factors for eggshell contamination by Bacillus cereus group bacteria in French laying farms.

Abstract 1. The growth of Bacillus cereus group bacteria often limits the shelf-life of pasteurised liquid egg products and is also a putative toxin producer. This study was performed to better understand the route of contamination by B. cereus in egg products by studying the factors affecting eggshell contamination on-farm. 2. Eggs were collected in warm and cold seasons in 50 conventional laying farms in Western France. Egg surfaces were analysed for the presence of B. cereus group bacteria, environmental measurements were recorded and production practices were identified through a questionnaire filled out by the farmers. 3. A total of 44% of the farms were contaminated by mesophilic and 10% by psychrotrophic B. cereus group bacteria. No significant effect of the season was observed, whatever the thermal type. Several procedures were associated with reduced eggshell contamination by mesophilic bacteria, including the limitation of dust formation from manure and feeding and efficient disinfection of the silo, houses and the sanitary wall between houses. 4. The research highlights the need to promote prevention strategies, from farm to fork, for the control of eggshell and putative subsequent egg product contamination by B. cereus group bacteria.

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages

BackgroundA remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity.ResultsWe obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or ‘clusters’, based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages.ConclusionsOverall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.