We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4°C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database (∼150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4°C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4°C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated
Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G؉C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium-and high-G؉C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of ϳ150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G؉C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G؉C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.
Bacillus thuringiensis (Bt) belongs to the Bacillus cereus (Bc) group, well known as an etiological agent of foodborne outbreaks (FBOs). Bt distinguishes itself from other Bc by its ability to synthesize insecticidal crystals. However, the search for these crystals is not routinely performed in food safety or clinical investigation, and the actual involvement of Bt in the occurrence of FBOs is not known. In the present study, we reveal that Bt was detected in the context of 49 FBOs declared in France between 2007 and 2017. In 19 of these FBOs, Bt was the only microorganism detected, making it the most likely causal agent. Searching for its putative origin of contamination, we noticed that more than 50% of Bt isolates were collected from dishes containing raw vegetables, in particular tomatoes (48%). Moreover, the genomic characterization of isolates showed that most FBO-associated Bt isolates exhibited a quantified genomic proximity to Bt strains, used as biopesticides, especially those from subspecies aizawai and kurstaki. Taken together, these results strengthen the hypothesis of an agricultural origin for the Bt contamination and call for further investigations on Bt pesticides.
Enterobacter sakazakii is an occasional contaminant of powdered infant formula that can cause rare but severe foodborne infections in infants. To determine optimal methods for the detection and identification of E. sakazakii, 38 naturally contaminated samples from infant formulae factories were analyzed by two PCR-based methods and by a method (TS 22964/RM 210) developed by the International Organization for Standardization and the International Dairy Federation (ISO-IDF) using three different commercial chromogenic agars. The ISO-IDF method includes two enrichment steps, plating of the second enrichment broth on E. sakazakii isolation agar (a chromogenic selective agar), picking of five typical colonies for transfer onto tryptone soy agar, and subsequent confirmation of yellow-pigmented colonies by biochemical characterization. Twenty-two of the 38 samples were positive by the culture method. E. sakazakii isolation agar (ESIA; AES Laboratoires), COMPASS agar (Biokar Diagnostics), and Druggan-Forsythe-Iversen agar (Oxoid) compared favorably with violet red bile glucose agar (VRBG, a selective medium for Enterobacteriaceae), with positive predictive values of 86.96, 88, and 74.07%, respectively, in contrast to 47.83% for VRBG. One additional positive sample was detected using the nonpatented real-time PCR method evaluated, and those results were in 97.3% concordance with the ISO-IDF results. Some discrepancies between the results of the DuPont Qualicon BAX system and those of the ISO-IDF method could be explained by heterogeneity of contamination and sampling. Thus, both PCR-based systems were suitable for detecting and specifically identifying E. sakazakii within 1 to 2 days, and COMPASS agar and ESIA could be used interchangeably as a first-step medium to isolate presumptive E. sakazakii colonies.
-Potential of TTGE for the bacterial study of several raw milks. Recently, novel molecular biological methods based on the direct analysis of DNA (or RNA) have been used to identify bacteria in the environment. Such studies contribute to a better knowledge of the microbial community of complex ecosystems such as dairy products, and therefore to a better understanding of the behaviour of some micro-organisms. The potential of one of these methods, temporal temperature gradient gel electrophoresis (TTGE), was evaluated by analysing the bacterial composition of several raw milks. Total bacterial DNA is extracted from raw milks, and a discriminating zone of the 16S rDNA (V3 region) is amplified by PCR. The resulting DNA fragments are separated using denaturing electrophoresis. The bacterial community is represented by an electrophoresis profile where each band corresponds to a defined species or to a group of species. Bacteria in the unknown ecosystem are assigned an identity by comparison with a bacterial reference database (83 species) previously determined using DNA fragments amplified from pure bacterial strains. Ten raw milks (five un-cooled samples from the morning milking and five cooled samples of pooled milks) were analysed by TTGE. Species with a low % GC V3 sequence (<55%) are reported in this study. TTGE is proven to be a powerful and simple method to rapidly identify (by assignment using the reference database) a broad range of bacterial species within raw milk samples. A combined TTGE and specific PCR approach is found to be necessary for an accurate and full species identification. Raw milk / temporal temperature gradient gel electrophoresis / 16S rDNA / microbial ecologyRésumé -Les nouvelles techniques de biologie moléculaire basées sur l'analyse directe de l'ADN (ou ARN) dans son milieu contribuent à l'heure actuelle à une meilleure connaissance de la communauté microbienne des écosystèmes complexes comme les produits laitiers, et ainsi à mieux appréhender le comportement de certains micro-organismes. Le potentiel d'une de ces techniques, la TTGE ou temporal temperature gradient gel electrophoresis, a été évalué pour l'étude bactérienne de quelques laits crus. Après extraction de l'ADN bactérien total des laits crus, une région discriminante de l'ADNr 16S (la région variable V3) est amplifiée par PCR. Les fragments d'ADN obtenus sont ensuite séparés par électrophorèse en conditions dénaturantes. La communauté bactérienne est ainsi représentée par un profil électrophorétique où chaque bande peut correspondre * Auteur correspondant : v.lafarge@afssa.fr 170 V. Lafarge et al.
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