Comparative phenotypic, genotypic and genomic analyses of Bacillus thuringiensis associated with foodborne outbreaks in France.

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.

Comparison of three chromogenic media and evaluation of two molecular-based identification systems for the detection of Enterobacter sakazakii from environmental samples from infant formulae factories.

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.

Evaluation of the International Organization for Standardization-International Dairy Federation (ISO-IDF) draft standard method for detection of Enterobacter sakazakii in powdered infant food formulas.

As a result of the growing recognition of Enterobacter sakazakii as an emergent pathogen, the International Dairy Federation (IDF) and the International Organization for Standardization (ISO) have standardized a reference method for the detection of E. sakazakii in milk powder products and powdered infant food formulas (IFF). The objectives of this study were to assess the applicability of the ISO-IDF draft standard, and to compare several chromogenic selective media for E. sakazakii [ready-to-use ESIATM, homemade E. sakazakii isolation agar, and Druggan-Forsythe-lversen (DFI) agar], and a selective media for Enterobacteriaceae Violet Red Bile Glucose (VRBG). We found that the method is sensitive, selective, and applicable to the analysis of powdered IFF, provided that some modifications are added. In particular, definition of typical colonies on chromogenic media should be less restrictive, and the possibility of using chromogenic media other than ESIA should be introduced. Any of the chromogenic media tested here could be used initially, since their performances were similar. In these media, alpha-glucosidase-positive but non-yellow-pigmented isolates should be also considered. Consequently, the yellow pigmentation test should be abandoned, or completed with another test in order to select colonies to confirm. Although the specificity of VRBG was relatively poor, it could be used as a second nonchromogenic medium.

Raw cow milk bacterial population shifts attributable to refrigeration.

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 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 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 degrees 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 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.