In this study, the performance of four alternative selective chromogenic B. cereus agar was compared to the reference mannitol-yolk polymyxin (MYP) agar (ISO 7932) using inclusion and exclusion test strains (n = 110) and by analyzing naturally contaminated milk and other food samples (n = 64). Subsequently, the panC group affiliation and toxin gene profile of Bacillus cereus senso lato (s.l.) isolates were determined. Our results corroborate that the overall best performing media CHROMagar™ B. cereus (93.6% inclusivity; 82.7% exclusivity) and BACARA® (98.2% inclusivity, 62.7% exclusivity) are more sensitive and specific compared to Brilliance™ B. cereus, MYP and ChromoSelect Bacillus Agar. Both media allow unequivocal detection of B. cereus with low risks of misidentification. Media containing ß-D-glucosidase for the detection of presumptive B. cereus may form atypical colony morphologies resulting in a false negative evaluation of the sample. Naturally contaminated samples presented high numbers of background flora, while numbers of presumptive B. cereus were below the detection limit (<10 CFU g−1 or mL−1). Recovery after freezing resulted in the highest detection of B. cereus s.l. on BACARA® (57.8%), CHROMagar™ B. cereus (56.3%) and MYP agar (54.7%). The panC/toxin profile combination IV/A was the most abundant (33.0%), followed by III/F (21.7%) and VI/C (10.4%). More panC and toxin combinations were present in 15.6% of samples when reanalyzed after freezing. In order to improve detection and confirmation of B. cereus s.l. in food samples, we recommend the parallel use of two complementary selective media followed by molecular characterization (e.g., panC typing combined with toxin gene profiling). When determining psychrotolerant or thermophilic members of the B. cereus group, the selective agar media should additionally be incubated at appropriate temperatures (5 °C, ≥45 °C). If high-risk toxin genes (e.g., ces or cytK-1) are detected, the strain-specific ability to produce toxin should be examined to decisively assess risk.

Unique among the retroviruses, mouse mammary tumor virus (MMTV) carries, in addition to the usual long terminal repeat (LTR) promoter, another promoter, P2, which is located in the central part of the proviral U3 sequence, within the LTR open reading frame (ORF). Using an in vitro reporter system based on a sensitive luciferase expression assay, we investigated the regulation of the P2 promoter in the context of the Mtv-2 and Mtv-8 genomes. Irrespective of the genomic source, the activity of the P2 promoter is regulated by a downstream-located enhancer and an upstream-located negative regulatory element (NRE), the activity of which overrides the activator. During this study, we unexpectedly detected another independent neighboring promoter that we called P3. The novel P3 promoter does not seem to be controlled by any NRE but is influenced by the same enhancer that modulates the P2 promoter. The respective transcription starts of the two promoters located in this tight cluster are only 61 bases apart. The transcripts originating from this promoter complex carry the same first intron, which is bound by canonical splice donor and splice acceptor sites located in the LTR. One novel doubly spliced transcript carrying a 459-nucleotide-long ORF was detected in several MMTVcarrying murine cells and could be successfully expressed in murine cells as a His-tagged fusion product. The novel viral protein, the function of which remains to be elucidated, has an apparent molecular mass of 20 kDa.In common with all other retroviruses, mouse mammary tumor virus (MMTV) contains a major promoter in the U3 region of the long terminal repeat (LTR) upstream of the R region (10,17,21). However, in addition to the usual LTR promoter, another promoter, P2, is located in the central part of the proviral LTR within the open reading frame (ORF) region (14,33,36). This region is well conserved among the different viral strains and endogenous variants of MMTV and contains several elements that variously repress viral transcription (4,11,20,27,40), stimulate it (12), are implicated in the response of the LTR to steroid hormones (6,8,12,13,19,22,34), and may control the tissue specificity of expression of the virus (40, 41). The importance of this region in transcriptional control and specificity is underscored by the finding that MMTV proviruses associated with T-cell lymphoma in mice show deletions or mutations in this area, resulting in the genetic reorganization of its sequence (1,16,26,28,35,38,39). Thus, the presence of the P2 promoter in this crucial regulatory region of the MMTV provirus suggests that its activity might be intimately linked to these effects. We first investigated, therefore, if some of the sequences reported previously as acting on the global transcriptional activity of the MMTV LTR influence the promoter activity of the central LTR P2promoter. An unexpected outcome of these studies on the regulation of the P2 promoter was the detection of another independent promoter in close proximity, thus revealing the presence of ...

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