No abstract

Bacillus toyonensis strain BCT-7112T (NCIMB 14858T) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol (catQ) and tetracycline (tetM) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis. The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group (sensu lato). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group (sensu lato). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context we discuss definitions of what are and are not intrinsic genes, an issue that is of fundamental importance to both Regulatory Authorities, and the animal feed and related industries.

Intracellular pathogens such as microsporidia can interact with host proteostasis pathways such as autophagy. Previous work done in Caenorhabditis elegans demonstrated involvement of autophagy in controlling microsporidia proliferation through ubiquitin labelling of the parasite and subsequent degradation by lysosome. However, it remains unknown if such mechanisms also play the role in mammalian models. Here we used immunochemistry assays, super-resolution confocal imaging, chemical and genetic modulation of the autophagy flux to elucidate the interplay between autophagy and microsporidia in mammalian cells. We show that despite targeting by early autophagy markers (ubiquitin and p62); the host cell was not able to complete autophagy-mediated removal of Encephalitozoon cuniculi. Furthermore, instead of helping to control microsporidia proliferation, the induction of autophagy dramatically increased proliferation of two microsporidia species in two different cell models. Finally, we showed that the reduction of the autophagy flux using siRNA treatment or microbiota-derived metabolites was able to reduce the parasite proliferation. Taken together our results indicate that microsporidia infecting mammalian cells developed strategies to evade the host autophagy and to divert the autophagy flux to promote their growth.