Taxonomic refinement of Bacillus thuringiensis

<i>Bacillus thuringiensis</i> is the most important biological control agent against various agricultural pests. The bacterium taxonomically belongs to the <i>Bacillus cereus</i> group, which also contains human pathogenic species, e.g., <i>Bacillus anthracis</i>. Thus, precise identification and taxonomic delineation of candidate strains for agricultural usage is of high importance in terms of both public health and biosecurity. By October 2023, whole genome sequences (WGS) of 885 bacterial strains were labeled as <i>B. thuringiensis</i> in the NCBI GenBank database. This study investigates the taxonomic authenticity of those strains using DNA similarity indexes, i.e., average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH). All strains were compared with the type strain of <i>B. thuringiensis</i> ATCC 10972^T. WGS-based phylotaxonomic investigations showed that out of 885 strains 803 strains authentically belonged to <i>B. thuringiensis</i> while 82 strains were mislabeled as <i>B. thuringiensis</i> having dDDH and ANI values less than the acceptable threshold of 70 and 95% respectively, for prokaryotic species definition in comparison with the <i>B. thuringiensis</i> type strain. Among these 82 mislabeled strains, 39 strains need to be reclassified within the <i>B. cereus</i> group in the species <i>B. anthracis</i> (33 strains), <i>Bacillus toyonensis</i> (five strains), and <i>Bacillus mycoides</i> (one strain). Furthermore, four strains were identified as <i>Bacillus tropicus</i> while one strain belonged to each of the species <i>Bacillus licheniformis</i>, <i>Bacillus paranthracis</i>, and <i>Bacillus weidmannii.</i> The remaining 36 strains did not match with any known <i>Bacillus</i> species nor the species of other bacterial genera, thus they could be assigned to hypothetical new species. Results of the present study, on the one hand, pave the way of comprehensive taxonomic refinements within <i>B. thuringiensis</i> species. On the other hand, highlight the role of taxonomic investigations in targeting authentic <i>B. thuringiensis</i> strains for biological control purposes.