Molecular Identification of Bacillus Isolated from Korean Water Deer (Hydropotes inermis argyropus) and Striped Field Mouse (Apodemus agrarius) Feces by Using an SNP-Based 16S Ribosomal Marker

Simple Summary Wildlife is a great concern because of its free-ranging movements. They carry bacterial zoonoses in their feces, such as Bacillus species. In this study, we developed a 16S Bacillus species-specific 16S ribosomal RNA (rRNA) molecular marker for species identification. For discrimination of genetically similar members of Bacillus cereus group, including Bacillus cereus, B. anthrax, and B. thuringiensis, a single nucleotide polymorphism (SNP)-based marker was developed. We altered an artificial base at the 3 '-end of SNP sites in each SNP-based 16S rRNA primer sequence to improve the ability of SNP-based primers to bind the PCR template sequence, thereby improving the allele-specific detection of target B. cereus strains. SNP analysis in high-quality DNA sequences could facilitate identification and discrimination of closely related bacterial species. Ambiguous, heterogeneous, endospore-forming Bacillus species, notably Bacillus cereus, often produce fatal toxins that threaten human health. We identified Bacillus from wild animal fecal samples (n = 80), including the Korean water deer (n = 25) and striped field mouse (n = 55). Using traditional culture-based methods, 25 animal fecal samples (31.25%; 25/80) were found to be positive for Bacillus species, whereas using molecular techniques, 19 samples (23.75%; 19/80) were found to be positive for the same. In addition, we designed a Bacillus species-specific 16S ribosomal RNA (rRNA) gene marker and utilized it to identify 19 samples by means of PCR amplification and sequencing, using at least one colony from the 19 Bacillus positive samples. The recovered sequences were matched to sequences of three Bacillus species (B. cereus, B. amyloliquefaciens, and B. megaterium) from the GenBank database. Moreover, the phylogenetic tree generated in this study established specific clades for the Bacillus group. In addition, to differentiate between B. cereus, B. anthracis, and B. thuringiensis, we designed a single nucleotide polymorphism (SNP)-based primer by identifying SNPs in the alignment of 16S rRNA gene sequences of B. cereus group strains. The SNPs were used to design primer sets for discrimination between highly similar species from the B. cereus group. The study could be used in surveillance of agricultural fresh-produce-associated Bacillus outbreaks, for accurate identification of each Bacillus species, and in the development of control measures.

Automated summary

This study developed a molecular marker and a primer based on single nucleotide polymorphism (SNP) for the identification and discrimination of Bacillus species from wildlife fecal samples. The study successfully identified three Bacillus species and could be used for surveillance and control of Bacillus outbreaks associated with agricultural fresh produce.