In this study, the bacterial community of galchi-baechu kimchi was determined using culture-based and culture-independent techniques (next generation sequencing:NGS), and showed discrepancies between results. Weissella koreensis and Pediococcus inopinatus were the dominant species according to the NGS results, while Bacillus species and P. inopinatus were dominant in the culture-dependent analysis. To identify safe starter candidates, sixty-five Bacillus strains isolated from galchi-baechu kimchi using culture-dependent methods were evaluated for their antibiotic resistance, presence of toxin genes, and hemolytic activity. Strains were then assessed for salt tolerance and protease and lipase activity. As a result, four strains- B. safensis GN5_10, B. subtilis GN5_19, B. velezensis GN5_25, and B. velezensis GT8-were selected as safe starter candidates for use in fermented foods.

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Bacillus subtilis is a spore-forming bacterium that can withstand a range of extreme environmental conditions [1]. B. subtilis has been detected in diverse habitats such as soil, air, and within plants [1]. Its spore-forming properties also permit entrance into the gastrointestinal tract of animals, where it can form vegetative cells from spores, thereby sporulating again [2,3]. Thus, research into the application of B. subtilis in vaccine delivery into the gastrointestinal tract or as a probiotic has been conducted [4][5][6] .B. subtilis has been detected in several types of fermented soybeans in East Asia, such as meju and doenjang in Korea, douchi in China, and natto and miso in Japan [7][8][9][10][11]. B. subtilis exhibits extracellular amylase and protease activities [12,13]. These activities influence the production of amino acids and flavor compounds during soybean fermentation [14][15][16]. It is well known that these enzymatic activities contribute toward the quality and sensory properties of fermented soybeans [14,17]. B. subtilis also produces several bacteriocins [18] and has therefore been used as a starter culture for soybean fermentation [19], as well as a commercial fungicide (Taegro; B. subtilis var. amyloliquefaciens strain FZB24; Novozymes, Denmark).B. subtilis is generally regarded as a safe bacterium because of its long history of use in the food industry. It also produces several industrially-important enzymes such as xylanase, lichenase, cellulose, and pectinase. These enzymes, produced from non-genetically-modified B. subtilis, can be applied in the food industry [20]. Although much research into the commercial value of B. subtilis has been conducted, including applications in the food industry and in vaccine development, [5,20], studies on methods to distinguish B. subtilis from other Bacillus species are lacking and most of them are identified methods after DNA purification such as restriction fragment length polymorphism or randomly amplified polymorphic DNA analysis [21][22][23][24]. For the commercial use of B. subtilis, it is necessary to ensure the specific isolation of this species from other related species. In this study, we developed a method to specifically distinguish B. subtilis and thereby ensure its purity as a resource.Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorph...

Staphylococcus equorum strain KM1031 is resistant to chloramphenicol, erythromycin and lincomycin. To shed light on the genetic factors underlying these antibiotic resistances, we determined the global gene expression profile of S. equorum KM1031 using RNA sequencing. During chloramphenicol, erythromycin and lincomycin treatment, 8.3% (183/2,336), 16.0% (354/2,336), and 2.9% (63/2,336) of S. equorum KM1031 genes exhibited significant differences in expression, respectively. These three antibiotics upregulated genes related to efflux and downregulated genes related to transporters. Antibiotic treatment also upregulated osmoprotectant-related genes involved in salt tolerance. To identify specific genes functionally related to antibiotic resistance, we compared the genome of strain KM1031 with those of three S. equorum strains that are sensitive to these three antibiotics. We identified three genes of particular interest: an antibiotic biosynthesis monooxygenase gene (abm, AWC34_RS01805) related to chloramphenicol resistance, an antibiotic ABC transporter ATP-binding protein gene (msr, AWC34_RS11115) related to erythromycin resistance, and a lincosamide nucleotydyltransferase gene (lnuA, AWC34_RS13300) related to lincomycin resistance. These genes were upregulated in response to the corresponding antibiotic; in particular, msr was upregulated more than fourfold by erythromycin treatment. Finally, the results of RNA sequencing were validated by quantitative real-time PCR. This transcriptomic analysis provides genetic evidence regarding antibiotic stress responses of S. equorum strain KM1031.

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