No abstract

We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.

Several types of fermented soybean food are consumed in Korea and representative examples are ganjang (soy sauce) and doenjang (soybean paste). The traditional production of ganjang and doenjang starts from the manufacture of meju. Meju is made by soaking, steaming, crushing, and molding soybeans into blocks, followed by spontaneous fermentation for 2-3 months. The ripened meju is mixed with brine and ripened for a further 2-3 months, then the liquid portion of the mixture is separated, resulting in a traditional type of ganjang. The remaining solid portion is subsequently mashed and fermented for >6 months and becomes quality doenjang [1]. Ripened meju is used as a starter culture as well as a nutrient and flavor source for fermented food production in Korea [2].Understanding of traditional fermented soybean foods has long been a research theme of food scientists in Korea. Several studies including microbial community analysis have provided basic insight for accelerated ripening, quality assurance, and flavor enhancement of fermented soybean foods. Early microbial studies isolated and identified microorganisms exhibiting strong amylase, protease, and lipase activities that can contribute to degradation of soybean macromolecules [3][4][5][6]. More recent culture-independent microbial community analysis techniques have revealed the presence of a wider variety of microorganisms in the food matrices [7][8][9][10][11][12][13][14][15]. However, regardless of the analytical methods used, most microbial studies reported that the bacterial genus Bacillus and the fungal genus Aspergillus were the most populous microorganisms.Recently, molecular biology techniques and sequence databases have contributed to identification, characterization, and typing of bacteria and increased the number of novel bacterial species [16]. Rapid advances in whole-genome sequencing technologies and analysis pipelines have further enhanced the resolution power of bacterial species and influenced the taxonomic status of closely related identities. This advanced bacterial identification methodology has affected the consolidation and assignment of new taxa in the genus Bacillus [17][18][19]. In this context, we isolated and identified Bacillus spp. from 12 meju samples collected from five regions of Korea to determine the predominant Bacillus species in meju based on current bacterial taxonomic standards [20]. One hundred and fifty-one Bacillus isolates were identified, in the following order of predominance: B. velezensis, B. sonorensis, B. subtilis, and B. licheniformis. Strains of four Bacillus spp. were respectively inoculated into sterilized soybeans and the free amino acid profiles of the resulting cultures were analyzed to discern their metabolic traits. After 30 days of culture, B. licheniformis showed the highest production of serine, threonine, and glutamic acid; B. subtilis exhibited the highest production of alanine, asparagine, glycine, leucine, proline, tryptophan, and lysine. B. velezensis increased the γ-aminobutyric acid (GABA) ...

We evaluated the antibiotic minimum inhibitory concentrations (MICs) of 123 Bacillus velezensis strains predominantly isolated from fermented soybean foods from Korea. When the 2018 European Food Safety Authority breakpoint values for Bacillus spp. were applied, all the strains were sensitive to chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, tetracycline, and vancomycin, and eight strains (6.5%) were resistant to streptomycin. The population distribution in MIC tests with streptomycin was continuous and the profile was clearly different from that expected for acquired antibiotic resistance. As of October 25th 2021, there were 181 complete published genomes of B. velezensis strains; 175 (96.7%) and 136 (75.2%) of these strains respectively possess potential tetracycline and streptomycin resistance genes tetL and ant(6) in the chromosome. In B. licheniformis, SpeG confers resistance to clindamycin and there is an ‘speG’ gene annotated in the genomes of 180 B. velezensis strains; however, the gene products exhibit ≤ 26.6% amino acid identity with that from B. licheniformis DSM 13T. All the potential antibiotic resistance genes in the 181 B. velezensis strains were intrinsic, and traits of lateral gene transfer were not found. In this context, B. velezensis may not present a high risk in terms of antibiotic resistance in food fermentation or human use.