Mechanistic insights into the antimicrobial activity of a novel essential oil blend against Bacillus subtilis using RNA sequencing.

The growing need for alternatives to conventional antibiotics has intensified interest in essential oils as multitarget antimicrobial agents. This study investigated the global transcriptional response of Bacillus subtilis to a novel essential oil blend (EOB1) composed of garlic, lemongrass, cumin, lavender, and nutmeg. The minimum inhibitory concentration (MIC) of EOB1 against B. subtilis was determined to be 25% (v/v), and a subinhibitory concentration of 12.5% (v/v) was used for transcriptomic analysis. Exposure to EOB1 caused widespread transcriptional changes, with 2178 genes identified as differentially expressed, including 1044 upregulated and 1134 downregulated transcripts. After filtering at a false discovery rate (FDR) of <0.05 and an absolute log2 fold change of >1, 1682 differentially expressed genes remained, comprising 981 upregulated and 701 downregulated genes. This corresponds to approximately 41% of the 4100 annotated protein-coding genes of the B. subtilis 168 genome. Principal component analysis showed clear separation between treated and control samples, with the first principal component explaining 97% of the total variance, indicating a strong global transcriptional effect of EOB1 on bacterial gene expression; because library preparation and sequencing were performed in a single batch and replicates within each condition clustered tightly, this separation is attributable to the treatment rather than to technical or batch-related variability. Among the most significantly induced genes were arsB, copA, cadA, pchE, htrB, cypX, arsC, and nfrAB, suggesting the activation of metal detoxification pathways, oxidative and envelope stress responses, secretion stress pathways, and secondary metabolite-associated responses. In contrast, genes involved in cell wall remodeling and nutrient uptake, including cwlO and gltT, were strongly downregulated. Gene Ontology enrichment analysis further supported these patterns, with significant overrepresentation of fatty acid β-oxidation, oxidoreductase activity acting on nicotinamide adenine dinucleotide phosphate (NAD(P)H), stress response pathways, amino acid biosynthetic processes, and cytoplasmic components. Collectively, these findings indicate that EOB1 disrupts multiple aspects of the physiology of B. subtilis and triggers coordinated metabolic, redox, and envelope-associated stress responses. This study provides mechanistic insight into the antimicrobial activity of EOB1 and supports the potential of essential oil blends as alternative antimicrobial agents.