Isolation, molecular characterization and antibacterial profiling of metabolites produced by Bacillus siamensis and Staphylococcus equorum isolated from traditionally fermented cow milk

Abstract

The increasing emergence of antibiotic-resistant pathogens has intensified the search for alternative and natural antimicrobial agents. This study investigated the isolation, molecular characterisation, and antibacterial profiling of metabolite-producing lactic acid-related bacteria from traditionally fermented cow milk (nono). Fermented milk samples were collected from the Zango (Tudun Wada) and Rafinguza areas of Kaduna State, Nigeria. Bacterial isolates were obtained using de Man, Rogosa, and Sharpe (MRS) agar plates. The bacterial isolates were characterised based on their morphological, biochemical, and physiological properties, including gram reaction, catalase activity, carbohydrate fermentation, salt tolerance, and temperature tolerance. The microbes were extracted using ammonium sulfate precipitation and evaluated for antibacterial activity against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. Molecular identification of selected potent isolates was carried out through PCR amplification and sequencing of the 16S rRNA gene, followed by NCBI Blast sequences. Gas chromatography–mass spectrometry (GC–MS) was used to characterise the bioactive compounds in the mixed culture. The lactic acid bacterial counts ranged from 25.5 × 10⁶ to 73.0 × 10⁶ CFU/mL, confirming that milk is a rich source of Lactic acids related bacteria All isolates were gram-positive and catalasenegative, with rod- and coccus-shaped morphologies, and demonstrated varying degrees of tolerance to NaCl concentrations (4–6.5%) and temperatures (10–45°C). The identified components exhibited concentration-dependent antibacterial activity against all test pathogens, with stronger inhibition observed against Staphylococcus aureus and Escherichia coli than against Pseudomonas aeruginosa. The MIC and MBC assays confirmed the bacteriostatic and bactericidal effects of the bacterial isolates. Molecular analysis identified the selected potent isolates was performed through genomic DNA extraction, PCR amplification of the 16S rRNA gene, agarose gel electrophoresis, purification, sequencing and BLAST analysis against NCBI Genbank database.The isolates were identified as Bacillus siamensis and Staphylococcus equorum, sequences and with a Query Cover (%) of 99, an E-value of 0.0, and Percentage Similarity (%) of 99.08 and 96.03, respectively, through NCBI Blast sequences confirming their taxonomic relationships. GC–MS profiling revealed the presence of several bioactive compounds, including fatty acids and terpenoid derivatives, associated with antimicrobial activity. Traditionally fermented cow milk harbours LAB isolates with significant antibacterial potential, which can serve as natural antimicrobial agents for food preservation and therapeutic applications.