Numrah Hammad 1 , Aqsa Ahmad 1 , Bushra Mumtaz 1 , Bakhtawar Maqbool 2 , Ishtiaq Ahmed 2 and Muhammad Imran Arshad 3*
1 Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan; 2Department of Pathobiology, University of Veterinary and Animal Sciences, Lahore, Sub-Campus Jhang, Jhang 35200, Pakistan; 3Department of Veterinary Preventive Medicine, College of Veterinary
*Corresponding author: m.arshad@qu.edu.sa
Bacillus subtilis (B. subtilis) is a ubiquitous soil bacterium recognized for its probiotic potential, biofilm-forming capacity, and promising bacterium for agricultural biocontrol applications. We investigated growth characteristics, biofilm formation, and antibacterial profile of B. subtilis isolated from diverse soil environments. Ten soil samples from each site including animal hospital site, fruit plant rhizosphere and laboratory surroundings were collected. The soil samples were cultured on nutrient agar, Luria-Bertani agar and rabbit blood agar. Identification was confirmed via Gram’s staining, spore staining and motility assays, revealing Gram-positive, rod-shaped, spore-forming, motile bacilli. Phenotypic characterization was performed using soil (0.7% agar), semi-solid (0.6% agar), and liquid (0.4% agar) media to assess colony morphology, motility and biofilm formation. Biofilm formation was quantitatively evaluated using a microtiter plate assay with crystal violet staining, demonstrating significantly higher mean absorbance values (0.37) compared to controls (0.086), indicative of robust biofilm production across isolates. Antibacterial activity was assessed using the spread plate method against Staphylococcus aureus, Salmonella typhi, Salmonella typhimurium, Pseudomonas aeruginosa, and Escherichia coli. Notably, selective inhibition was observed against E. coli, with no detectable activity against other tested pathogens. Spearman’s rank correlation analysis revealed a weak association (0.434-0.789) between biofilm formation and antibacterial activity.