Time-kill kinetics antibacterial activity of ethyl acetate extract of Bacillus subtilis subsp. subtilis 168

Abstract

The rise of antibiotic-resistant pathogens underscores the urgent need to fortify existing antimicrobials. Novel antibiotics are products of microbes, as two-thirds of the currently available antibiotics are sourced from them. Understanding the killing rate of the potential antibiotic-producing microbes is essential for the development stage of effective antimicrobials. Our preliminary investigation identified a potential antibiotic-producing Bacillus subtilis subsp. subtilis 168. This study investigated the inhibitory activity and time-kill kinetics of extract of the candidate isolate against selected pathogenic strains. Broth microdilution and time-kill assays were performed to evaluate the inhibitory activity of the Bacillus subtilis subsp. subtilis 168 extract isolated by chemical technique. The extract showed antibacterial activity against Staphylococcus aureus (ATCC 25923) (MIC = 28.70 mg/mL), Escherichia coli (ATCC 25922) (MIC = 28.70 mg/mL), Pseudomonas aeruginosa (ATCC 27853) (MIC = 28.70 mg/mL), and Klebsiella pneumoniae (MIC = 14.35 mg/mL). At 0.5× MIC, the extract was bacteriostatic against all test bacteria for a 24-hour incubation period. At 1x MIC (28.70 mg/mL), the extract achieved bactericidal endpoints (≥99.9% killing) of 3.87 log10 CFU/mL reduction of S. aureus (ATCC 25923) at 6 h incubation, 6.81 log10 CFU/mL reduction of E. coli (ATCC 25922) at 6 h (1x MIC: 28.70 mg/mL), 6.87 log10 CFU/mL reduction of P. aeruginosa (ATCC 27853) at 8 h (1x MIC: 28.70 mg/mL), and 6.90 log10 CFU/mL reduction at 8 h incubation of K. pneumoniae (1x MIC: 14.35 mg/mL). At 2x MIC, the extract showed bactericidal endpoints of at least 3 log10 CFU/mL reduction of all the test strains at lower exposure times. The killing kinetics against each test strain were statistically significant (P ≤ 0.05) at 1x MIC, and 2x MIC. The killing kinetics demonstrated time-and concentration-dependent bactericidal activity against the selected panel of pathogenic strains, achieving a minimum three-log reduction at 1× MIC and 2× MIC within 2-8 h of exposure. These findings suggest a promising therapeutic potential of the extract.