Introduction: Cholera is a serious infectious disease caused by the bacterium Vibrio cholerae. The rising antibiotic resistance of this pathogen highlights the urgent need for alternative antimicrobial strategies. Postbiotics—bioactive compounds produced by probiotics—have gained increasing attention for their antimicrobial activity. This study aimed to evaluate and optimize the antimicrobial effects of cell-free supernatant derived from the probiotic Bifidobacterium bifidum (CFS_BB) against the standard strain Vibrio cholerae El Tor O1 ATCC 14,035.
Methods: In this in vitro experimental study conducted in 2024, an overnight culture of Bifidobacterium bifidum was grown in de Man, Rogosa, and Sharpe (MRS) broth at varying concentrations (10⁴ to 10⁹ colony-forming units per milliliter [CFU/mL]). After centrifugation and filtration to eliminate viable cells, the resulting supernatant was sterilized. The antimicrobial activity of the CFS_BB against Vibrio cholerae was evaluated using the microbroth dilution method. Protein content in the CFS_BB was measured using the bicinchoninic acid (BCA) assay. Statistical analysis was performed using a significance level of p < 0.05.
Results: An increase in the initial concentration of Bifidobacterium bifidum led to a higher protein content in the CFS_BB. CFS_BB from cultures at 10⁸ and especially 10⁹ CFU/mL exhibited the strongest inhibitory effects on Vibrio cholerae growth, with significant activity observed at concentrations between 400 and 800 mg/mL. As the initial Bifidobacterium bifidum concentration increased from 10⁴ to 10⁹ CFU/mL, the culture medium’s pH dropped markedly from 6.3 to 4.3, indicating enhanced inhibitory potency. Correspondingly, the MIC decreased from over 800 to 400 mg/mL, and the overall inhibitory effect intensified from “low” to “strong.” The effectiveness of CFS_BB was highly dependent on both the initial bacterial concentration and storage temperature, with the greatest inhibition observed at higher concentrations (10⁸–10⁹ CFU/mL) and under cold storage (4 °C), particularly within the first 7 days.
Conclusion: CFS_BB demonstrates significant antibacterial activity against Vibrio cholerae El Tor O1, CFU/mL which increases with higher bacterial densities and greater production of antimicrobial metabolites. A starting concentration of 10⁹ CFU/mL is recommended as optimal for preparing effective CFS_BB. These findings support further development of postbiotics as effective and safe alternatives to traditional antibiotics, especially against antibiotic-resistant pathogens.