Co-Culturing Bacillus Strains for Enhanced Antimicrobial Activity

Abstract

Background/Objectives: Bacillus probiotic mixtures have been used as a novel source of antimicrobial production. However, there is a need to emphasize the potential of co-cultured Bacillus strains and to understand the underlying mechanisms involved in their combination formulation. Methods: Bacillus strains BPR-11, BPR-16, and BPR-17, were cultured either as mono or multi-cultures. The bacterial growth and density were evaluated by measuring their optical density. The chemical profiles of their bioactive extracts were analysed by LC-MS, and their antimicrobial activity were tested against selected pathogens. Results: Our results reveal that co-cultured strains significantly increased bacterial growth, with an optical density level of, 2.67 which was significantly higher than the monocultures of BPR-11 (2.24), BPR-16 (2.32), and BPR-17 (2.22). Furthermore, the ethyl acetate extracts from these bacterial cultures showed that the combined co-culture (F1) exhibited the highest antimicrobial activity, with MICs of 25 µg/mL against C. perfringens, E. coli, and S. aureus, and 50 µg/mL against P. aeruginosa and S. enterica. In contrast, the monocultures BPR-16 and BPR-17 showed moderate activity with MICs of 50 µg/mL against C. perfringens, E. coli, and S. aureus, and 100 µg/mL against P. aeruginosa and S. enterica. BPR-11 had the lowest antimicrobial activity, with MICs of 100 µg/mL against C. perfringens, E. coli, and S. aureus, and no activity against P. aeruginosa and S. enterica. Metabolite profiling showed that the extract from the co-culture had a marked increase in the production of antimicrobial metabolites, including C₁₃-C₁₆ surfactin C. Lastly, the metabolism study of surfactin C analogues suggested that they were highly stable (99%) when incubated with cytochromes P450 over 120 min. Conclusions: Together, these findings highlight the potential for multi-strain co-culturing to develop new antimicrobials and provide valuable insights into the synergistic effects for antimicrobial production.

Keywords: Bacillus; antimicrobials; co-culture.

Figure 1

Fermentation OD₆₀₀ over time with sigmoidal bacterial growth phases. The graph shows the optical density (OD) measurements over time for bacterial strains BPR-11, BPR-16, BPR-17, and F1. OD₆₀₀ was monitored every 60 min for 8 h. Values represent the mean ± SD values of 3 replicates. Strain F1 exhibited the highest final OD₆₀₀ followed by BPR-17, BPR-16 and BPR-11 respectively. The differences between F1 and individual monocultures were statistically significant. **** indicates p < 0.001 by one-way ANOVA followed by Tukey’s post hoc test.

Figure 2

HPLC Chromatograms of EtOAc extracts of bacterial strains BPR-17, BPR-16, BPR-11, and F1. The peaks between 1–2 min were associated with the metabolites from the culture medium. Peaks at 4–5 min represent maculosin, maculosine 2, genistein and daidzein, while peaks at 6–7 min represent C₁₃, C₁₄, C₁₅ and C₁₆ surfactin C.

Figure 3

Standard concentration curve of pure C₁₃, C₁₄, C₁₅ and C₁₆ surfactin C by LC-MS. The measured R² values measured were with C₁₃ surfactin C (0.9906), C₁₄ surfactin C (0.9994), C₁₅ surfactin C (0.9984) and C₁₆ surfactin C (0.9893). These R² values were approximately 0.99, highlighting the linearity between the measured concentration and response.

Figure 4

Representative chromatograms displaying the production of C₁₃ (A), C₁₄ (B), C₁₅ (C), and C₁₆ (D) surfactin homologues produced by Bacillus strains BPR-11, BPR-16, BPR-17, and their multistrain-culture F1. These results are consistent across multiple analyses. The highest peak intensities were consistently observed in F1 throughout each spectrum, with lesser amounts detected for BPR-17 and BPR-16. BPR-11 consistently showed little to no detection of the surfactin homologues across repeated experiments.

Figure 5

Quantification of surfactin C analogues in co-culture and monocultures. The bar graph illustrates the quantification of C₁₃-C₁₆ surfactin C produced by the co-cultured Bacillus strain F1 and individual mono-cultured strains BPR-17, BPR-16, and BPR-11. The data reveals significant variations in surfactin C production among the strains, with F1 exhibiting the highest levels across all analogues, followed by BPR-17, BPR-16, and BPR-11.

Figure 6

Metabolic stability of C13-C16 surfactin C analogues in chicken liver microsomes over 120 min. Data points represent mean ± standard deviation (n = 3 independent replicates). The slowly decreasing lines indicate minimal degradation of the surfactin C analogues over the incubation period, suggestive of high metabolic stability within the gut. No statistically significant differences were observed between time points for any analogue (p > 0.05, one-way ANOVA with Tukey’s post hoc test). Quantification was performed using LC-MS/MS analysis, with results expressed as percentage of initial concentration remaining.