<i>Bacillus licheniformis</i> 809 and <i>Bacillus subtilis</i> 810 promote in vitro supportive effects against potentially harmful microorganisms under a feed matrix-based assay

We developed a series of in vitro studies to assess the effects of Bacillus licheniformis 809 (BL) and B. subtilis 810 (BS) on the growth of potentially harmful bacteria. The BL and BS as single strains (experiment 1) or a 1:1 ratio combination of BL and BS (experiment 2) were inoculated in treatment samples to an estimated concentration of 5 × 105 CFU/g of cattle feed. The strains of E. coli and S. enterica were inoculated and incubated aerobically at 37 °C for 18 to 24 h, whereas C. jejuni was incubated microaerophilic at 42 °C for 18 to 24 h. After 18 h incubation of the feed samples, each individual pathogenic strain was inoculated to a rate of 1 × 105 CFU for E. coli and S. enterica and 1 × 106 CFU/g feed for C. jejuni, respectively. Immediately after incubating all feed samples aerobically at 37 °C, 120 rpm vortex for 24 h for E. coli and S. enterica or microaerophilic at 42 °C, no vortex for 32 to 36 h for C. jejuni. Aliquots from all feed samples containing E. coli and S. enterica were incubated aerobically at 37 °C for 24 h and for C. jejuni incubated microaerophilic 42 °C for 24 h before pathogen enumeration and CFU/g feed calculation. In the first assay, BL significantly reduced the C. jejuni counts at 8, 24, and 36 h (P < 0.001). Similarly, a treatment × hour interaction was also observed (P < 0.001) on the E. coli O149:F4 and O138:F18 counts. For both E. coli, BL and BS reduced the counts of E. coli vs. CON at 4 and 8 h post-DFM addition (P < 0.001) compared to CON, but no differences were observed at 24 h (P ≥ 0.27). Moreover, BL inoculation yielded less E. coli O149:F4 counts when compared to BS at 8 h (P = 0.02), but also at 4 and 8 h when E. coli O138:F18 was evaluated (P ≤ 0.04). For S. enterica Enteritidis, counts were less in BL + BS at 4, 8, and 24 h vs. CON (P ≤ 0.001). Similarly, C. jejuni counts were less for BL + BS at 24 and 32 h (P ≤ 0.0001). Lastly, the counts of E. coli O149:F4 and O138:F18 were less (P < 0.0001) at 6 and 24 h for BL + BS vs. CON. In summary, Bacillus licheniformis 809 and B. subtilis 810, alone or in combination, reduced the in vitro counts of potentially harmful bacteria. Additional work is warranted to evaluate if such responses may be replicated under in vivo challenge models.