Immunostimulatory activity of inactivated environmental Bacillus isolates and their endospores

The spore-forming capacity of Bacillus spp. enables environmental persistence and stable product formulations, yet the interactions of environmental Bacillus spores and vegetative cells with the human immune system are not fully understood. We investigated the immunostimulatory potential of seven environmental Bacillus isolates (B. subtilis, B. velezensis, B. licheniformis, B. pumilus) and optimized their inactivation methods to preserve surface integrity and immunostimulatory activity. Inactivation was evaluated using heat/UV-C and 10-100% formalin, followed by scanning electron microscopy and human reporter cell assays (THP1-Dual monocytes, HEK-Blue hTLR2/6 and hTLR4). Heat/UV-C treatment retained Bacillus morphology and enhanced immune activation compared to 10-100% formalin treatment. Spores and vegetative cells activated key immune transcription factors (nuclear factor kappa B and interferon regulatory factors) in human monocytes, with spores inducing 1.5- to 8-fold lower responses compared to vegetative cells, and strain- and species-dependent effects observed. All tested Bacillus isolates significantly activated Toll-like receptor TLR2/6, but not TLR4. Genome analysis identified Bacillus spore envelope components (SpsAEIK, CotBCGQ) that may influence differences in immune responses. Our findings improve understanding of Bacillus-human immune interactions, revealing strain- and species-specific immunostimulatory activity. These results support further exploration of Bacillus isolates for immune-related and environmental applications.