Abstract
Chronic inflammatory disorders of the gastrointestinal tract are characterized by gut dysbiosis, which refers to an imbalance of intestinal microbiota influenced by diet, genetic background, or antibiotics. One important example is Inflammatory Bowel Disease (IBD) which affects millions of people worldwide and approximately 25-30% of individuals have been affected by IBD before the age of 20 [1]. Live cultures of probiotics are often recommended to promote gut health and diversity of intestinal microbiota however spore-based probiotics, including Bacillus licheniformis (B. licheniformis), to withstand the gastrointestinal tract [1, 2]. In addition, vitamin B12 is also prescribed by physicians as a novel treatment to remodel the mammalian gut microbiota by coadministration with probiotic. Therefore, we aimed to elucidate the role of vitamin B12 in aerobic and anaerobic environments for B. licheniformis (1) spore germination, (2) growth rate, and (3) biofilm formation through the following specific aims: I) To elucidate the role of vitamin B12 in aerobic and anaerobic environments for B. licheniformis germination. II) To determine the effects of vitamin B12 in aerobic and anaerobic environments for B. licheniformis biofilm growth and formation. We performed germination assays in the presence of vitamin B12 and determined that B. licheniformis is unable to germinate regardless of oxygen levels. The growth of B. licheniformis was determined by the growth curve assay in aerobic and anaerobic conditions for total of 72 hours. We show that all concentrations of vitamin B12 are able to modulate the growth of B. licheniformis in aerobic conditions but in anaerobic conditions, the growth was significantly decreased in all concentrations of vitamin B12. Moreover, biofilm for aerobic condition was determined by quantifying the pellicle formation at the air-liquid interface. Pellicle formation of B. licheniformis in aerobic conditions only showed a significant decrease at 48 hours whereas the pellicle-associated planktonic cells increased at 24 hours. In anaerobic conditions, we observed that B. licheniformis formed a biofilm at the liquid-solid interface of the test tubes which suggests that mechanism for biofilm in aerobic and anaerobic conditions are different. In anaerobic conditions, vitamin B12 also modulated the formation of biofilm at 24 and 48 hours. However, in biofilm-associated planktonic cells, significant differences were observed in all timepoints tested. Collectively, our results demonstrated that vitamin B12 can modulate the growth of B. licheniformis during the early phase of bacterial growth regardless of oxygen levels. In addition, vitamin B12 can modulate the growth and formation of pellicles, biofilm, and planktonic differentially in aerobic and anaerobic conditions. We also established that vitamin B12 is not able to initiate spore germination as a sole germinant. Future work could further elucidate the role of vitamin B12 in spore germination and characterize the mechanism by which vitamin B12 is able to modulate growth and biofilm formation.