Abstract
The consortia of gut-associated microbes and their metabolic activities is collectively referred to as the gut microbiome. It is the subject of intense research toward elucidating roles during health and dysbiosis, including using species commonly found in the microbiome for medical intervention in a variety of contexts including fecal transplants to resolve persistent infections with antibiotic-resistant bacteria. To that end, probiotics are live, non-pathogenic bacterial cells advertised to offer direct metabolic benefits to their host or even competitively exclude more harmful species. Bacillus licheniformis (B. licheniformis) is a soil-borne, spore-forming Gram-positive bacteria that has been used as an industrial organism to produce a wide range of proteases and antibiotics. More recently, it is being used as a probiotic, despite limited research into its efficacy in this role or its ability to survive in the human body. The overarching goal of this project was to evaluate the ability of B. licheniformis 14580 to tolerate bile, an important antimicrobial component of the human gut, in growth and biofilm assays. Based on the existing body of literature, I hypothesized that treatment with 0.05%, 0.5%, and 5% bile would decrease planktonic growth and increase biofilm formation. These hypotheses were investigated using the following specific aims:
• Aim 1: To determine the effects of 0.05%, 0.5%, and 5% bile on the growth of Bacillus licheniformis cultures starting from 0.200 OD600 with a growth curve assay
• Aim 2: To determine the effects of 0.05%, 0.5%, and 5% bile on the establishment and growth of Bacillus licheniformis biofilms starting from 0.200 OD600 cultures with a pellicle formation assay
Growth of B. licheniformis was measured via spectrophotometer in the presence and absence of bile at concentrations of 0.05%, 0.5%, or 5% over a 72-hour time course with nine measured time points. Biofilm growth was monitored at 24 and 48 hours in the presence and absence of 0.05%, 0.5%, and 5% bile via measurement of homogenized pellicles with a spectrophotometer. Additionally, real-time PCR (qPCR) was performed on biofilm and stress related genes with cDNA generated from 24-hour biofilms grown in 0% and 0.5% bile. For each experiment (biological N = 3, technical replicates for each N = 3), individual Student’s T-tests (2 tailed, paired) were used to compare pairs (2 conditions) to ascertain which bile concentration(s) increased or decreased growth, biofilm formation, or differential gene expression compared to the untreated control (p<0.05 indicating significance for growth and biofilm, p<0.01 for differential expression). One-way ANOVA tests were also used to detect significant differences in the growth rate and biofilm formation of three bile concentrations (0%, 0.05%, 0.5%) and a bacterial control. ANOVA p-values under 0.05 resulted in rejection of null hypotheses. The results of these experiments showed that the presence of 0.05% bile significantly decreased growth in the first 12 hours of growth curves and 5% bile decreased growth for the first 48 hours. In 24-hour biofilms, 0.05%, 0.5%, and 5% bile all significantly increased biofilm formation. At 48 hours, 0.05% and 0.5% bile significantly increased biofilm formation. qPCR data showed that the biofilm genes yveM and yuaB, superoxide dismutase encoding sodF, and regulatory element abrB were all significantly upregulated in response to 5% bile. These findings indicate that 5% bile decreases the growth of B. licheniformis in liquid culture while also increasing biofilm formation at 24 hours. They also indicate that B. licheniformis grown in 0.05% and 0.5% bile can reach the stationary phase of a growth curve at the same time as a 0% bile control, and that bile in these concentrations significantly increased biofilm formation at 24 and 48 hours. This data can be interpreted as supporting the possibility of B. licheniformis surviving in bile concentrations within the range of a healthy human gut.