The overall goal of this procedure is to assess the influence of bacteriocins on gut microbiota composition using a mouse model. This method will help determine the real effect of bacteriocins from probiotics on the delicate balance of bacterial populations in the gut. The main advantage of this technique is that it allows the analysis of physiological parameters to infer global effects on the host organism.
After growing bacterial cultures according to the text protocol, count the cells by first using ice-cold, 0.9%sodium chloride solution to make ten fold serial dilutions. Spread 100 microliters of each dilution onto a BHI Agar plate. Incubate the plates at 30 degrees Celsius overnight for cell growth and colony formation to determine the CFU per milliliter.
To prepare bacteria-containing drinking water, use 100 milliliters of sterile, filtered drinking water to dilute the cells from each bacterial stock culture for a final average of ten to the ninth per milliliter CFU. For bacterial survival assessment, count live cells just after dilution and after 24 hours once a week during the first two weeks of bacteria treatment. After ear labeling and acclimating BALB/c young female mice according to the text protocol, on day one of the experiment, collect fecal samples for each mouse and mark them as time zero, or t zero.
Place the samples in previously prepared storage boxes and refrigerate the samples at four degrees Celsius until their transport to the laboratory, where they'll be frozen at minus 80 degrees Celsius, then weigh the mice and record the weight. Prepare bacteria-containing drinking water as demonstrated earlier in the video and place drinking water bottles in the corresponding independent cages that are clearly identified so that the mice in each cage share the same water bottle. Prepare new bottles with 100 milliliters of fresh drinking water every day for 15 days preferably using mineral water to avoid chlorine or bacterial inhibiting compounds, and add freshly defrosted bacterial suspensions.
Collect fecal samples on days seven and 14. Then, for the next two weeks, allow the mice to drink bacteria-free water and collect additional fecal samples on days 21 and 28. To count bacteriocin-producing bacteria cells in fecal samples, transfer diluted cells of the bacteriocin producer to four milliliters of pre-warmed MRS soft agar.
Mix the agar by vortexing and pour the cells onto an MRS 1.5%agar plate. This layer is referred to as the first layer. Transfer another four milliliters of cell-free 0.8%MRS soft agar onto the first layer to embed all the cells within the soft agar.
This layer is meant to prevent cells from growing as colonies on the surface of the agar plates. In the sterile hood, dry the plates by taking the lid off for five to 10 minutes before incubating the plates at 30 degrees Celsius for 20 to 24 hours for cell growth and colony formation. To identify bacteriocin producing colonies, mix 40 microliters of an overnight culture of an appropriate indicator strain with four milliliters of pre-warmed soft agar for each plate.
Pour the mixture onto the plates. This layer is referred to as the third layer. Dry and incubate the plates as before for cell growth and colony formation.
Bacteriocin-producing colonies have clear zones of inhibition around the colonies. Using a commercial DNA extraction kit, suspend one to two fecal mice pellets in 300 microliters of re-suspension solution. Transfer the sample to a two milliliter microtube with 0.5 grams of glass beads.
Add one microliter of 10 units per microliter Mutanolysin and two microliters of 20 milligrams per milliliter Lysozyme to each sample and incubate the tubes at 37 degrees Celsius for 40 to 60 minutes. Disrupt the samples using vibration and glass beads, and then proceed with one of the standard protocols for DNA extraction from fecal samples. After isolating and cleaning the DNA according to the text protocol, quantify the samples.
Then, normalize and pool each sample from mice sharing the same cage at each time point. The aim of this work was to show the capacity of bacteriocins to modulate gut microbiota populations in a mouse model. The tested bacteriocin-producing lactic acid bacteria, or LAB, as well as the respective counts in drinking water and counts in feces are summarized here.
An example of the bacteriocin production by LAB recovered from feces is illustrated in this figure. This PCoA plot reveals that there were no significant differences between the bacteriocin treatments and the isogenic strains when all samples and all bacteria groups were considered together. However, there were likely differences when particular bacterial genera were studied independently, as shown here where changes in the relative abundances of genera were compared to the control group.
Furthermore, Pearson's correlation analysis revealed significant linkage between different bacterial genera and a correlation between bacterial genera and triglycerides and LDL in serum, as shown by this network built in CoNet. While attempting this procedure, it's important to remember the use of isogenic strains as controls and a control without bacterial treatment. Following this procedure, other methods like hepatic function, moe-coe-sic transcriptomics, and fecal water metabolomics can be performed and all at once while additional questions, like how do microbiota fluctuations affect the bile salts, cholesterol cycle, digestibility of nutrients, and immune response.
