The authors wish to thank the EEA Grant NILS Science and Sustainability Coordinated Mobility of Researchers (reference 017-ABEL-CM-2013). C.B. and G.P.-M. were supported by the AGL2015-70487-P grant from the Spanish Ministry of Economy and Competitiveness. O.C.O.U. and D.B.D. were supported by a strategic scholarship program for food science research from the Norwegian University of Life Sciences (NMBU) (project 1205051025). We would also like to thank Inmaculada Noguera for her assistance with animal care and sampling and Jesus Dehesa for his help with ensuring the availability of laboratory materials in the animal facility. We also appreciate Professor Lars-Gustav Snipen for his advice on statistics.

Care and handling must be carried out at a specialized animal care unit. Procedures described here were approved by the corresponding Ethics Committee of the University of Valencia and local authorities, following the principles of laboratory animal care mandatory by European Union Law and 2010/63/EU and the Spanish Government RD 53/2013 on the protection of animals used for scientific purposes, in order to respect the 3R principle in animal experimentation (Replacement, Reduction, Refinement).
1. Frozen Bacterial Cultures Used to Inoculate Mice
<ol>
	<li>Inoculate individual strains of LAB in 5 mL of brain-heart infusion (BHI) medium and grow them for 20 - 24 h (overnight) at 30 &#176;C.</li>
	<li>Dilute each overnight bacterial culture 100-fold in BHI by transferring 2 mL of overnight culture to 200 mL of BHI. Grow them at 30 &#176;C for 20 - 24 h. 
	NOTE: Strains used in this study are listed in Table 1.</li>
	<li>Harvest cells by centrifugation at 5,000 x g for 20 min at 4 &#176;C.</li>
	<li>Remove the supernatant and wash the cells twice by suspending each cell pellet in 100 mL of ice-cold phosphate-buffered saline (PBS) and collecting the cells as in step 1.3.</li>
	<li>After the second wash, suspend each cell pellet in 20 mL of ice-cold 15% glycerol in PBS.</li>
	<li>Aliquot the cell suspension into 1-mL volumes in tubes and then store them at -80 &#176;C until use. 
	NOTE: Cells should be used within 1-2 months after this point.</li>
	<li>Determine the cell number before storage and prior to administration so that the number of live cells given to mice is known.
	<ol>
		<li>For cell counting, make ten-fold serial dilutions of cells in ice-cold 0.9% sodium chloride (NaCl) solution. Spread 100 &#181;L of each dilution onto a BHI agar plate. Incubate overnight (20 - 24 h) at 30 &#176;C for cell growth and colony formation, the latter to determine colony-forming units (cfu)/mL.</li>
	</ol>
	</li>
	<li>To make bacteria-containing drinking water, dilute cells of each bacterial stock culture in 100 mL of sterile, filtered drinking water, with a final average cfu of 109/mL of water. For bacterial survival assessment, count live cells just after dilution and after 24 h once a week during the first two weeks of bacteria treatment.</li>
</ol>
2. Mice Assay and Experimental Design
Note: Specific pathogen-free (SPF) BALB/c young female mice (6 - 8 weeks) are needed for this experiment; here, a total of 100 animals were purchased.
<ol>
	<li>Provide the mice with free access to pelleted food and water throughout the experiment.</li>
	<li>Design the experiment and calculate the power.
	<ol>
		<li>If each treatment has its own control, apply a paired case-control design (t- test). Use a preliminary assay to calculate the mean and standard error of the most important effect(s) to be determined.</li>
		<li>Optimize the power of the assay and the number of animals needed per group using different methods and freely available programs11.</li>
		<li>To test for the effects of bacteriocin producer versus non-producer strains, use 9 animals per experimental condition. 
		NOTE: This number was determined based on the experimental standard error and provided a satisfactory power (0.7 - 1.0) and a significance level below 0.05. 
		NOTE: In the given example, 11 groups of mice were made, one per cage. Ten cages corresponded to the treatments with 5 bateriocin-producing strains and the corresponding 5 isogenic non-producing strains; the 11th cage had 10 mice and constituted the untreated control.</li>
	</ol>
	</li>
	<li>After the arrival of the mice to the husbandry facilities, distribute them in cages and ear-label the animals to allow for individual tracking. Acclimatize the mice to the husbandry facilities and conditions for a week before proceeding.</li>
	<li>Prepare bacteria-containing drinking water, as described in step 1.8, 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. 
	NOTE: The control group must be kept in a separate cage, with no contact with the tested bacteria.</li>
	<li>Prepare a new bottle with fresh drinking water (100 mL) every day and add freshly defrosted bacterial suspensions. Do this for 15 consecutive days.</li>
	<li>Follow the bacteria treatment with a period of two weeks, during which time mice drink bacteria-free water (Figure 1).</li>
</ol>
3. Collecting Samples
<ol>
	<li>Collect fecal samples.
	<ol>
		<li>Prepare autoclaved cages (empty) and sterile forceps and label sterile 1.5-mL tubes for each mouse.</li>
		<li>Collect samples at the same time of day to avoid variations in microbiota composition during the course of a day. For optimal results, collect fresh samples separately. 
		NOTE: Preferably, collect feces early in the morning, when mice tend to defecate spontaneously.</li>
		<li>Clean an empty cage with 70% alcohol and place a mouse inside. Allow it to defecate, and collect 2 - 4 fecal pellets using sterile forceps, placing them into 1.5 mL tubes. 
		NOTE: Sometimes it is enough to hold the mouse tail upwards; the stool can then be directly collected from the anus into a tube.</li>
		<li>Collect fecal samples from each mouse once a week during the four-week experiment (Figure 1). Collect the first samples on day one (T0, time zero), just before the exposure of the mice to bacteria. Collect the following fecal samples on days 7, 14, 21, and 28.</li>
		<li>Refrigerate the samples at 4 &#176;C until their transport to the laboratory, where they are frozen at -80 &#176;C. Plan in advance and provide enough storage boxes, as a large number of fecal samples will be collected during the experiment.</li>
	</ol>
	</li>
	<li>Weigh all mice every week on fecal collection day.</li>
	<li>Collect blood samples from the facial vein from all mice on 15th day, the last day of bacteria administration, and determine the levels of triglycerides, total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in the blood serum. 
	NOTE: Experienced personnel should collect the blood to reduce stress in the animals.</li>
</ol>
4. LAB Counting and Bacteriocin Activity
<ol>
	<li>Weigh one fecal pellet of each sample in a 1.5 mL tube and add an adequate volume of ice-cold 0.9% NaCl to achieve a 10% (w/v) solution. Use sterile micropestles for 1.5 mL tubes to homogenize the fecal suspension and prepare ten-fold serial dilutions in ice-cold 0.9% NaCl.</li>
	<li>Count the total number of LAB cells.
	<ol>
		<li>Transfer diluted cells (100 &#181;L) to 4 mL of prewarmed Man Rogosa Sharpe (MRS) soft agar (50 &#176;C, 0.8% agar). Mix by vortexing before pouring the cells onto a solid MRS agar plate (1.5% agar).</li>
		<li>Dry the plate by taking the lid off for 5 - 10 min in the sterile hood before incubating the cells for 20 - 24 h at 30 &#176;C for cell growth and colony formation.</li>
	</ol>
	</li>
	<li>Count bacteriocin-producing cells.
	<ol>
		<li>Transfer diluted cells (100 &#181;L) of the bacteriocin producer to 4 mL of prewarmed MRS soft agar (50 &#176;C, 0.8% agar), mix by vortexing, and pour the cells onto an MRS agar plate (1.5% agar); this layer is referred to as the first layer.</li>
		<li>Transfer another 4 mL of cell-free MRS soft agar (0.8% agar) onto the first layer to embed all cells within the soft agar. 
		NOTE: This layer is meant to prevent cells from growing as colonies on the surface of the agar plates. Cells growing on the surface are easily displaced when pouring indicator cells on the top (step 4.3.4) and will therefore complicate the interpretation of results. This layer is referred to as the second layer.</li>
		<li>Dry the plate by taking the lid off for 5 - 10 min in the sterile hood before incubating for 20 - 24 h at 30 &#176;C for cell growth and colony formation.</li>
		<li>To identify bacteriocin-producing colonies, mix 40 &#181;L of an overnight culture of an appropriate indicator strain, as shown in Table 1, with 4 mL of prewarmed soft agar. Pour the mixture onto the plate; this layer is referred to as the third layer.</li>
		<li>Dry the plate by taking the lid off for 5 - 10 min in the sterile hood before incubating for 20 - 24 h at 30 &#176;C for cell growth and colony formation. 
		NOTE: Bacteriocin-producing colonies have clear (inhibition) zones around the colonies (Figure 2).</li>
	</ol>
	</li>
</ol>
5. DNA Extraction, 16S rDNA Amplification, and Sequencing
NOTE: Steps for DNA extraction are described for the use of a commercial kit (e.g., Realpure &#34;SSS&#34; Kit).
<ol>
	<li>Suspend 1 - 2 fecal mice pellets (~200 mg) in 300 &#181;L of lysis solution.</li>
	<li>Transfer the sample to a 2 mL microtube in which about 0.5 g of glass beads (diameter: 0.1 mm) have previously been placed.</li>
	<li>Add 1 &#181;L of mutanolysin at 10 U/&#181;L and 2 &#181;L of lysozyme at 20 mg/mL to each sample and incubate at 37 &#176;C for 40 - 60 min. Proceed with one of the standard protocols for DNA extraction from fecal samples12.</li>
	<li>Apply two cycles of bead-beater steps, 1 min each.</li>
	<li>Add 2 &#181;L of proteinase K and mix thoroughly.</li>
	<li>Incubate for 20 min and vortex every 5 min.</li>
	<li>Cool the samples to 37 &#176;C and add 1 &#181;L of 5 &#181;g/mL RNase A to the sample. Mix thoroughly.</li>
	<li>Incubate at 37 &#176;C for 30 - 60 min.</li>
	<li>Cool the samples to room temperature and add 180 &#181;L of protein precipitation solution. Vortex vigorously at maximum speed for 20 - 30 s.</li>
	<li>Centrifuge at 16,000 x g for 5 min. If there are any particles floating in the supernatant, incubate the samples in ice for 5 min and then centrifuge again.</li>
	<li>Transfer the supernatant containing the DNA to a new 1.5-mL microtube containing 300 &#181;L of isopropanol.</li>
	<li>Mix by inverting the tubes 20-30 times and incubate for 1 h at -20 &#176;C. Alternatively, incubate the tubes for longer a period (e.g., overnight).</li>
	<li>Centrifuge at 16,000 x g for 2 min.</li>
	<li>Remove the supernatant and dry the tube on absorbent paper. Add 300 &#181;L of 70% ethanol to wash the DNA pellet.</li>
	<li>Centrifuge at 16,000 x g for 1 min, remove the supernatant, and leave the pellet to dry for about 15 min.</li>
	<li>Once the pellet is dry, add 100 &#181;L of sterile water or Tris (10 mM)/EDTA (1 mM) buffer and resuspend the pellet.</li>
	<li>To eliminate any possible inhibitory compounds during subsequent steps, clean the DNA. Mix extracted DNA with 2 volumes (200 &#181;L) of buffer NTI to adjust the binding conditions for step 5.15.</li>
	<li>Place a silica membrane containing PCR clean-up column into a collection tube (2 mL) and load the sample.</li>
	<li>Centrifuge for 30 s at 11,000 x g. Discard the flow-through.</li>
	<li>Wash the silica membrane with 700 &#181;L of buffer NT3 and repeat step 5.16.</li>
	<li>Dry the silica membrane by centrifugation for 1 min at 11,000 x g to remove any residual ethanol containing wash buffer NT3.</li>
	<li>Transfer the column to a new 1.5-mL microtube and incubate for 2 - 5 min at 70 &#176;C for the total removal of the ethanol.</li>
	<li>Add 30 &#181;L of PCR-grade water and incubate for 5 min at 70 &#176;C. Elute by centrifugation for 1 min at 11,000 x g.</li>
	<li>Quantify the DNA using a UV/Vis spectrophotometer or a fluorometric method.</li>
	<li>Normalize and pool the DNA samples from mice sharing same cage at each time point.</li>
	<li>To observe individual variations during time, sequence DNA samples from three randomly selected mice from the control and two other random cages on days 0, 14, and 28.</li>
</ol>
6. 16S rRNA Gene Amplification and Sequencing
<ol>
	<li>Prepare a library of amplified 16S rRNA genes. Amplify the V3-V4 region of the bacterial 16S rRNA gene5 using forward and reverse primers with appropriate overhang adaptors: 
	5&#39;-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3&#39; 
	5&#39;-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3&#39;</li>
	<li>Check the amplified bands using electrophoresis with 1% agarose gel.</li>
	<li>Clean up the polymerase chain reaction (PCR) products using magnetic beads for amplicon purification.</li>
	<li>Proceed with the following steps as indicated by the manufacturer according to the massive sequencing platform used6.</li>
</ol>
7. Data Analysis and Statistics
<ol>
	<li>Quality filtering (usually performed at sequencing facilities).
	<ol>
		<li>Filter the raw reads data and de-multiplex using the software supplied with the equipment.</li>
		<li>Process the paired-end reads using the UPARSE pipeline13 implemented in USEARCH14 (version 7.0.1090). Merge the paired ends and apply quality filtering using a maximum expected error (maxee) value of 1.0. Discard sequences less than 150 nt. Dereplicate sequences to discard singletons.</li>
		<li>Cluster the sequences into operational taxonomic units (OTUs) on a 97% sequence identity and use UCHIME15 to filter chimeric sequences.</li>
	</ol>
	</li>
	<li>OTU picking, taxonomic assignment and phylogenetic reconstruction, and diversity analyses and visualizations.
	<ol>
		<li>Process the OTUs using Quantitative Insights Into Microbial Ecology (QIIME)16.</li>
		<li>Pick and align the representative OTUs against the Greengenes core set database17 using PyNAST18 with a minimum identity of 75% (default).</li>
		<li>Assign taxonomy to the aligned sequences using the Ribosomal Database Project (RDP) classifier program19 with a confidence of 0.8.</li>
		<li>Normalize the OTU table to the sequence count of the sample with the lowest sequence depth.</li>
		<li>Build a phylogenic tree from aligned sequences using Fast Tree20 after the filtration step in order to remove highly variable regions and positions that are all gaps. Use this tree to calculate alpha and beta diversities and to calculate rarefaction curves and Shannon indexes.</li>
		<li>To generate and visualize unweighted UniFrac distance metrics21, use the principle coordinate analysis (PCoA). 
		NOTE: For statistical analysis, different software packages may be used, such as R22 or the statistical tools implemented within QIIME16.</li>
		<li>For the comparison of Shannon indexes of diversity between the different treatments, use an ANCOVA, considering time as a continuous dependent variable in R.</li>
		<li>Compare the distances between treatments in PCoA in QIIME using a two-sided Student&#39;s t-test and calculate the nonparametric p-values with 1,000 Monte Carlo permutations using the Bonferroni correction.</li>
		<li>Analyze for correlations between the relative abundance of specific OTUs and other measured parameters, such as serum levels of cholesterol, HDL, LDL, etc., using Pearson correlation analysis. To this end, use CoNet23 with the subsequent visualization of correlation networks using Cytoscape 3.1.124.</li>
	</ol>
	</li>
</ol>

<ol><li>Papagianni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ribosomally+synthesized+peptides+with+antimicrobial+properties%3A+biosynthesis%2C+structure%2C+function%2C+and+applications%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications.</a> Biotechnol Adv. 21 (6), 465-499 (2003).</li>
<li>Gillor, O., Kirkup, B. C., Riley, M. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGillor%2C+O+%22Advances+in+Applied+Microbiology%22">Advances in Applied Microbiology</a>. 54, Academic Press. 129-146 (2004).</li>
<li>Dzung, B. D., Ingolf, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ribosomally+Synthesized+Antibacterial+Peptides+in+Gram+Positive+Bacteria%5BTitle%5D)+AND+%22Curr+Drug+Targets%22%5BJournal%5D)">Ribosomally Synthesized Antibacterial Peptides in Gram Positive Bacteria.</a> Curr Drug Targets. 3 (2), 107-122 (2002).</li>
<li>Dobson, A., Cotter, P. D., Ross, R. P., Hill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteriocin+Production%3A+a+Probiotic+Trait%3F%5BTitle%5D)+AND+%22Appl+Environ+Microb%22%5BJournal%5D)">Bacteriocin Production: a Probiotic Trait?</a> Appl Environ Microb. 78 (1), 1-6 (2012).</li>
<li>Li, D., Wang, P., Wang, P., Hu, X., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+gut+microbiota%3A+A+treasure+for+human+health%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">The gut microbiota: A treasure for human health.</a> Biotechnol Adv. 34 (7), 1210-1224 (2016).</li>
<li>Millette, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capacity+of+Human+Nisin-+and+Pediocin-Producing+Lactic+Acid+Bacteria+To+Reduce+Intestinal+Colonization+by+Vancomycin-Resistant+Enterococci%5BTitle%5D)+AND+%22Appl+Environ+Microb%22%5BJournal%5D)">Capacity of Human Nisin- and Pediocin-Producing Lactic Acid Bacteria To Reduce Intestinal Colonization by Vancomycin-Resistant Enterococci.</a> Appl Environ Microb. 74 (7), 1997-2003 (2008).</li>
<li>Cotter, P. D., Ross, R. P., Hill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteriocins+%5Bmdash%5D+a+viable+alternative+to+antibiotics%3F%5BTitle%5D)+AND+%22Nat+Rev+Microb%22%5BJournal%5D)">Bacteriocins [mdash] a viable alternative to antibiotics?</a> Nat Rev Microb. 11 (2), 95-105 (2013).</li>
<li>Leroy, F., Foulquié Moreno, M. R., De Vuyst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enterococcus+faecium+RZS+C5%2C+an+interesting+bacteriocin+producer+to+be+used+as+a+co-culture+in+food+fermentation%5BTitle%5D)+AND+%22Int+J+Food+Microb%22%5BJournal%5D)">Enterococcus faecium RZS C5, an interesting bacteriocin producer to be used as a co-culture in food fermentation.</a> Int J Food Microb. 88 (2-3), 235-240 (2003).</li>
<li>Ananou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+effect+of+enterocin+AS-48+and+high+hydrostatic+pressure+to+control+food-borne+pathogens+inoculated+in+low+acid+fermented+sausages%5BTitle%5D)+AND+%22Meat+Sci%22%5BJournal%5D)">Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages.</a> Meat Sci. 84 (4), 594-600 (2010).</li>
<li>Riboulet-Bisson, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+Lactobacillus+salivarius+Bacteriocin+Abp118+on+the+Mouse+and+Pig+Intestinal+Microbiota%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota.</a> PLoS One. 7 (2), e31113(2012).</li>
<li>Charan, J., Kantharia, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+calculate+sample+size+in+animal+studies%3F%5BTitle%5D)+AND+%22J+Pharmacol+Pharmacother%22%5BJournal%5D)">How to calculate sample size in animal studies?</a> J Pharmacol Pharmacother. 4 (4), 303-306 (2013).</li>
<li>Umu, ÖC. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Potential+of+Class+II+Bacteriocins+to+Modify+Gut+Microbiota+to+Improve+Host+Health%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health.</a> PLoS One. 11 (10), e0164036(2016).</li>
<li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UPARSE%3A+highly+accurate+OTU+sequences+from+microbial+amplicon+reads%5BTitle%5D)+AND+%22Nat+Meth%22%5BJournal%5D)">UPARSE: highly accurate OTU sequences from microbial amplicon reads.</a> Nat Meth. 10 (10), 996-998 (2013).</li>
<li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Search+and+clustering+orders+of+magnitude+faster+than+BLAST%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Search and clustering orders of magnitude faster than BLAST.</a> Bioinformatics. 26 (19), 2460-2461 (2010).</li>
<li>Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UCHIME+improves+sensitivity+and+speed+of+chimera+detection%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">UCHIME improves sensitivity and speed of chimera detection.</a> Bioinformatics. 27 (16), 2194-2200 (2011).</li>
<li>Caporaso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((QIIME+allows+analysis+of+high-throughput+community+sequencing+data%5BTitle%5D)+AND+%22Nat+Meth%22%5BJournal%5D)">QIIME allows analysis of high-throughput community sequencing data.</a> Nat Meth. 7 (5), 335-336 (2010).</li>
<li>DeSantis, T. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Greengenes%2C+a+Chimera-Checked+16S+rRNA+Gene+Database+and+Workbench+Compatible+with+ARB%5BTitle%5D)+AND+%22Appl+Environ+Microb%22%5BJournal%5D)">Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB.</a> Appl Environ Microb. 72 (7), 5069-5072 (2006).</li>
<li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PyNAST%3A+a+flexible+tool+for+aligning+sequences+to+a+template+alignment%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">PyNAST: a flexible tool for aligning sequences to a template alignment.</a> Bioinformatics. 26 (2), 266-267 (2010).</li>
<li>Wang, Q., Garrity, G. M., Tiedje, J. M., Cole, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naive+Bayesian+Classifier+for+Rapid+Assignment+of+rRNA+Sequences+into+the+New+Bacterial+Taxonomy%5BTitle%5D)+AND+%22Appl+Environ+Microb%22%5BJournal%5D)">Naive Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy.</a> Appl Environ Microb. 73 (16), 5261-5267 (2007).</li>
<li>Price, M. N., Dehal, P. S., Arkin, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FastTree+2+-+Approximately+Maximum-Likelihood+Trees+for+Large+Alignments%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">FastTree 2 - Approximately Maximum-Likelihood Trees for Large Alignments.</a> PLoS One. 5 (3), e9490(2010).</li>
<li>Lozupone, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UniFrac%3A+a+New+Phylogenetic+Method+for+Comparing+Microbial+Communities%5BTitle%5D)+AND+%22Appl+Environ+Microb%22%5BJournal%5D)">UniFrac: a New Phylogenetic Method for Comparing Microbial Communities.</a> Appl Environ Microb. 71 (12), 8228-8235 (2005).</li>
<li>R Core Team. R Foundation for Statistical Computing. , Vienna, Austria. Available from:
 <a target="_blank" href="http://www.R-project.org">http://www.R-project.org</a> (2014).</li>
<li>Faust, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+co-occurrence+relationships+in+the+human+microbiome%5BTitle%5D)+AND+%22PLoS+Comput+Biol%22%5BJournal%5D)">Microbial co-occurrence relationships in the human microbiome.</a> PLoS Comput Biol. 8 (7), e1002606(2012).</li>
<li>Shannon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytoscape%3A+A+Software+Environment+for+Integrated+Models+of+Biomolecular+Interaction+Networks%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks.</a> Genome Res. 13 (11), 2498-2504 (2003).</li>
<li>Klindworth, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+general+16S+ribosomal+RNA+gene+PCR+primers+for+classical+and+next-generation+sequencing-based+diversity+studies%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.</a> Nucleic Acids Res. 41 (1), e1(2013).</li>
<li>Edwards, A. N., Suárez, J. M., McBride, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culturing+and+Maintaining+Clostridium+difficile+in+an+Anaerobic+Environment%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Culturing and Maintaining Clostridium difficile in an Anaerobic Environment.</a> J Vis Exp. (79), e50787(2013).</li>
<li>Delves-Broughton, J., Blackburn, P., Evans, R. J., Hugenholtz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+of+the+bacteriocin%2C+nisin%5BTitle%5D)+AND+%22Antonie+Van+Leeuwenhoek%22%5BJournal%5D)">Applications of the bacteriocin, nisin.</a> Antonie Van Leeuwenhoek. 69 (2), 193-202 (1996).</li>
<li>Allende, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+and+bacteriocin+production+by+lactic+acid+bacteria+in+vegetable+broth+and+their+effectiveness+at+reducing+Listeria+monocytogenes+in+vitro+and+in+fresh-cut+lettuce%5BTitle%5D)+AND+%22Food+Microb%22%5BJournal%5D)">Growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing Listeria monocytogenes in vitro and in fresh-cut lettuce.</a> Food Microb. 24 (7-8), 759-766 (2007).</li>
<li>Strompfová, V., Lauková, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+study+on+bacteriocin+production+of+Enterococci+associated+with+chickens%5BTitle%5D)+AND+%22Anaerobe%22%5BJournal%5D)">In vitro study on bacteriocin production of Enterococci associated with chickens.</a> Anaerobe. 13 (5-6), 228-237 (2007).</li>
<li>Caballero-Guerrero, B., Jiménez Díaz, R., Maldonado-Barragán, A., Ruiz-Barba, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coculture+with+specific+bacteria+enhances+survival+of+Lactobacillus+plantarum+NC8%2C+an+autoinducer-regulated+bacteriocin+producer%2C+in+olive+fermentations%5BTitle%5D)+AND+%22Food+Microb%22%5BJournal%5D)">Coculture with specific bacteria enhances survival of Lactobacillus plantarum NC8, an autoinducer-regulated bacteriocin producer, in olive fermentations.</a> Food Microb. 27 (3), 413-417 (2010).</li>
<li>Drider, D., Fimland, G., Héchard, Y., McMullen, L. M., Prévost, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Continuing+Story+of+Class+IIa+Bacteriocins%5BTitle%5D)+AND+%22Microb.+Mol+Biol+Rev%22%5BJournal%5D)">The Continuing Story of Class IIa Bacteriocins.</a> Microb. Mol Biol Rev. 70 (2), 564-582 (2006).</li>
<li>Gao, Y., Jia, S., Gao, Q., Tan, Z. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0956713509001224">A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage.</a> Food Control. 21 (1), 76-81 (2010).</li>
</ol>

The authors have nothing to disclose.

The procedure described here has been used to determine whether changes in the microbiota are bound to health or age. Different parts of the protocol are important, but among them, sampling the feces, choosing the DNA fragment to be sequenced and analyzed, and performing the DNA extraction and bioinformatic analysis could certainly be the most critical points. Sampling is crucial because, for ethical reasons, mice should not be stressed and because it is known to change the proportion of bacteria in the gut. Samples must...

Bacteriocins are antimicrobial peptides produced by a wide range of bacterial species1,2. These compounds and their producers, especially LAB, have been explored and exploited worldwide for decades for their potential applications in food preservation and medicine3. Several bacteriocins are known to kill important pathogens, including species of Listeria, Enterococcus, Staphylococcus, and Bacillus. Some bacteriocins even have the ability to modulate the immune response4. Many bacteriocins have relatively narrow spectra, a property that is much appreciated in some applications. For example, some narrow-spectrum bacteriocins can be used to direct specific activity against selected groups of problematic bacteria, without much disturbance on the commensal or beneficial flora sharing the same niche; this is especially essential in the gut environment, where numerous beneficial microbes thrive in an interactive and dynamic manner5. Bacteriocins are also very attractive for prophylactic or probiotic use, as they can suppress the (out)growth of pathogens, pathobionts, or opportunistic bacteria that may unbalance the gut homeostasis6,7.
In terms of their nature and physicochemical properties, bacteriocins are very diverse, as they have different structures, target specificities, modes of action, etc. Most bacteriocins have been studied in great detail in in vitro settings, but very few have been tested in food matrices8,9 or in vivo, such as in an animal gut6,10. The in vitro properties can differ to a great extent when assessed in vivo due to the complexity of the gut environment and also to putative unintended effects on beneficial bacteria. Most probiotics are LAB. They produce an array of other metabolites, including short-chain fatty acids, which are known to influence the physiology of the host, as well as to demonstrate antimicrobial properties toward certain bacteria. Therefore, in the case of probiotic strains that produce bacteriocins, it is best to establish realistic assays, such as healthy animals with normal microbiota.
In the present study, we provide a strategy that allows for the assessment of the effect of different bacteriocin-producing strains, whose bacteriocins have different inhibitory spectra, on healthy mice. Our strategy includes feeding mice with isogenic non-bacteriocin mutants, which enables the differentiation of bacteriocin-mediated effects from non-bacteriocin-mediated effects. Sequencing 16S rDNA allows for following the dynamic changes of the bacterial population in the gut. Subsequent statistical analysis deciphers correlations between bacterial species and also between bacterial species and measured physiological parameters (e.g., bodyweight, serum biochemical parameters, etc.). We believe that the protocol presented in this study is also applicable to other probiotic or prebiotic applications beyond the study of bacteriocins in live animals.

<table><tbody><tr><td>Balb/c mice (female)</td><td>Harlan</td><td></td><td>Mice should be 6 - 8 weeks of age</td></tr><tr><td>Plastic Petri dish</td><td>Thermo Scientific</td><td>101VR20</td><td></td></tr><tr><td>Brain-Heart-Infusion broth</td><td>Conda</td><td>1400.00</td><td></td></tr><tr><td>European Bacteriological Agar</td><td>Pronadisa</td><td>1800.00</td><td></td></tr><tr><td>Agarose D1 Low EEO</td><td>Pronadisa</td><td>8010.00</td><td></td></tr><tr><td>1x TAE buffer</td><td>Thermo Scientific</td><td>15558042</td><td></td></tr><tr><td>MRS broth</td><td>Difco</td><td>288130</td><td></td></tr><tr><td>PBS tablets</td><td>Sigma</td><td>P4417-100TAB</td><td></td></tr><tr><td>scale</td><td>Mettler Toledo</td><td>PB602-S</td><td></td></tr><tr><td>sterile forceps</td><td>Levantina de Laboratorios S.L.</td><td>260-3710014</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Centrifuge</td><td>Hermle</td><td>Z383K</td><td></td></tr><tr><td>sodium chloride</td><td>AppliChem Panreac</td><td>121659.1211</td><td></td></tr><tr><td>Realpure SSS Kit</td><td>Real Life Science Solutions, Durviz, Spain</td><td>RBME04 (300 ml)</td><td></td></tr><tr><td>Isopropanol</td><td>AppliChem Panreac</td><td>131090.1611</td><td></td></tr><tr><td>Ethanol</td><td>AppliChem Panreac</td><td>131086.1214</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Invitrogen</td><td>Q32851</td><td></td></tr><tr><td>AMPure XP beads</td><td>Beckman Coulter Genomics, USA</td><td>A63881 (60 ml)</td><td></td></tr><tr><td>PerfeCta NGS library quantification kit</td><td>Quanta BioSciences, Maryland, USA</td><td>733-2300</td><td></td></tr><tr><td>MiSeq v3 reagent kit</td><td>Illumina, San Diego, California, USA</td><td>MS-102-3003</td><td></td></tr><tr><td>Primers for 16S rRNA gene amplification</td><td></td><td></td><td>Primers contain V3-V4 region of bacterial 16S rRNA gene and Illumina overhang adaptors:5&amp;rsquo;-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3&amp;rsquo; and 5&#039;-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3&amp;rsquo;</td></tr><tr><td>Nextera XT Index kit</td><td></td><td>FC-131-1002</td><td>Indices and Illumina sequencing adaptors</td></tr><tr><td>Micropestle for 1.5 mL tubes, Eppendorf / Sigma , Ref.</td><td>Sigma</td><td>Z317314-1PAK</td><td></td></tr><tr><td>Glass beads, 0.1 mm diameter</td><td>Biospec Products</td><td>11079-101</td><td></td></tr><tr><td>NucleoSpin Gel and PCR Clean-up Kit</td><td>Macherey-Nagel</td><td>740609.25</td><td></td></tr><tr><td>Omni Bead Ruptor 24</td><td>Omni International Inc.</td><td>19-040</td><td></td></tr><tr><td>mutanolysin</td><td>Sigma</td><td>M9901-10KU</td><td></td></tr><tr><td>lysozyme</td><td>Roche</td><td>10837059001</td><td></td></tr><tr><td>proteinase K</td><td>Roche</td><td>3115887001</td><td></td></tr><tr><td>RNase A</td><td>Sigma</td><td>R4875</td><td></td></tr></tbody></table>

a method to assess bacteriocin effects on the gut microbiota of mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

The production of bacteriocins has been considered a positive probiotic feature in LAB, as it was assumed to prevent the growth of opportunistic bacteria and pathogens. The aim of this work was to show the capacity of bacteriocins to modulate gut microbiota populations in a mouse model. For this purpose, a procedure was developed to compare the effect of the intake of bacteriocin-producing strains and their isogenic non-producing strains. The procedure for the inoculation and the time ext...

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A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

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Research

JoVE Journal

Biology

14.2K Views.  Consejo Superior de Investigaciones Científicas (CSIC).  Bacteriocinen zijn van mening dat ze een sleutelrol spelen bij het definiëren van microbiële diversiteit in verschillende ecologische niches. Hier beschrijven we een efficiënte procedure om te beoordelen hoe bacteriocinen invloed hebben op de darmmicrobiota samenstelling in een diermodel. 

Video: A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Immunology and Infection

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Reducing Research Animal Stress with Micropipette-Guided Drug Administration

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the stomach of research animals. Unfortunately, this method comes with difficulties for both the operator and the research animal. Studies have shown that OG may lead to complications, including esophagitis, perforation of the esophagus, and inadvertent tracheal drug administration. In addition, OG is associated with increased plasma and fecal corticosterone levels (due to stress), altered blood pressure, and increased heart rate, which could negatively influence or bias study results. A previously developed alternative method termed micropipette-guided drug administration (MDA) incentivizes the animal to consume treatments readily in a minimally invasive manner. Herein, we present examples of the use of the MDA technique with treatments reconstituted in different vehicles and demonstrate effective delivery of the varied treatments to multiple different mouse strains. We further demonstrate that MDA is a technique that decreases the timing and invasiveness of drug administration and does not affect the gut microbiome composition as assessed by quantitative analysis of core gut microbial species. Overall, MDA may offer a less stressful and effective alternative to OG.

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the ...

Cancer Research

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease.

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Exploring Gut Phage-Bacteria Dynamics in Murine Models Using T4 Phage

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host&#39;s health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli,&#160;in the context of the murine gastrointestinal tract. The methods described here outline&#160;how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels.&#160;This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

Author Spotlight: Investigating Bacteriophage-Induced Immune Responses in Gnotobiotic Mice

Bacteriophages (phages), viruses that infect bacteria, are an integral component of the gut microbiome. Though these symbiotic inhabitants drive bacterial fitness and population dynamics, little is understood about how they impact gut homeostasis and disease. This protocol studies isolated T4 phages within a mouse model, adaptable to other phage-bacterial pairs.

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across ...

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the biological function of intestinal bacteria as a whole or at the species level. Several different FMT methods have been published. Here, we present an FMT protocol that successfully depletes gut microbiota in a matter of days, followed by transplantation of fecal microbiota from fresh or frozen donor intestinal contents to conventional mice. Real time-PCR is applied to test the efficacy of bacterial depletion. Sequencing of the 16S ribosomal RNA (rRNA) is then applied to test the relative abundance and identity of gut microbiota in recipient mice. We also present a flow cytometry-based detection method of immunoglobulin A (IgA)-coated bacteria in the gut.

We established an efficient way to deplete intestinal bacteria in three days, and subsequently transplant fecal microbiota via gavage of fecal fluid prepared from fresh or frozen intestinal contents in mice. We also present an optimized method to detect the frequency of IgA-coated bacteria in the gut.

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the ...

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Gut microbiota dysbiosis is thought to exert a role in the progression of colitis. However, the precise standards for probiotic administration in alleviating colitis remain undefined. Most analysis methods rely on limited diversity and abundance of gut microorganisms. Therefore, observational studies cannot establish causation. In this study, we applied antibiotic-induced pseudo-germ-free mice to investigate the role of gut microbiota in regulating the probiotic effects of Bacillus&#160;cereus (B. cereus)&#160;on dextran sulfate sodium (DSS)-induced colitis in mice. This process allows for evaluating the bidirectional regulating effect of B. cereus&#160;supplementation on health and provides stable and reproducible results. Here, the detailed protocols for B. cereus cultivation, gavage operation, stool collection, and antibiotic clearance treatment on colitis mice are provided. The optimization methods are also applicable for other chronic inflammatory-associated disorders. The results showed that B. cereus administration decreased body weight loss, colon length shortening, disease activity index, and histopathological scores. However, treatment with antibiotics suppressed the positive effect of B. cereus on colitis. These results indicate that gut microbiota are needed for the alleviating effects of B. cereus on colitis. Therefore, exploring the beneficial effects of probiotics in this research is a promising approach for developing novel treatment strategies for alleviating the symptoms of chronic inflammatory-associated disorders.

Investigating the Alleviating Effects of Bacillus cereus Administration on Colitis through Gut Microbiota Modulation

Here, we present a protocol to establish the antibiotic-induced pseudo-germ-free dextran sulfate sodium-induced colitis mouse model to investigate the role of gut microbiota in regulating the positive effects of Bacillus cereus on colitis.

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

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