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DSS‐induced Ulcerative Colitis and Gut Microbiota in Mice: Evaluation of the Temporal Effects of Chronic DSS Exposure Utilizing Improved Sample Preparation and Analysis Protocols
Author(s) -
Stewart Amanda,
Harold Abby,
Minor Emily,
Nickerson Andrew,
Rottgen Trey,
Rajendran Vazhaikkurichi
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.03558
Subject(s) - feces , microbiome , ulcerative colitis , microbiology and biotechnology , biology , gut flora , colitis , inflammatory bowel disease , immunology , medicine , disease , bioinformatics
Changes in gut microbiota play a major role in the development of inflammatory bowel diseases, including ulcerative colitis (UC). To elucidate changes in this complex microbial community structure, analysis of fecal microbial DNA is necessary as the majority of gut microbes are unculturable or unknown.Previous studies in mice have shown changes in the gut microbiome due to experimentally‐induced UC, commonly induced by administration of dextran sulfate sodium (DSS).Although several studies have reported changes in the fecal microbiome due to short‐duration DSS exposure, neither temporal changes due to chronic UC from long‐term DSS exposure nor the potential loss of gut microbes has been reported.Improved methods for sample preparation and analysis of the 16s rRNA gene via terminal restriction fragment length polymorphism (T‐RFLP) are now available.Utilizing a combination of previously reported sample preparation and T‐RFLP analysis protocols, the current study examined changes in the fecal microbiome due to chronic DSS exposure.Thirty‐two BALB/c mice were divided into control and DSS treatment groups.DSS‐treatments were administered in 5 alternating weeks. Feces was collected twice weekly from individual mice over a 10‐week sampling period; disease activity index was determined as in Dieleman and co‐workers (1997). To maximize bacterial cell wall fracture, and thus bacterial DNA recovery, samples were snap‐frozen in liquid nitrogen, stored at 80°C for >24h, and lyophilized.To determine changes in total fecal DNA content, DNA was extracted from a known dry weight of feces via a bead‐beating protocol. Degenerate 5’‐ and 3’ fluorescently labeled PCR primers were used in a three‐step PCR protocol with 50 ng template and 6% formamide to simultaneously maximize amplification and primer specificity.PCR products were visualized on a 2% agarose gel and subsequently subjected to restriction digestion with the enzyme HpyCH4V or Hpy188l, which were chosen via in silico Primer Sequence Prevalence and Enzyme Resolving Power Analyses of MiCA 3 Microbial Community Analysis III. This protocol successfully generated a single PCR product suitable for downstream T‐RFLP analysis.Disease activity index was greater in DSS‐treated mice ( p <0.0001) and was positively correlated with stool consistency (r 2 = 0.88).Likewise, fecal dry weight was reduced in DSS‐treated mice ( p <0.01). In addition, DNA content was greater in feces from DSS‐treated (762 ± 141 ng DNA/mg dry wt feces) than control mice (449 ± 31 ng/mg dry wt feces; p <0.01).This may indicate total fecal microbiome loss as a primary contributor to changes in microbial community structure in inflammatory bowel disease.