292 In vitro fermentation characteristics of dietary fibers using fecal inocula from dogs treated with metronidazole
Authors: Belchik Sara E , Oba Patrícia M , Geary Elizabeth L , Bauer Laura , Dilger Ryan N , Swanson Kelly S 
Published: September 2024

Abstract
Metronidazole is a potent antibiotic often prescribed to treat gastrointestinal enteropathies, but is known to induce loose stools, negatively alter the fecal microbiome, and affect fecal metabolite production. Dietary intervention may aid in post-antibiotic recovery, but little research has been conducted regarding the potential of fiber utilization for microbial recovery in canines. Using an in vitro fermentation assay, the objective was to investigate the fermentation characteristics of dietary fibers using fecal inocula from dogs treated with metronidazole. All procedures were approved by the University of Illinois IACUC prior to experimentation. Healthy male beagles (n = 4) were fed a commercial canned diet for 2 wk, then administered metronidazole [20 mg/kg body weight (BW) twice a day] for 2 wk. Fresh fecal samples were collected at wk 2 [diet only (CTL)] and 4 [antibiotic treatment (ANT)], stabilized in a 20% glycerol solution, and then frozen. On the in vitro fermentation day, feces from each time point were thawed and used to inoculate tubes. At baseline and after 6, 12, and 18 h of fermentation, pH, short-chain fatty acid (SCFA) concentrations, and microbiota populations were measured. SCFA were measured by gas chromatography and fecal microbiota populations were analyzed by 16S rRNA sequencing. All blank tube-corrected changes from baseline data were analyzed using repeated measures and the MIXED procedure of SAS 9.4, with significance set at P &lt; 0.05. QIIME2 analysis was used for bacterial diversity measures. As expected, metronidazole administration heavily influenced microbiota populations and fiber fermentability characteristics. Pectin fermentation reduced (P &lt; 0.001) pH and increased (P &lt; 0.001) SCFA concentrations over time, but responses were less (P &lt; 0.001) in ANT tubes than CTL tubes. Beet pulp fermentation also reduced (P &lt; 0.001) pH and increased (P &lt; 0.001) SCFA concentrations over time. Whereas the magnitude of pH change was small between inoculum sources, SCFA concentrations were different (P &lt; 0.001) between ANT tubes and CTL tubes. Propionate and butyrate concentrations were greater (P &lt; 0.001) in CTL tubes than ANT tubes at all time points, but acetate concentrations varied over time. Chicory pulp fermentation reduced (P &lt; 0.001) pH over time, with greater (P &lt; 0.01) reductions in ANT tubes than CTL tubes. Chicory pulp fermentation increased SCFA concentrations, but had different patterns based on inoculum source. Metronidazole altered microbiota populations by reducing (P &lt; 0.001) bacterial alpha diversity. The analysis of bacterial beta diversity revealed separate clusters for dogs in the absence and presence of metronidazole. Beta diversity analysis also showed that tubes containing chicory pulp clustered separately from those containing the other fibers. Relative abundances of over 50 bacterial genera differed (P &lt; 0.05) among inoculum sources. In summary, interesting fermentation patterns in response to varying fiber sources were observed, allowing for improved insight into their potential abilities when provided to antibiotic-treated dogs.