In vitro fermentation characteristics of dietary fibers using fecal inocula from dogs treated with metronidazole

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 metabolites. Dietary intervention may aid in the recovery following antibiotic cessation, 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 of this study was to investigate the fermentation characteristics of dietary fibers using fecal inocula from dogs treated with metronidazole. Four healthy male beagles were fed a commercial kibble diet for 2 weeks, then administered metronidazole (20 mg/kg body weight twice a day) for 2 weeks. Fresh fecal samples were collected at weeks 2 and 4, stabilized in a 20% glycerol solution, and then frozen. For the in vitro fermentation experiment, feces from each time point (ABX-= pre-metronidazole collection; ABX+ = post-metronidazole collection) were thawed, diluted in an anaerobic diluting solution, and used to inoculate tubes. Tubes contained sterile medium and either cellulose, pectin, beet pulp, or chicory pulp fiber to test fermentation potential, with additional tubes used without fiber inclusion for blank corrections. At baseline (0 h) and after 6, 12, and 18 h of fermentation, pH, short-chain fatty acid (SCFA) production, and microbiota were measured. Data was analyzed within each fiber using the Mixed Models procedure of SAS version 9.4, with effects of antibiotic treatment, time and treatment*time interactions reported, accounting for the random effect across replicates. As expected, antibiotic administration had large effects on fiber fermentability characteristics, slowing pH reduction, lowering SCFA production, and altering SCFA molar ratios. Butyrate production was minimal among all fibers tested in ABX+ inocula tubes. Additionally, ABX+ inoculum lowered bacterial alpha diversity, affected bacterial beta diversity and the relative abundances of over 50 bacterial genera. Increased Bifidobacterium and Lactobacillus was observed in tubes containing ABX + inoculum (P < 0.0001) during pectin or beet pulp fermentation. Additionally, increased Faecalibacterium, Streptococcus, and Bacteroides was observed in tubes containing ABX- inoculum during chicory pulp fermentation (P < 0.0001). Beta diversity plots during beet pulp and chicory pulp fermentation demonstrated positive shifts toward ABX- inoculum tubes, but pectin fermentation did not yield the same shifts. The data presented here demonstrate that metronidazole administration can elicit unique responses to various fiber sources by reducing microbial diversity and negatively altering microbial fermentative activity (i.e., lower SCFA production). Both beet pulp and chicory pulp increased SCFA production and microbial diversity over time, with ABX+ inoculum tubes approaching that of ABX- inoculum tubes after 18 h of fermentation. More research is necessary but this data suggests that functional fibers promote microbial activity and recovery, and shed light on the potential effects of functional fibers in antibiotic-treated dogs.