The effect of phenobarbital treatment on behavioral comorbidities and on the composition and function of the fecal microbiome in dogs with idiopathic epilepsy

Abstract

Phenobarbital (PB) is one of the most important antiseizure drugs (ASDs) to treat canine idiopathic epilepsy (IE). The effect of PB on the taxonomic changes in gastrointestinal microbiota (GIM) and their functions is less known, which may explain parts of its pharmacokinetic and pharmacodynamic properties, especially its antiseizure effect and drug responsiveness or drug resistance as well as its effect on behavioral comorbidities. Fecal samples of 12 dogs with IE were collected prior to the initiation of PB treatment and 90 days after oral PB treatment. The fecal samples were analyzed using shallow DNA shotgun sequencing, real-time polymerase chain reaction (qPCR)-based dysbiosis index (DI), and quantification of short-chain fatty acids (SCFAs). Behavioral comorbidities were evaluated using standardized online questionnaires, namely, a canine behavioral assessment and research questionnaire (cBARQ), canine cognitive dysfunction rating scale (CCDR), and an attention deficit hyperactivity disorder (ADHD) questionnaire. The results revealed no significant changes in alpha and beta diversity or in the DI, whereas only the abundance of Clostridiales was significantly decreased after PB treatment. Fecal SCFA measurement showed a significant increase in total fecal SCFA concentration and the concentrations of propionate and butyrate, while acetate concentrations revealed an upward trend after 90 days of treatment. In addition, the PB-Responder (PB-R) group had significantly higher butyrate levels compared to the PB-Non-Responder (PB-NR) group. Metagenomics of functional pathway genes demonstrated a significant increase in genes in trehalose biosynthesis, ribosomal synthesis, and gluconeogenesis, but a decrease in V-ATPase-related oxidative phosphorylation. For behavioral assessment, cBARQ analysis showed improvement in stranger-directed fear, non-social fear, and trainability, while there were no differences in ADHD-like behavior and canine cognitive dysfunction (CCD) scores after 90 days of PB treatment. While only very minor shifts in bacterial taxonomy were detected, the higher SCFA concentrations after PB treatment could be one of the key differences between PB-R and PB-NR. These results suggest functional changes in GIM in canine IE treatment.

Keywords: behavioral comorbidities; butyrate; canine idiopathic epilepsy; gastrointestinal microbiota; phenobarbital; short-chain fatty acids.

Figure 1

(A–C) Comparison of alpha diversity parameters in box and whiskers plots. (A) Species number, (B) Shannon–Wiener index, and (C) Pielou's evenness demonstrated no significant changes between pre-phenobarbital treatment (D0) and 90-day post-treatment (D90). Lines in the boxes represent the median of each group and the boxes represent the interquartile range, whereas the whiskers represent the minimum and maximum data. (D) Rarefaction curve based on the number of observed operational taxonomic units (OTUs). The red circles and blue triangles represent the mean of sequences per sample at D0 and D90, respectively. The error bars represent standard deviations.

Figure 2

Principal coordinates analysis (PCoA) of weighted UniFrac distances of taxa diagrams demonstrating beta diversity (A) between the day prior to phenobarbital (PB) treatment (D0; red) and 90 days post-PB treatment (D90; blue), and (B) between PB-R group (light blue) and PB-NR group (dark blue) at D90. There was no significant microbiome clustering. (C) Changes in PCoA weighted UniFrac distances of taxa after PB treatment. The arrows show the direction from D0 (arrow tail) to D90 (arrow head) of each dog.

Figure 3

Relative abundance of each order prior to phenobarbital treatment (D0) and 90 days post-treatment (D90) in fecal samples of each dog (n = 12).

Figure 4

Significant decrease in order Clostridiales in percentage after 90 days of phenobarbital treatment (D90) compared to pre-treatment (D0). Lines in dark and light gray boxes represent the median at D0 and D90, respectively. The box limits indicate the interquartile range, while the whiskers represent the minimum and maximum data. *P < 0.05 (one-tailed Wilcoxon matched-paired signed-ranks test).

Figure 5

(A) The total short-chain fatty acids (SCFAs), (C) propionate, and (D) butyrate levels in fecal samples increased significantly when comparing 90 days after phenobarbital (PB) treatment (D90) to the day before PB treatment (D0). (B) The graph shows an upward trend of acetate at D90 compared to D0. *P < 0.05 (one-tailed Wilcoxon matched-paired signed-ranks test). (E) Butyrate level at D90 differed between PB-R and PB-NR. *P < 0.05 (one-tailed Mann–Whitney U-test). The lines in the boxes indicate a median value and the box limits represent the interquartile range. The whiskers indicate minimum and maximum data.