Background Metronidazole has a substantial impact on the gut microbiome. However, the recovery of the microbiome after discontinuation of administration, and the metabolic consequences of such alterations have not been investigated to date. Objectives To describe the impact of 14‐day metronidazole administration, alone or in combination with a hydrolyzed protein diet, on fecal microbiome, metabolome, bile acids (BAs), and lactate production, and on serum metabolome in healthy dogs. Animals Twenty‐four healthy pet dogs. Methods Prospective, nonrandomized controlled study. Dogs fed various commercial diets were divided in 3 groups: control group (no intervention, G1); group receiving hydrolyzed protein diet, followed by metronidazole administration (G2); and group receiving metronidazole only (G3). Microbiome composition was evaluated with sequencing of 16S rRNA genes and quantitative polymerase chain reaction (qPCR)‐based dysbiosis index. Untargeted metabolomics analysis of fecal and serum samples was performed, followed by targeted assays for fecal BAs and lactate. Results No changes were observed in G1, or G2 during diet change. Metronidazole significantly changed microbiome composition in G2 and G3, including decreases in richness (P < .001) and in key bacteria such as Fusobacteria (q < 0.001) that did not fully resolve 4 weeks after metronidazole discontinuation. Fecal dysbiosis index was significantly increased (P < .001). Those changes were accompanied by increased fecal total lactate (P < .001), and decreased secondary BAs deoxycholic acid and lithocholic acid (P < .001). Conclusion and Clinical Importance Our results indicate a minimum 4‐week effect of metronidazole on fecal microbiome and metabolome, supporting a cautious approach to prescription of metronidazole in dogs.

The intestinal microbiota plays an important role in health and disease and produces, through fermentative reactions, several metabolic products, such as lactate, that can affect the host. The microbiota also interacts with and metabolizes compounds produced by the host, such as primary bile acids. Lactate and bile acids (BA) are of particular interest in gastrointestinal diseases because they have been associated with metabolic acidosis and bile acid diarrhea, respectively. The objectives of this study were to validate an enzymatic assay to quantify D-, L-, and total lactate in canine feces, and to characterize fecal lactate and BA concentrations as well as bacterial abundances in healthy dogs and dogs with gastrointestinal diseases. Fecal samples were collected from 34 healthy dogs, 15 dogs with chronic enteropathy (CE), and 36 dogs with exocrine pancreatic insufficiency (EPI). Lactate was quantified with an enzymatic assay, BA with gas chromatography-mass spectrometry, and 11 bacterial groups with qPCR. A fecal lactate reference interval was established from 34 healthy dogs and was 0.7–1.4 mM, 0.3–6.0 mM, and 1.0–7.0 mM for D-, L-, and total lactate, respectively. The assay to measure D-, L-, and total lactate in canine fecal samples was linear, accurate, precise, and reproducible. Significant increases in fecal lactate and decreases in secondary BA concentrations were observed in dogs with CE and dogs with EPI. Dogs with EPI had an increased abundance of Escherichia coli, Lactobacillus, and Bifidobacterium; a decreased abundance of Fusobacterium and Clostridium hiranonis; and a higher Dysbiosis Index when compared to healthy dogs. Further studies are necessary to determine the clinical utility of lactate and BA quantification in canine feces. These metabolites suggest functional alterations of intestinal dysbiosis and may become promising targets for further elucidating the role of the microbiota in health and disease.

Copyright © 2016. . © 2016 Blake and Suchodolski

Background The fecal microbiota, fecal bile acid concentrations, and abundance of Clostridium perfringens and Clostridium difficile are altered in acute and chronic gastrointestinal disease in adult dogs. However, less is known in young puppies. Hypothesis/Objectives To determine composition of the fecal microbiota, assess development of fecal bile acid profiles, and determine the abundance of Clostridial species in puppies, young adult dogs, and adult dogs. Animals Healthy puppies from a whelping kennel (n = 53) and healthy client‐owned dogs <1 year old (n = 20) were separated into 6 age groups, then compared to client‐owned dogs over 1 year of age (n = 13). Methods Prospective observational study. Naturally voided fecal samples were analyzed by quantitative polymerase chain reaction to measure bacterial abundances. Fecal bile acids were quantified using gas chromatography‐mass spectrometry. Results Puppies up to 5 to 6 weeks of age had increased Dysbiosis Index (median [min‐max]: 5.39 [1.32‐8.6], P < .001), increased abundance of C. difficile (4.1 [0.01‐4.85] log DNA, P < .001), decreased secondary bile acid concentrations (0.61 [0.28‐5.06] μg/mg, P = .006), and decreased abundance of C. hiranonis (0.84 [0.01‐6.71], P = .005) compared to adult dogs (−4.62 [−8.36 to −0.61], 0.01 [0.01‐0.01], 4.12 [0.32‐8.94], and 6.02 [5.06‐7.00], respectively). Secondary bile acid concentration positively correlated with C. hiranonis abundance (ρ = 0.77; P < .001). Conclusions and Clinical Importance The increase in secondary bile acids and simultaneous decrease of C. difficile and C. perfringens after 5 to 6 weeks of age warrants further investigation into regulatory impacts that secondary bile acids could have on clostridial species in dogs.

The long-term impact of treatment of dogs with steroid-responsive enteropathy (SRE) on the fecal microbiome and metabolome has not been investigated. Therefore, this study aimed to evaluate the fecal microbiome and metabolome of dogs with SRE before, during, and following treatment with standard immunosuppressive therapy and an elimination diet. We retrospectively selected samples from 9 dogs with SRE enrolled in a previous clinical trial, which received treatment for 8 weeks, and had achieved remission as indicated by the post-treatment clinical scores. Long-term (1 year) samples were obtained from a subset (5/9) of dogs. Samples from 13 healthy dogs were included as controls (HC). We evaluated the microbiome using 16S rRNA sequencing and qPCR. To evaluate the recovery of gut function, we measured fecal metabolites using an untargeted approach. While improvement was observed for some bacterial taxa after 8 weeks of treatment, several bacterial taxa remained significantly different from HC. Seventy-five metabolites were altered in dogs with SRE, including increased fecal amino acids and vitamins, suggesting malabsorption as a component of SRE. One year after treatment, however, all bacterial species were evaluated by qPCR and 16S rRNA gene sequencing, and all but thirteen metabolites were no longer different from healthy controls.

Background: In previous studies, only a subset of dogs with chronic enteropathy (CE) had an increased dysbiosis index (DI) or altered fecal metabolites or both, suggesting differences in underlying intestinal pathophysiology between these subsets. Objectives: To compare microbial functional genes and fecal metabolites between healthy dogs with DI < 0 (HC) and dogs with CE and DI > 0 (increased DI-CE) or DI < 0 (normal DI-CE). Animals: Retrospective cross-sectional study including 78 HC and 138 CE dogs. Methods: Fecal microbiome was assessed by DNA shotgun sequencing. Dysbiosis index was quantified by qPCR. Targeted fecal metabolites, long-chain fatty acids, sterols, bile acids (BAs), and carbohydrates were measured using gas chromatography–mass spectrometry (GC–MS). Results: In permutational analysis of variance (PERMANOVA), functional gene profiles showed larger shifts in increased DI-CE (median R2 [95% confidence interval (CI)] = 0.12 [0.08–0.17]) than normal DI-CE (0.02 [0.01–0.04]) compared with HC (adjusted-p < 0.02), characterized by increased counts of carbohydrate and lipid degradation genes. Similarly, increased DI-CE (PERMANOVA, median R2 [95% CI] = 0.23 [0.14–0.34]) had larger shifts in fecal metabolome than normal DI-CE (0.10 [0.04–0.20]; adjusted-p < 0.02). Increased DI-CE had lower fecal unconjugated secondary BAs percentage (95% CI; HC, 88.4%–96.4%; normal DI-CE, 79.8%–99.0%; increased DI-CE, 28.1%–64.1%) and transporter-independent carbohydrates (combined ribose, xylose, rhamnose, and arabinose) concentrations (1.6–2.6; 0.7–1.8; 0.3–1.3 ng/mg; adjusted-p < 0.01). Conclusions: Results indicate differences in fecal microbial gene profiles and metabolome in increased DI-CE versus normal DI-CE and HC, suggesting dogs with an increased DI have more severe intestinal changes in metabolic functions.