Sara E. Martini

Background/Objectives: Canine athletes have a higher energy requirement and are more susceptible to nutrient depletion, electrolyte imbalance, and metabolic stress than sedentary pets. The objective of this study was to characterize the plasma metabolome of American Foxhound dogs following a bout of unstructured exercise. Methods: Thirty-nine adult American Foxhound dogs (32 intact males, 7 spayed females; age: 6.2 ± 3.1 yr; BW: 36.3 ± 5.3 kg) were allotted to a standard performance diet (CTRL) or NUTRO^® Natural Choice^® Adult High Endurance Formula (TEST). After 80 d in the study, blood samples were collected prior to (0 h), and 3 h and 25 h post-exercise (average: 17.7 km run over 2–3 h). Plasma samples of the 10 top performers of each treatment group were analyzed for untargeted metabolite profiling. Results: Of the 566 named metabolites identified, >200 and >185 metabolites were impacted (p < 0.05) by exercise and diet, respectively. Principal component analysis indicated distinct clustering by diet. Random forest analysis highlighted several metabolites having a high degree of predictive accuracy based on diet and exercise, with most related to amino acid, lipid, xenobiotic, and cofactor and vitamin metabolism. Relating to exercise, glycolytic end-products and citric acid cycle intermediates were increased at 3 h post-exercise. Similarly, tocopherols and omega-3 polyunsaturated fatty acids were higher in dogs fed TEST than those fed CTRL during recovery, indicating a lower oxidative stress and anti-inflammatory response. Conclusions: Overall, the data suggest a protective effect (lower susceptibility to oxidative stress and muscle fatigue) of feeding a nutrient-fortified diet for dogs undergoing unstructured exercise.

Antibiotics may be used for gastrointestinal enteropathies but research has demonstrated significant microbiota dysmetabolism, fermentation pattern alterations, and prolonged dysbiosis following treatment. The objective of this study was to determine how dietary fiber or fecal microbial transplant (FMT) treatments impacted the fecal characteristics, metabolite concentrations, and microbiota populations of cats treated with metronidazole. Twenty-five healthy adult cats (6.75 ± 1.20 yr) were fed a commercial kibble diet for 2 wk, administered metronidazole (20 mg/kg BW BID) for 2 wk, then monitored for 4 wk. Cats were allotted to one of three interventions (diet, diet + beet pulp, diet + FMT) for 1 wk, interventions ceased, then recovery was monitored for 4 wk. Fresh fecal samples were collected at the end of each phase and at the mid-points of recovery. As anticipated, metronidazole increased fecal scores and moisture (p < 0.05), reduced fecal bacterial alpha diversity (p < 0.0001), and reduced fecal metabolite concentrations. Few treatment effects were detected, with antibiotic recovery contributing to many of the results observed. Dysbiosis was persistent throughout the study, with 4/25 cats still demonstrating mild dysbiosis after 9 wk. Overall, dietary or FMT treatments may aid in accelerated antibiotic recovery in cats but further research is needed to refine treatments for greater efficacy.

Antibiotics are commonly used to aid in the remission of gastrointestinal diseases, but usage may lead to prolonged dysbiosis. The objective of this study was to evaluate the effects of metronidazole on fecal microbiota, fecal metabolites, and serum bile acids and uremic toxins of healthy adult cats. Twelve healthy adult cats (4.7 ± 0.4 yr) received metronidazole (20 mg/kg BW PO BID) for 14 days (day 0–14) and were monitored during a 28-day recovery period (day 15–42). Fecal and blood samples were collected at baseline (day 0), after metronidazole (day 14), and weekly during recovery (on days 21, 28, 35, and 42). Fecal samples were analyzed for microbiota and bacterial metabolites. Serum samples were analyzed for bile acids and uremic toxins. Metronidazole increased dysbiosis index and fecal lactate concentrations (p < 0.0001) and decreased fecal propionate, butyrate, and secondary bile acid concentrations (p < 0.0001) for up to 28 days. Prolonged dysbiosis and Peptacetobacter (Clostridium) hiranonis reductions were observed in 10/12 (83%) cats. Serum uremic toxins were also reduced (p < 0.0001) after metronidazole administration. The observed changes after metronidazole administration illustrate how changes in the gut microbiome alter microbial metabolism and its relation to host dysmetabolism. In conclusion, metronidazole is a potent antibiotic with persistent effects observed in the microbiome and metabolome, even up to one month after administration.

Antibiotics are commonly used to aid in the remission of gastrointestinal diseases, but usage may lead to prolonged dysbiosis. The objective of this study was to evaluate the effects of metronidazole on fecal microbiota, fecal metabolites, and serum bile acids and uremic toxins of healthy adult cats. Twelve healthy adult cats (4.7±0.4 yr) received metronidazole (20 mg/kg BW PO BID) for 14 days (day 0–14) and were monitored during a 28-day recovery period (day 15–42). Fecal and blood samples were collected at baseline (day 0), after metronidazole (day 14), and weekly during recovery (days 21, 28, 35, and 42). Fecal samples were analyzed for microbiota and bacterial metabolites. Serum samples were analyzed for bile acids and uremic toxins. Metronidazole increased dysbiosis index and fecal lactate concentrations (P<0.0001), and decreased fecal propionate, butyrate, and secondary bile acid concentrations (P<0.0001) for up to 28 days. Prolonged dysbiosis and Peptacetobacter (Clostridium) hiranonis reductions were observed in 10/12 (83%) cats. Serum uremic toxins were also reduced (P<0.0001) after metronidazole administration. The observed changes after metronidazole administration illustrate how changes in the gut microbiome alter microbial metabolism and its relation to host dysmetabolism. In conclusion, metronidazole is a potent antibiotic with persistent effects observed in the microbiome and metabolome, even up to one month after administration.

Antibiotics are known to cause loose stools, disrupt the fecal microbiota, and alter fecal bile acid (BA) profiles of dogs. Recovery may be aided by diet, but little research has been conducted. The objective of this study was to determine how a veterinary low-fat diet affected the fecal characteristics, metabolites, BA, and microbiota of dogs receiving antibiotics. Twenty-four healthy adult dogs [7.38 ± 1.95 yr; 7.67 ± 0.76 kg body weight (BW)] were used in an 8-wk completely randomized design study. During a 2-wk baseline, all dogs were fed a leading grocery brand diet (GBD). Over the next 2 wk, dogs were fed GBD and received metronidazole orally (20 mg/kg BW twice daily). At week 4, dogs were randomly allotted to one of two treatments [GBD or Blue Buffalo Natural Veterinary Diet GI Gastrointestinal Support Low-Fat (BB)] and fed for 4 wk. Fecal scores were recorded daily and fresh fecal samples were collected at weeks 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter content, and metabolite and BA concentrations. Fecal microbiota populations were analyzed using 16S rRNA gene amplicon sequencing and qPCR-based dysbiosis index (DI). All data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, testing for effects of treatment, time, and treatment*time and significance set at P < 0.05. Metronidazole increased (P < 0.0001) fecal scores (looser stools), reduced fecal short-chain fatty acid, branched-chain fatty acid, phenol, and indole concentrations, increased primary BA concentrations, and decreased secondary BA concentrations. Metronidazole also reduced fecal bacterial alpha diversity, altered the abundance of 58 bacterial genera, and increased DI. During antibiotic recovery, changes in fecal pH, dry matter percentage, and metabolite and immunoglobulin A concentrations were altered (P < 0.05) by diet. Fecal BA concentrations recovered quickly for all dogs. Change in lithocholic acid was affected (P < 0.0001) by diet, but other BA were not. Recovery of over 25 bacterial genera was impacted by diet (P < 0.05). While many bacterial taxa returned to baseline levels after 4 wk, others did not fully recover. DI and bacterial alpha diversity measures recovered quickly for all dogs but were not impacted by diet. In conclusion, metronidazole drastically altered the fecal microbiota and metabolites of dogs. While most variables returned to baseline by week 8, diet may be used to aid in recovery.

Antibiotics are used to treat gastrointestinal diseases or infections but are known to negatively affect stool quality and gut microbiota in cats and dogs. Therefore, identifying dietary strategies that may aid in antibiotic recovery is of interest. The objective of this study was to determine how a veterinary gastrointestinal diet affected the fecal characteristics, microbiota, and metabolite and bile acid (BA) concentrations of cats recovering from metronidazole administration. Twenty-four healthy adult cats were used in an 8-wk completely randomized design study. During a 2-wk baseline, all cats consumed a leading grocery brand diet (GBD). Over the next 2 wk, cats consumed GBD and received metronidazole (20 mg/kg body weight twice daily). At week 4, cats were randomly allotted to one of 2 treatments [GBD; BLUE Natural Veterinary Diet GI Gastrointestinal Support (BB)] and fed for 4 wk. Fecal scores were recorded daily and fresh fecal samples were collected at weeks 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter (DM) %, metabolites, and microbiota. Microbiota was analyzed by 16S rRNA gene sequencing and qPCR, which was used to calculate dysbiosis index. Data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, testing for effects of diet, time and diet*time. Metronidazole had dramatic effects on all outcomes, including increased fecal scores (looser stools), reduced fecal pH and DM%, reduced fecal short-chain fatty acid, branched-chain fatty acid, ammonia, phenol, and indole concentrations, and altered fecal BA concentrations (increased primary BA; reduced secondary BA). Metronidazole reduced fecal bacterial alpha diversity, increased dysbiosis index, and altered the relative abundance of 78 bacterial genera. Fecal outcomes partially recovered over the next 4 wk, with some being impacted by diet. Fecal acetate concentrations were higher after metronidazole in cats fed BB. Dysbiosis index and alpha diversity measures slowly recovered over 4 wk, without diet differences. Recovery of 16 bacterial genera was impacted by diet. Fecal BA profiles demonstrated a prolonged impairment of primary to secondary BA conversion, with cholic acid being lower after metronidazole in cats fed BB. In conclusion, our data demonstrate that metronidazole is a powerful antibiotic that has long-lasting effects on the fecal microbiota and metabolites of cats. Outcome variables slowly recovered over time, but a gastrointestinal diet may aid in recovery.

DNA shotgun sequencing is an untargeted approach for identifying changes in relative abundances, while qPCR allows reproducible quantification of specific bacteria. The canine dysbiosis index (DI) assesses the canine fecal microbiota by using a mathematical algorithm based on qPCR results. We evaluated the correlation between qPCR and shotgun sequencing using fecal samples from 296 dogs with different clinical phenotypes. While significant correlations were found between qPCR and sequencing, certain taxa were only detectable by qPCR and not by sequencing. Based on sequencing, less than 2% of bacterial species (17/1190) were consistently present in all healthy dogs (n = 76). Dogs with an abnormal DI had lower alpha-diversity compared to dogs with normal DI. Increases in the DI correctly predicted the gradual shifts in microbiota observed by sequencing: minor changes (R = 0.19, DI < 0 with any targeted taxa outside the reference interval, RI), mild-moderate changes (R = 0.24, 0 < DI < 2), and significant dysbiosis (R = 0.54, 0.73, and 0.91 for DI > 2, DI > 5, and DI > 8, respectively), compared to dogs with a normal DI (DI < 0, all targets within the RI), as higher R-values indicated larger dissimilarities. In conclusion, the qPCR-based DI is an effective indicator of overall microbiota shifts observed by shotgun sequencing in dogs.