ABSTRACT The bile acid-converting Peptacetobacter hiranonis is a Gram-positive, anaerobic, potentially spore-forming bacterium. It was first isolated from human feces and was subsequently shown to convert bile acids (BA) in both in vitro and in vivo experiments. The conversion of BA relies on the presence of the 7α-dehydroxylation multi-step pathway, encoded by the BA-inducible (bai) operon, harbored by P. hiranonis . In companion animals, P. hiranonis has been characterized as a biomarker for intestinal health, with its loss associated with dysbiosis. However, characterization of P. hiranonis cultured from companion animals is limited. An in-depth characterization of P. hiranonis was published by Chen et al. recently, including the proposal of a new species, Peptacetobacter hominis . We have sequenced the whole genome of both canine- and feline-derived strains of P. hiranonis , characterized these strains biochemically, and assessed their in vitro BA-converting ability as well as their antimicrobial resistance profiles. The strains described here can convert primary into secondary BAs and are whole-genome inhibited by low concentrations of amoxicillin-clavulanate, cefepime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, and metronidazole. Based on whole genome analysis, we propose dividing P. hiranonis into two host-adapted subspecies: P. hiranonis subsp. deconjugans and P. hiranonis subsp. nondeconjugans , based on their genomic differences and divergent ability to deconjugate BAs – a function that appears widely distributed among P. hiranonis strains cultured from dogs, but absent from those cultured from cats. Taken together, our results confirmed the BA conversion ability of P. hiranonis cultured from dogs and cats and reveal host-associated genomic and functional differences within the species.

Objective:To evaluate the effect of cholestyramine on the fecal dysbiosis index (DI) and the fecal excretion of conjugated and unconjugated bile acids (BAs) in dogs. Methods:This was a prospective, pre-post experimental study. Individually housed research colony dogs were screened for inclusion. Included dogs were deemed healthy based on a lack of gastrointestinal signs and evaluation of annual blood work and fecal flotation. Cholestyramine was orally administered (53 to 75 mg/kg/d of active drug) to healthy dogs for 12 weeks. Dogs were maintained on the same maintenance diet for the entirety of the study. Feces were collected at 5 time points: 12 and 6 weeks prior to treatment, time 0, and 6 and 12 weeks during treatment. Repeated-measures 1-way ANOVAs were used to test differences over time for fecal conjugated and unconjugated BAs (time 0 and weeks 6 and 12 of treatment) and the fecal DI (all time points), including its individual bacterial taxa. Results:12 dogs completed the study. No adverse events following cholestyramine treatment were observed. The DI was significantly lower following cholestyramine treatment compared to before treatment and was not outside of the reference range. Cholestyramine administration significantly increased total fecal BAs, which was largely driven by a significant increase in secondary unconjugated BAs. Conclusions:Cholestyramine increased the total fecal BA excretion in colony dogs and had little to no clinical effect on the DI. Clinical Relevance:These findings support the investigation of the use of cholestyramine in controlled clinical trials of dogs with suspected BA diarrhea.

OBJECTIVE To determine breed-specific reference intervals for whole blood (WB) and plasma taurine concentrations in adult, overtly healthy Cavalier King Charles Spaniels (CKCSs) and determine whether taurine concentrations differ across preclinical myxomatous mitral valve disease (MMVD) stages or between CKCSs eating diets that meet World Small Animal Veterinary Association (WSAVA) nutritional guidelines versus other diets. ANIMALS 200 privately owned CKCSs. PROCEDURES Clinically healthy adult CKCSs were recruited prospectively. Diet and supplement history was collected. Dogs were staged by echocardiography using MMVD consensus guidelines. Taurine concentrations were measured in deproteinized lithium heparin WB and plasma samples with the postcolumn ninhydrin derivatization method on a dedicated amino acid analyzer. RESULTS There were 12 stage A (6%), 150 stage B1 (75%), and 38 stage B2 (19%) CKCSs. Seventy-eight dogs (39%) were reported by their owners to be eating diets meeting WSAVA nutritional guidelines; 116 (58%) were not. Taurine concentrations in plasma ( P = .444) and WB ( P = .073) were not significantly different across MMVD stages or between CKCSs eating diets meeting WSAVA nutritional guidelines versus other diets ( P = .345 and P = .527, respectively). Reference intervals for WB taurine (152 to 373 µM) and plasma taurine (51 to 217 µM) concentrations in CKCSs were generated. CLINICAL RELEVANCE In CKCSs, taurine concentrations do not differ significantly based on preclinical MMVD stage, nor do they differ significantly based on consumption of a diet that does or does not meet WSAVA nutritional guidelines.

Amino acids play an important role in metabolism. Comprehensive analytical validation of an assay for the concurrent measurement of a large number of amino acids in dogs is lacking, which precludes its usefulness in a clinical setting. Amino acids are often measured in plasma or whole blood. However, serum is commonly used for gastrointestinal diagnostic testing in dogs and is therefore convenient to use. This study aimed to analytically validate an assay for the concurrent measurement of amino acids in dog serum and to evaluate differences in amino acid concentrations in whole blood, plasma, and serum in dogs. Analytical validation of the assay (Biochrom 30+ Amino Acid Analyzer) was performed on fresh or banked serum samples from dogs. Whole blood, plasma, and serum from 36 healthy dogs were analyzed, and concentrations of the three sample types were compared. The assay was demonstrated to be precise, reproducible, accurate, linear, and stable for the measurement of the majority of compounds detected in dog serum. Cystine, glutamic acid, and ethanolamine were shown to be unstable at conditions commonly encountered in clinical settings. Significant differences in concentrations were identified between whole blood, plasma, and serum for 33 of 42 compounds. Amino acid profiles in serum and plasma were more similar to each other than to those in whole blood. While some amino acids are present in similar concentrations in whole blood, plasma, and serum, others are highly dependent on the type of biofluid, and measurements warrant strict adherence to sample type-based reference intervals.

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: The fecal microbiota, fecal bile acid concentrations, and abundance ofClostridium perfringensandClostridium difficileare 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 ofC. difficile(4.1 [0.01-4.85] log DNA,P < .001), decreased secondary bile acid concentrations (0.61 [0.28-5.06] mu g/mg,P= .006), and decreased abundance ofC. 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 withC.hiranonisabundance (rho = 0.77;P < .001). Conclusions and Clinical Importance: The increase in secondary bile acids and simultaneous decrease ofC. difficileandC. perfringensafter 5 to 6 weeks of age warrants further investigation into regulatory impacts that secondary bile acids could have on clostridial species in dogs.

The aim was to characterize differences in fecal consistency, and fecal microbiota and metabolome profiles in dogs with acute diarrhea (AD) treated with either fecal microbiota transplantation as enema (FMT; n = 11) or oral metronidazole (MET; n = 7) for 7 days. On days 0, 7, and 28 fecal samples were obtained. Fecal samples from healthy dogs (HC; n = 14) were used for comparison. Samples were analyzed by the previously validated qPCR based canine Dysbiosis Index (DI; increased values indicate microbiota dysbiosis) and 16S rRNA gene sequencing. The fecal metabolome was analyzed using a previously validated targeted canine assay for fecal unconjugated bile acids, and untargeted metabolomics. Fecal consistency improved significantly in dogs treated with FMT and MET by day 7 and day 28 (p < 0.01) compared to day 0. However, on day 28 fecal consistency was significantly better in FMT compared to MET (p = 0.040). At day 0, dogs with AD had an altered microbiota indicated by significantly increased DI, decreased alpha-diversity, and altered beta-diversity. In the FMT group, the DI decreased over time, while MET led to a significant increase in the dysbiosis index at day 7 and 28 compared to FMT. Sequencing data revealed that in FMT microbial diversity and beta-diversity was similar to HC at day 28, while in MET these parameters were still significantly different from HC. In dogs treated with FMT, a decrease in cholic acid and the percentage of primary bile acids was observed, whereas treatment with metronidazole led to an increase in cholic acid at day 7 and an increase in percentage of primary bile acids over time. Based on untargeted metabolomics, dogs with AD had an altered fecal metabolome compared to HC. Dogs treated with FMT clustered closer to HC at day 28, while dogs treated with MET did not. In this pilot study, dogs with AD had significant differences in fecal microbiota and metabolome profiles. Dogs treated with MET still had altered microbial and metabolic profiles at day 28 compared to dogs treated with FMT or healthy dogs.

Abstract 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.

Abstract Background Tylosin is commonly prescribed to dogs with diarrhea. Orally administered antibiotics may alter the intestinal microbiota, which is responsible for crucial key bile acid (BA) biotransformation reactions. Objectives To prospectively evaluate the impact of tylosin administration on fecal microbiota and unconjugated bile acids (UBAs) over time. Animals Sixteen healthy adult dogs. Methods Prospective, randomized controlled clinical trial. Dogs were randomized to receive 20 mg/kg of tylosin or a placebo capsule PO q12h for 7 days while undergoing daily fecal scoring. Fecal samples were collected on days 0, 7, 21, and 63. The microbiota was assessed using quantitative PCR and 16S rRNA gene sequencing. Unconjugated BAs were assessed using gas chromatography‐mass spectrometry (GC‐MS). Results Fecal scores were unchanged during placebo and tylosin administration. In the placebo group, no significant changes were observed in fecal microbiota or UBA concentrations. Day 7 samples from tylosin‐exposed dogs exhibited decreased bacterial diversity (observed species, Chao1, Shannon, P < .001) characterized by decreases in anaerobes Fusobacteriaceae (linear discriminant analysis [LDA] score, 5.03) and Veillonellaceae (LDA score, 4.85). Primary UBA concentrations were increased at day 21 (median, [range]; 7.42, [0.67‐18.77] μg/kg; P = .04) and day 63 (3.49 [0‐28.43] μg/kg; P = .02) compared to day 0 (.14 [.03‐1.19] μg/kg) in dogs receiving tylosin. At day 63, bacterial taxa were not significantly different compared to day 0, but the extent of microbial recovery was individualized. Conclusions and Clinical Importance Tylosin causes fecal dysbiosis in healthy dogs with corresponding shifts in fecal UBAs. Changes did not uniformly resolve after discontinuation of tylosin.

Plant proteins are oft thought to confer health benefits over animal proteins. In a prior study, lipoprotein profiles improved and body weight gain reduced in mice fed milk protein rather than soy protein fed in a purified diet (PD) matrix. Non‐nutrient components such as fiber can alter macro‐ and micro‐nutrient utilization; this study tested the effect of dietary matrix on lipoprotein profiles, fecal lipids and bile acids. Weanling C57Bl/6 mice (n=15/diet) were fed either PD containing isolated soy protein (ISP) or dried whole milk powder (DWMP) as the protein source, or natural ingredient diets (NID) containing soybean meal (SPC), or dried whole milk powder (DMC) as protein sources in a 14 wk feeding trial. Diets provided 50%EN CHO, 20% PRO, 30% FAT. ISP & DWMP provided 14% neutral detergent fiber primarily from cellulose, while SPC & DMC contained 11.4% and 8.4% NDF, respectively, from wheat mids, corn, corn gluten meal and soybean meal (SPC only). Terminal measures: bodyweight, lipoprotein density distribution (https://doi.org/10.1177/1040638718793677), and fecal fatty acids (FA), sterols, primary (PBA) and secondary (SBA) bile acids by IS‐controlled GC/MS. Values are means with differences tested by one‐way ANOVA and Tukey's HSD. Energy intake was similar among groupsaveraging 12 kcal/day. Diet Weight, g Fatty acids mg/mg feces Fecal content / 48 hours Body, final Feces, 48h Sterols, mg (%phyto‐) Bile acids, ug Primary Secondary ISP 35.0a 1.14a 17.6b 2.66c(52.6%) 378b 426a DWMP 31.9ab 1.22a 14.8b 1.87d(31.7%) 510ab 370ab SPC 28.8b 0.88b 39.7a 4.82a(68.6%) 398b 295bc DMC 29.7b 0.66c 31.1a 3.69b(60.1%) 551a 263c Mice fed ISP & DWMP were heavier than SPC & DMC fed mice (p<0.001). ISP‐fed mice were heaviest (p<0.0002 vs. SPC & DMC). SPC & DMC excreted 46.0 ug fecal FA/mg feces, 3‐fold greater than the 13.5 ug fecal FA/mg feces excreted by ISP&DWMP (p<0.000). Total fecal FA excretion in 48h was ≤ 1 kcal; unable to account for differences in body weight. Total fecal sterol excretion was 1.9‐fold greater in NID vs PD, p<0.0001. Phytosterols comprised a greater fraction of fecal sterols in NID that in PD; soy diets (ISP & SPC) excreted 2.5 and 1.5‐fold more than dairy protein comparators. Zoosterols were increased 17% in NID fed mice (1.49 mg avg) compared to PD mice (1.27 mg avg) and unaffected by phytosterols. Bile acid excretion in DWMP was 9% greater than ISP, and DMC was 17% greater than SPC. Secondary bile acids avg 22.4% lower in mice fed milk‐based diets compared to soy. When expressed as % of total, PBAs were highest in NIDs (61.0 vs 50.7%, p<0.0002), whereas SBAs were lower compared to purified diets (38.3 vs 48.4%, p<0.0002). Compared to all other diets, mice fed ISP had a 30% increase in small, dense LDL (d=1.05 – 1.063 g/mL) (p<0.000). Carbohydrate refinement has a greater effect fecal lipids than protein source. Milk protein‐containing diets resulted in improved lipoprotein profiles in PD and NID diets.Support or Funding InformationSupported in part by Texas A&M AgriLife Research project 8738 (to R.L.W.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Background Intestinal absorption of bile acids is mediated by the apical sodium‐dependent bile acid transporter (ASBT). Fecal bile acid dysmetabolism has been reported in dogs with chronic inflammatory enteropathy (CIE). Objective Characterization of ASBT distribution along the intestinal tract of control dogs and comparison to dogs with CIE. Animals Twenty‐four dogs with CIE and 11 control dogs. Methods The ASBT mRNA and protein expression were assessed using RNA in situ hybridization and immunohistochemistry, respectively. The concentrations of fecal bile acids were measured by gas chromatography‐mass spectrometry. The fecal microbiota dysbiosis index was assessed with a quantitative polymerase chain reaction panel. Results In control dogs, ASBT mRNA expression was observed in enterocytes in all analyzed intestinal segments, with highest expression in the ileum. The ASBT protein expression was restricted to enterocytes in the ileum, cecum, and colon. Dogs with CIE had significantly decreased expression of ASBT protein in the ileum ( P = .001), which was negatively correlated with histopathological score (ρ = −0.40; P corr = .049). Additionally, dogs with CIE had a significantly increased percentage of primary bile acids in feces compared to controls ( P = .04). The fecal dysbiosis index was significantly higher in dogs with CIE than in control dogs ( P = .01). Conclusions and Clinical Importance These findings indicate that ileal protein expression of ASBT is downregulated in dogs with CIE. This change may be linked to the inflammatory process, intestinal dysbiosis, and fecal bile acid dysmetabolism observed in these patients.

increased after treatment (p < 0.05).Before the treatment, valerate levels in TGD300 and TGD900 groups were significantly lower than in placebo group (p < 0.05).Following TGD900 therapy, fecal acetate and valerate levels were significantly increased, while succinate and lactate were significantly reduced (p < 0.05).Total SCFAs were marginally increased after this treatment (p = 0.09).Formate and valerate levels were significantly increased after TGD300 treatment; however, significant increase of the former was also observed in the placebo group (p < 0.05).No significant differences were observed in the control, BMs-C (mostly constipation), BMs-D (mostly diarrhea), and BMs-M (mixed type, constipation and diarrhea) groups.Clostridium cluster IV, Clostridium subcluster XIVa, Clostridium cluster XVIII and fecal pH increased significantly and order Lactobacillales decreased in patients with BMD compared with control.TGD treatment significantly improved BMs compared with placebo treatment (46.2%, 95% confidence interval (CI): 19.2-74.9% vs. 0%, 95% CI: 0-33.6%, p < 0.05).This effect was not observed in patients without BMD.Conclusion: Patients with BMD suffer from gut dysbiosis.TGD treatment alleviates BMD symptoms in T2DM patients by increasing fecal acetate level.

of a patient or linked to inflammation is unknown.We investigated if these genetic risk variants associate to intestinal microbiota composition differences.Methods: Fecal and blood samples were collected from 30 CD patients from whom Immunochip data were available.A genetic risk score (GRS) was calculated for each patient, taking into account the risk allele frequency and odds ratio of each single nucleotide polymorfism (SNP).The GRS associated with CD was calculated with 197 SNPs available on the Immunochip.Autophagy, ER stress and NOD2 associated GRS were also generated.Patients were divided into 4 groups according to quartiles of the general GRS.16S rDNA paired-end sequencing targeting the V4 hypervariable region was performed using Illumina MiSeq sequencer.Sequencing depth was downsized to 10000 reads/sample.The Ribosomal Database Project classifier was used for taxonomic assignment.Statistical analyses were performed with R package phyloseq, using parametric and non-parametric tests, with multiple testing correction (FDR).Correlation between genera abundances and genetic risk scores was performed with Spearman correlation.Results: The microbiota richness (alpha diversity) and overall microbiota composition were not significantly different between patients belonging to Q1 (n=8) or Q4 (n=8) of their GRS.At genus level, no differences were observed.When looking specifically to particular pathways, we observed that microbiota richness (anova p value 0.045) and community composition (Bray-Curtis dissimilarity adonis p value 0.03) differed according to autophagy GRS.However, no significant taxon abundance differences were observed at phylum, genus or operational taxonomic unit (OTU) level.Noteworthy, some differences at genus level (eg.Anaerostipes, Roseburia and Megamonas) were observed between the different groups of autophagy GRS before multiple testing correction.Conclusion: Host-genetics seem to influence the intestinal microbiota composition through pathways associated with host-microbiome interactions, particularly autophagy.However, this influence is small.Larger sample size may be needed to detect small differences at genus level.Environmental factors seem to have a larger impact on gut microbiota than host-genetics.

Molecular tools for bacterial identification and metagenomics, and mass spectroscopy for metabolomics allow a better understanding of the role of intestinal microbiota in health and disease