Long-read metagenomics retrieves complete single-contig bacterial genomes from canine feces.

BACKGROUND: Long-read sequencing in metagenomics facilitates the assembly of complete genomes out of complex microbial communities. These genomes include essential biologic information such as the ribosomal genes or the mobile genetic elements, which are usually missed with short-reads. We applied long-read metagenomics with Nanopore sequencing to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a dog fecal sample. RESULTS: We used nanopore long-read metagenomics and frameshift aware correction on a canine fecal sample and retrieved eight single-contig HQ MAGs, which were > 90% complete with < 5% contamination, and contained most ribosomal genes and tRNAs. At the technical level, we demonstrated that a high-molecular-weight DNA extraction improved the metagenomics assembly contiguity, the recovery of the rRNA operons, and the retrieval of longer and circular contigs that are potential HQ MAGs. These HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Catenibacterium, Blautia, and Enterococcus genera. Linking our results to previous gastrointestinal microbiome reports (metagenome or 16S rRNA-based), we found that some bacterial species on the gastrointestinal tract seem to be more canid-specific -Succinivibrio, Prevotellamassilia, Phascolarctobacterium, Blautia_A sp900541345-, whereas others are more broadly distributed among animal and human microbiomes -Sutterella, Catenibacterium, Enterococcus, and Blautia sp003287895. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Moreover, we show that long reads are essential to detect mobilome functions, usually missed in short-read MAGs. CONCLUSIONS: We recovered eight single-contig HQ MAGs from canine feces of a healthy dog with nanopore long-reads. We also retrieved relevant biological insights from these specific bacterial species previously missed in public databases, such as complete ribosomal operons and mobilome functions. The high-molecular-weight DNA extraction improved the assembly's contiguity, whereas the high-accuracy basecalling, the raw read error correction, the assembly polishing, and the frameshift correction reduced the insertion and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.

Effects of a Veterinary Gastrointestinal Low-Fat Diet on Fecal Characteristics, Metabolites, and Microbiota Concentrations of Adult Dogs Treated with Metronidazole.

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 dry kibble diet (GBD). Over the next 2 wk, dogs were fed GBD and received metronidazole orally (20 mg/kg BW twice daily). At wk 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 wk 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter content, and metabolite and BA concentrations. Fecal microbiota populations were analyzed by 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, change 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 wk 8, diet may be used to aid in recovery.

Effects of commercial and traditional kefir supplementation on apparent total tract macronutrient digestibility and the fecal characteristics, metabolites, and microbiota of healthy adult dogs.

Kefir is a fermented dairy beverage that has been consumed by humans for centuries, but poorly studied in pets. The objective of this study was to determine the effects of commercial or traditional kefir supplementation on apparent total tract macronutrient digestibility (ATTD) and fecal characteristics, microbiota populations, and metabolite and immunoglobulin (Ig) A concentrations of healthy adult dogs. Twelve healthy adult dogs (5.67 ±â€…1.72 yr, 7.27 ±â€…1.15 kg) were used in a replicated 3 × 3 Latin square design (n = 12/group). All dogs were fed a commercial diet and allotted to 1 of 3 treatments (60 mL/d): 2% reduced-fat milk treated with lactase [CNTL; 4.57E + 03 lactic acid bacteria (LAB) colony-forming units (CFU)/mL], commercial kefir (C-Kefir; 6.95E + 04 LAB CFU/mL), or traditional kefir brewed daily from 2% reduced-fat milk and kefir grains (T-Kefir; 1.79E + 09 LAB CFU/mL). The experiment was composed of three 28-d periods, with each consisting of a 22-d transition phase, a 5-d fecal collection phase, and 1 d for blood collection. Fecal samples were collected for determination of ATTD and fecal pH, dry matter, microbiota, and metabolite, and IgA concentrations. Data were analyzed using the Mixed Models procedure of SAS 9.4. The main effects of treatment were tested, with significance set at P ≤ 0.05 and trends set at P ≤ 0.10. Kefir products differed in microbial density and profile, but fecal microbiota populations were weakly impacted. Bacterial alpha diversity tended to be greater (P = 0.10) in dogs fed T-Kefir than those fed CNTL. Bacterial beta diversity analysis identified a difference (P < 0.0004) between dogs-fed CNTL and those fed C-Kefir. Dogs-fed C-Kefir tended to have a greater (P = 0.06) relative abundance of Fusobacteriota than those fed CNTL or T-Kefir. Dogs-fed T-Kefir had a greater (P < 0.0001) relative abundance of Lactococcus than those fed CNTL or C-Kefir. Dogs-fed T-Kefir also tended to have a lower (P = 0.09) relative abundance of Escherichia Shigella and greater (P = 0.09) relative abundance of Candidatus stoquefichus than dogs-fed CNTL or C-Kefir. Dogs-fed C-Kefir tended to have lower (P = 0.08) fecal valerate concentrations than those fed CNTL or T-Kefir. All other measures were unaffected by kefir treatments. Our results suggest that kefir supplementation had minor effects on the fecal microbiota populations and fecal metabolite concentrations of healthy adult dogs without impacting ATTD, fecal characteristics, or fecal IgA concentrations.

Kefir is a fermented dairy beverage that has been consumed by humans for centuries, but poorly studied in pets. Our objective was to determine the effects of commercial or traditional kefir supplementation on apparent total tract macronutrient digestibility and fecal characteristics, microbiota populations, and metabolite and immunoglobulin A concentrations of healthy adult dogs. Using a replicated Latin square design, milk (control; CNTL), commercial kefir (C-Kefir), and traditional kefir (T-Kefir) treatments were tested. Kefir products differed in microbial density and profile, but fecal microbiota populations were weakly impacted. Bacterial alpha diversity tended to be greater in T-Kefir than CNTL. Bacterial beta diversity analysis identified differences between CNTL and C-Kefir. Dogs-fed C-Kefir tended to have greater relative abundance of Fusobacteriota than those fed CNTL or T-Kefir. Dogs-fed T-Kefir had a greater relative abundance of Lactococcus than those fed CNTL or C-Kefir. Dogs-fed T-Kefir tended to have a lower relative abundance of Escherichia-Shigella and greater relative abundance of Candidatus stoquefichus than dogs-fed CNTL or C-Kefir. Dogs-fed C-Kefir tended to have lower fecal valerate than those fed CNTL or T-Kefir. Our results suggest that kefir supplementation had minor effects on the fecal microbiota and metabolites of healthy adult dogs.

Effects of a milk oligosaccharide biosimilar on fecal characteristics, microbiota, and bile acid, calprotectin, and immunoglobulin concentrations of healthy adult dogs treated with metronidazole.

In recent dog and cat experiments, a novel milk oligosaccharide biosimilar (GNU100) positively modulated fecal microbiota and metabolite profiles, suggesting benefits to gastrointestinal health. The objective of this study was to investigate the effects of GNU100 on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Twelve healthy adult female dogs (mean age: 3.74 ± 2.4 yr) were used in an 8-wk crossover design study (dogs underwent both treatments). All dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were orally administered metronidazole (20 mg/kg BW) twice daily. Fecal scores were recorded daily and fresh fecal samples were collected at weeks 2, 4, 5, 6, and 8 for measurement of pH, dry matter, microbiota populations, and BA, immunoglobulin A, and calprotectin concentrations. On weeks 0, 4, and 8, blood samples were collected for serum chemistry and hematology analysis. All data were analyzed as repeated measures using the Mixed Models procedure of SAS version 9.4, with significance considered P < 0.05. Metronidazole increased (P < 0.0001) fecal scores (looser stools) and modified (P < 0.05) fecal microbiota and BA profiles. Using qPCR, metronidazole reduced fecal Blautia, Fusobacterium, Turicibacter, Clostridium hiranonis, and Faecalibacterium abundances, and increased fecal Streptococcus and Escherichia coli abundances. DNA sequencing analysis demonstrated that metronidazole reduced microbial alpha diversity and influenced the relative abundance of 20 bacterial genera and families. Metronidazole also increased primary BA and reduced secondary BA concentrations. Most antibiotic-induced changes returned to baseline by week 8. Fecal scores were more stable (P = 0.01) in GNU100-fed dogs than controls after antibiotic administration. GNU100 also influenced fecal microbiota and BA profiles, reducing (P < 0.05) the influence of metronidazole on microbial alpha diversity and returning some fecal microbiota and secondary BA to baseline levels at a quicker (P < 0.05) rate than controls. In conclusion, our results suggest that GNU100 supplementation provides benefits to dogs treated with antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and secondary BA profiles which play an essential role in gut health.

Our objective was to test the effects of a novel milk oligosaccharide biosimilar (GNU100) on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were given an oral antibiotic. Fecal scores were recorded and fresh fecal samples were collected over time to assess fecal characteristics, microbiota populations, and BA concentrations. The antibiotic was shown to increase fecal scores (looser stools) and modify fecal microbiota populations (altered diversity and ~20 bacterial genera and families) and BA profiles (increased primary and reduced secondary BA). Most antibiotic-induced changes returned to baseline by week 8. In dogs fed GNU100, fecal scores were more stable and changes to microbial diversity were lower than controls after antibiotic administration. Fecal microbiota and secondary BA of GNU100-fed dogs also returned to baseline levels at a quicker rate than controls. These results suggest that GNU100 provides benefits to dogs given antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and BA profiles.

Effects of weight loss and feeding specially formulated diets on the body composition, blood metabolite profiles, voluntary physical activity, and fecal metabolites and microbiota of obese dogs.

Canine obesity negatively influences health and well-being, but can be managed by altering diet composition and caloric intake. Restricted feeding, dietary intervention, and consequent weight loss may be used to improve health and modify gastrointestinal microbiota. In this study, we aimed to determine the effects of restricted feeding of specially formulated foods on weight loss, body composition, voluntary physical activity, serum hormones and oxidative stress markers, and fecal metabolites and microbiota populations of obese dogs. Twenty-four obese dogs [body weight (BW) = 15.2 ±â€…1.7 kg; body condition score (BCS) = 8.7 ±â€…0.4; muscle condition score (MCS) = 3.5 ±â€…0.3; age = 7.2 ±â€…1.6 yr] were used in a 24-wk study. A control (OR) food was fed during a 4-wk baseline to identify intake needed to maintain BW. After baseline, dogs were allotted to one of two diets: OR or test (FT), and then fed to lose 1.5% BW/wk. Food intake, BW, BCS, and MCS were measured, blood and fecal samples were collected, DEXA scans were performed, and voluntary physical activity was measured over time. Microbiota data were evaluated using QIIME2 and change from baseline data from other measures were evaluated using the Mixed Models procedure of SAS, with P < 0.05 being significant. Restricted feeding led to reduced BW, BCS, fat mass, and blood cholesterol, triglyceride, glucose, and leptin concentrations, and increased MCS and lean body mass percentage. Blood cholesterol reduction was greater in dogs fed FT vs. OR. Fecal metabolites and bacterial alpha-diversity were affected by diet and weight loss. Dogs fed FT had greater reductions in fecal short-chain fatty acid, branched-chain fatty acid, and ammonia concentrations than those fed OR. Dogs fed OR had a higher alpha-diversity than those fed FT. Weight loss increased alpha-diversity (weeks 16, 20, and 24 > weeks 0 and 4). Beta-diversity showed separation between dietary groups and between week 0 and all other time points after week 8. Weight loss increased fecal Allobaculum and Ruminococcus torques. Weight loss also increased fecal Bifidobacterium, Faecalibaculum, and Parasutterella, but were greater in dogs fed OR. Weight loss decreased fecal Collinsella, Turicibacter, Blautia, Ruminococcus gnavus, Faecalibacterium, and Peptoclostridium, but were greater in dogs fed OR. In summary, restricted feeding promoted safe weight and fat loss, reduced blood lipid and leptin concentrations, and altered fecal microbiota of obese dogs.

In this study, we aimed to determine the effects of restricted feeding of specially formulated foods on weight loss, body composition, voluntary physical activity, serum hormones and oxidative stress markers, and fecal metabolites and microbiota populations of obese dogs. A control (OR) food was fed during a 4-wk baseline to identify intake needed to maintain the body weight (BW). After baseline, dogs were allotted to one of two diets: OR or test (FT) and then fed to lose 1.5% BW per week for 24 wk. Restricted feeding and weight loss led to reduced BW, body condition score, fat mass, and blood cholesterol, triglyceride, glucose and leptin concentrations and increased muscle condition score and lean body mass percentage. The reduction in blood cholesterol was greater in dogs fed FT vs. OR. Fecal metabolites and bacterial alpha-diversity were affected by diet and weight loss, with dogs fed with OR having a higher alpha-diversity than those fed with FT. Restricted feeding and weight loss increased alpha-diversity, affected beta-diversity, and impacted the relative abundances of nearly 20 bacterial genera. In summary, restricted feeding with high-protein, low-starch kibble diets promoted safe weight and fat loss, reduced blood lipid and leptin concentrations, and altered fecal microbiota of obese dogs.

Heterogeneity of gut microbial responses in healthy household dogs transitioning from an extruded to a mildly cooked diet.

BACKGROUND: The gut microbiota (GM) is associated with canine health and can be impacted by diet. Dog owners in the U.S. have increasingly shown an interest in feeding their dogs a mildly cooked (MC) diet. However, its impact on canine GM and health remains largely unknown. METHODS: Healthy household dogs were tracked upon switching from various brands of extruded to MC diets for four weeks. A health assessment was completed and stool samples were collected by each owner before (day 0) and after the diet transition (day 28). Shotgun metagenomic sequencing was performed at both time points to characterize the GM. RESULTS: Dogs completed the study by either completing the health assessments (n = 31) or providing stool samples at both time points (n = 28). All owners reported either better or no change in overall health at the end of the study (61% and 39%, respectively), and none reported worse overall health. Defecation frequency was also reported to be lower (58%) or about the same (35%). Principal coordinate (PCo) analysis showed a significant shift (p = 0.004) in the ß-diversity of the GM upon diet transition (34.2% and 10.3% explained by the first two axes). The abundances of 70 species increased after the diet change (adjusted p < 0.05), 67% and 24% of which belonged to the Lactobacillales and the Enterobacterales orders respectively. The abundances of 28 species decreased (adjusted p < 0.05), 46%, 18%, and 11% of which belonged to the Clostridiales, Bacillales, and Bacteroidales orders, respectively. Lower Lactobacillales and Enterobacterales, and higher Bacteroidales at baseline were associated with a greater shift along the PCo1 axis. Protein content of the baseline diet was correlated with the shift along the PCo1 axis (ρ = 0.67, p = 0.006). CONCLUSION: Owners reported either improvement or no change in health in dogs transitioning from extruded kibble to MC diets for 4 weeks, but this report of health perception requires further exploration in a controlled trial. Diet change also led to a significant shift in the GM profile of healthy dogs. The magnitude of shift was associated with baseline GM and dietary protein, and warrants further examination of individualized responses and personalized nutrition in companion dogs. These results also support future investigation of the impact of a MC diet on health maintenance given its increasing popularity.