Jan S. Suchodolski

Massive parallel 16S rRNA gene pyrosequencing reveals highly diverse fecal bacterial and fungal communities in healthy dogs and cats

This study evaluated the fecal microbiota of 12 healthy pet dogs and 12 pet cats using bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing. A total of 120,406 pyrosequencing reads for bacteria (mean 5017) and 5359 sequences (one pool each for dogs and cats) for fungi were analyzed. Additionally, group-specific 16S rRNA gene clone libraries for Bifidobacterium spp. and lactic acid-producing bacteria (LAB) were constructed. The most abundant bacterial phylum was Firmicutes, followed by Bacteroidetes in dogs and Actinobacteria in cats. The most prevalent bacterial class in dogs and cats was Clostridia, dominated by the genera Clostridium (clusters XIVa and XI) and Ruminococcus. At the genus level, 85 operational taxonomic units (OTUs) were identified in dogs and 113 OTUs in cats. Seventeen LAB and eight Bifidobacterium spp. were detected in canine feces. Ascomycota was the only fungal phylum detected in cats, while Ascomycota, Basidiomycota, Glomeromycota, and Zygomycota were identified in dogs. Nacaseomyces was the most abundant fungal genus in dogs; Saccharomyces and Aspergillus were predominant in cats. At the genus level, 33 different fungal OTUs were observed in dogs and 17 OTUs in cats. In conclusion, this study revealed a highly diverse bacterial and fungal microbiota in canine and feline feces.

Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice

This study is the first to use a metagenomics approach to characterize the phylogeny and functional capacity of the canine gastrointestinal microbiome. Six healthy adult dogs were used in a crossover design and fed a low-fiber control diet (K9C) or one containing 7.5% beet pulp (K9BP). Pooled fecal DNA samples from each treatment were subjected to 454 pyrosequencing, generating 503 280 (K9C) and 505 061 (K9BP) sequences. Dominant bacterial phyla included the Bacteroidetes/Chlorobi group and Firmicutes, both of which comprised ∼35% of all sequences, followed by Proteobacteria (13–15%) and Fusobacteria (7–8%). K9C had a greater percentage of Bacteroidetes, Fusobacteria and Proteobacteria, whereas K9BP had greater proportions of the Bacteroidetes/Chlorobi group and Firmicutes. Archaea were not altered by diet and represented ∼1% of all sequences. All archaea were members of Crenarchaeota and Euryarchaeota, with methanogens being the most abundant and diverse. Three fungi phylotypes were present in K9C, but none in K9BP. Less than 0.4% of sequences were of viral origin, with >99% of them associated with bacteriophages. Primary functional categories were not significantly affected by diet and were associated with carbohydrates; protein metabolism; DNA metabolism; cofactors, vitamins, prosthetic groups and pigments; amino acids and derivatives; cell wall and capsule; and virulence. Hierarchical clustering of several gastrointestinal metagenomes demonstrated phylogenetic and metabolic similarity between dogs, humans and mice. More research is required to provide deeper coverage of the canine microbiome, evaluate effects of age, genetics or environment on its composition and activity, and identify its role in gastrointestinal disease.

The Fecal Microbiome in Dogs with Acute Diarrhea and Idiopathic Inflammatory Bowel Disease

Background Recent molecular studies have revealed a highly complex bacterial assembly in the canine intestinal tract. There is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (IBD). The aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders. Methodology/Principal Findings Fecal samples from healthy dogs (n = 32), dogs with acute non-hemorrhagic diarrhea (NHD; n = 12), dogs with acute hemorrhagic diarrhea (AHD; n = 13), and dogs with active (n = 9) and therapeutically controlled idiopathic IBD (n = 10) were analyzed by 454-pyrosequencing of the 16S rRNA gene and qPCR assays. Dogs with acute diarrhea, especially those with AHD, had the most profound alterations in their microbiome, as significant separations were observed on PCoA plots of unweighted Unifrac distances. Dogs with AHD had significant decreases in Blautia, Ruminococcaceae including Faecalibacterium, and Turicibacter spp., and significant increases in genus Sutterella and Clostridium perfringens when compared to healthy dogs. No significant separation on PCoA plots was observed for the dogs with IBD. Faecalibacterium spp. and Fusobacteria were, however, decreased in the dogs with clinically active IBD, but increased during time periods of clinically insignificant IBD, as defined by a clinical IBD activity index (CIBDAI). Conclusions Results of this study revealed a bacterial dysbiosis in fecal samples of dogs with various GI disorders. The observed changes in the microbiome differed between acute and chronic disease states. The bacterial groups that were commonly decreased during diarrhea are considered to be important short-chain fatty acid producers and may be important for canine intestinal health. Future studies should correlate these observed phylogenetic differences with functional changes in the intestinal microbiome of dogs with defined disease phenotypes.

Molecular-phylogenetic characterization of microbial communities imbalances in the small intestine of dogs with inflammatory bowel disease

An association between luminal commensal bacteria and inflammatory bowel disease (IBD) has been suggested in humans, but studies investigating the intestinal microbial communities of dogs with IBD have not been published. The aim of this study was to characterize differences of the small intestinal microbial communities between dogs with IBD and healthy control dogs. Duodenal brush cytology samples were endoscopically collected from 10 dogs with IBD and nine healthy control dogs. DNA was extracted and 16S rRNA gene was amplified using universal bacterial primers. Constructed 16S rRNA gene clone libraries were compared between groups. From a total of 1240 selected clones, 156 unique 16S rRNA gene sequences were identified, belonging to six phyla: Firmicutes (53.4%), Proteobacteria (28.4%), Bacteroidetes (7.0%), Spirochaetes (5.2%), Fusobacteria (3.4%), Actinobacteria (1.1%), and Incertae sedis (1.5%). Species richness was significantly lower in the IBD group (P=0.038). Principal component analysis indicated that the small intestinal microbial communities of IBD and control dogs are composed of distinct microbial communities. The most profound difference involved enrichment of the IBD dogs with members of the Enterobacteriaceae family. However, differences involving members of other families, such as Clostridiaceae, Bacteroidetes and Spirochaetes, were also identified. In conclusion, canine IBD is associated with altered duodenal microbial communities compared with healthy controls.

Analysis of bacterial diversity in the canine duodenum, jejunum, ileum, and colon by comparative 16S rRNA gene analysis

The study aim was to describe the diversity of the intraluminal intestinal microbial community in dogs by direct sequence analysis of the 16S rRNA gene. Intestinal content was collected from the duodenum, jejunum, ileum, and colon from six healthy dogs. Bacterial 16S rRNA gene was amplified with universal bacterial primers. Amplicons were ligated into cloning vectors and near-full-length 16S rRNA gene inserts were analyzed. From a total of 864 clones analyzed, 106 nonredundant 16S rRNA gene sequences were identified. Forty-two (40%) sequences showed<98% sequence similarity to 16S rRNA gene sequences reported previously. Operation taxonomic units were classified into four phyla: Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria. Clostridiales predominated in the duodenum (40% of clones) and jejunum (39%), and were highly abundant in the ileum (25%) and colon (26%). Sequences affiliated with Clostridium cluster XI and Clostridium cluster XIVa dominated in the proximal small intestine and colon, respectively. Fusobacteriales and Bacteroidales were the most abundant bacterial order in the ileum (33%) and colon (30%). Enterobacteriales were more commonly observed in the small intestine than in the colon. Lactobacillales occurred commonly in all parts of the intestine.

The effect of the macrolide antibiotic tylosin on microbial diversity in the canine small intestine as demonstrated by massive parallel 16S rRNA gene sequencing

BackgroundRecent studies have shown that the fecal microbiota is generally resilient to short-term antibiotic administration, but some bacterial taxa may remain depressed for several months. Limited information is available about the effect of antimicrobials on small intestinal microbiota, an important contributor to gastrointestinal health. The antibiotic tylosin is often successfully used for the treatment of chronic diarrhea in dogs, but its exact mode of action and its effect on the intestinal microbiota remain unknown. The aim of this study was to evaluate the effect of tylosin on canine jejunal microbiota. Tylosin was administered at 20 to 22 mg/kg q 24 hr for 14 days to five healthy dogs, each with a pre-existing jejunal fistula. Jejunal brush samples were collected through the fistula on days 0, 14, and 28 (14 days after withdrawal of tylosin). Bacterial diversity was characterized using massive parallel 16S rRNA gene pyrosequencing.ResultsPyrosequencing revealed a previously unrecognized species richness in the canine small intestine. Ten bacterial phyla were identified. Microbial populations were phylogenetically more similar during tylosin treatment. However, a remarkable inter-individual response was observed for specific taxa. Fusobacteria, Bacteroidales, and Moraxella tended to decrease. The proportions of Enterococcus-like organisms, Pasteurella spp., and Dietzia spp. increased significantly during tylosin administration (p < 0.05). The proportion of Escherichia coli-like organisms increased by day 28 (p = 0.04). These changes were not accompanied by any obvious clinical effects. On day 28, the phylogenetic composition of the microbiota was similar to day 0 in only 2 of 5 dogs. Bacterial diversity resembled the pre-treatment state in 3 of 5 dogs. Several bacterial taxa such as Spirochaetes, Streptomycetaceae, and Prevotellaceae failed to recover at day 28 (p < 0.05). Several bacterial groups considered to be sensitive to tylosin increased in their proportions.ConclusionTylosin may lead to prolonged effects on the composition and diversity of jejunal microbiota. However, these changes were not associated with any short-term clinical signs of gastrointestinal disease in healthy dogs. Our results illustrate the complexity of the intestinal microbiota and the challenges associated with evaluating the effect of antibiotic administration on the various bacterial groups and their potential interactions.

Assessment of microbial diversity along the feline intestinal tract using 16S rRNA gene analysis

The aim of this study was to describe the microbial communities along the gastrointestinal tract in healthy cats based on analysis of the 16S rRNA gene. Gastrointestinal content (i.e. content from the stomach, duodenum, jejunum, ileum, and colon) was collected from four healthy conventionally raised colony cats and one healthy specific pathogen-free (SPF) cat. Bacterial 16S rRNA genes were amplified using universal bacterial primers and analyzed by comparative sequence analysis. A total of 1008 clones were analyzed and 109 nonredundant 16S rRNA gene sequences were identified. In the four conventionally raised cats, five different bacterial phyla were observed, with sequences predominantly classified in the phylum Firmicutes (68%), followed by Proteobacteria (14%), Bacteroidetes (10%), Fusobacteria (5%), and Actinobacteria (4%). The majority of clones fell within the order Clostridiales (54%), followed by Lactobacillales, Bacteroidales, Campylobacterales, and Fusobacteriales (14%, 11%, 10%, and 6%, respectively). Clostridiales were predominantly affiliated with Clostridium clusters I (58%) and XIVa (27%). The intestinal microbiota of the SPF cat displayed a reduced bacterial diversity, with 98% of all clones classified in the phylum Firmicutes. Further classification showed that the Firmicutes clones belonged exclusively to the class Clostridiales and were predominantly affiliated with Clostridium cluster I.

16S rRNA Gene Pyrosequencing Reveals Bacterial Dysbiosis in the Duodenum of Dogs with Idiopathic Inflammatory Bowel Disease

Background Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Methodology/Principal Findings Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p = 0.010), Bacteroidaceae (p = 0.015), Prevotellaceae (p = 0.022), and Clostridiales (p = 0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044) and Acinetobacter (p = 0.040), were either more abundant or more frequently identified in IBD dogs. Conclusions/Significance In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.

Molecular analysis of the bacterial microbiota in duodenal biopsies from dogs with idiopathic inflammatory bowel disease.

An association between mucosa-adherent commensal bacteria and inflammatory bowel disease (IBD) has been proposed for humans. There are no reports characterizing the mucosa-adherent duodenal microbiota in dogs with idiopathic IBD using molecular methods. The aim of this study was to investigate differences in the mucosa-adherent duodenal microbiota between dogs with idiopathic IBD and healthy dogs. Duodenal biopsy samples were collected from seven dogs with IBD and seven healthy control dogs. DNA was extracted, 16S ribosomal RNA genes were amplified and 16S rRNA gene clone libraries were constructed and compared between groups. A total of 1035 clones were selected, and based on a 98% similarity criterion, 133 unique phylotypes were identified across all dogs. These phylotypes belonged to seven bacterial phyla: Proteobacteria (52.9%), Firmicutes (26.1%), Bacteroidetes (7.7%), Actinobacteria (8.6%), Fusobacteria (4.4%), Tenericutes (0.2%) and Verrucomicrobia (0.1%). Significant differences were identified in the relative abundance of several bacterial groups between dogs with IBD and healthy dogs (p<0.001). Healthy dogs and dogs with IBD clustered according to their disease status. Dogs with IBD had a significantly higher abundance of clones belonging to Alpha-, Beta-, and Gamma-proteobacteria (p<0.0001 for all classes), and a significantly lower abundance of Clostridia (p<0.0001). Bacteria of the genera Pseudomonas, Acinetobacter, Conchiformibious, Achromobacter, Brucella, and Brevundimonas, were significantly more abundant in dogs with IBD. In conclusion, significant differences of the mucosa-adherent duodenal microbiota were observed between dogs with idiopathic IBD and healthy dogs in this study. These results warrant further investigations into the role of the intestinal microbiota in the pathophysiology of canine IBD.

Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis.

BackgroundThe nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis.ResultsFecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks.ConclusionsOur exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis.