Effect of high contents of dietary animal-derived protein or carbohydrates on canine faecal microbiota.

BACKGROUND: Considerable evidence suggests that food impacts both the gastro-intestinal (GI) function and the microbial ecology of the canine GI tract. The aim of this study was to evaluate the influence of high-carbohydrate (HC), high-protein (HP) and dry commercial (DC) diets on the canine colonic microbiota in Beagle dogs. Diets were allocated according to the Graeco-Latin square design. For this purpose, microbial DNA was isolated from faecal samples and separated by density gradient centrifugation, resulting in specific profiling based on the guanine-cytosine content (%G+C). In addition, 16 S rRNA gene amplicons were obtained from the most abundant %G + C peaks and analysed by sequence analysis, producing a total of 720 non-redundant sequences (240 sequences per diet). RESULTS: The DC diet sample showed high abundance of representatives of the orders Clostridiales, Lactobacillales, Coriobacteriales and Bacteroidales. Sequence diversity was highest for DC diet samples and included representatives of the orders Lactobacillales and Bacteroidales, which were not detected in samples from the HP and HC diets. These latter two diets also had reduced levels of representatives of the family Lachnospiraceae, specifically Clostridial cluster XIVa. The HC diet favoured representatives of the order Erysipelotrichales, more specifically the Clostridial cluster XVIII, while the HP diet favoured representatives of the order Fusobacteriales. CONCLUSIONS: This study detected Coriobacteriales in dog faeces, possibly due to the non-selective nature of the %G + C profiling method used in combination with sequencing. Moreover, our work demonstrates that the effect of diet on faecal microbiota can be explained based on the metabolic properties of the detected microbial taxa.

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

BACKGROUND: Recent 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. RESULTS: Pyrosequencing 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. CONCLUSION: Tylosin 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.

Prevalence and identification of fungal DNA in the small intestine of healthy dogs and dogs with chronic enteropathies.

Limited information is available about the prevalence and phylogenetic classification of fungal organisms in the gastrointestinal tract of dogs. Also, the impact of fungal organisms on gastrointestinal health and disease is not well understood. The aim of this study was to evaluate the prevalence of fungal DNA in the small intestine of healthy dogs and dogs with chronic enteropathies. Small intestinal content was analyzed from 64 healthy and 71 diseased dogs from five different geographic locations in Europe and the USA. Fungal DNA was amplified with panfungal primers targeting the internal transcriber spacer (ITS) region. PCR amplicons were subjected to phylogenetic analysis. Fungal DNA was detected in 60.9% of healthy dogs and in 76.1% of dogs with chronic enteropathies. This prevalence was not significantly different between the two groups (p=0.065). Fungal DNA was significantly more prevalent in mucosal brush samples (82.8%) than in luminal samples (42.9%; p=0.002). Sequencing results revealed a total of 51 different phylotypes. All sequences belonged to two phyla and were classified as either Ascomycota (32 phylotypes) or Basidiomycota (19 phylotypes). Three major classes were identified: Saccharomycetes, Dothideomycetes, and Hymenomycetes. The most commonly observed sequences were classified as Pichia spp., Cryptococcus spp., Candida spp., and Trichosporon spp. Species believed to be clinically more important were more commonly observed in diseased dogs. These results indicate a high prevalence and diversity of fungal DNA in the small intestine of both healthy dogs and dogs with chronic enteropathies. The canine gastrointestinal tract of diseased dogs may harbor opportunistic fungal pathogens.

Relatedness of Escherichia coli strains with different susceptibility patterns isolated from beagle dogs during ampicillin treatment.

The aim of this study was to investigate the effects of ampicillin treatment on selection and diversity of ampicillin-resistant intestinal Escherichia coli in beagles treated with ampicillin, ampicillin + beta-lactamase (targeted to degrade antibiotic residues in the gut) or placebo. Selected faecal (n = 339) and jejunal (n = 63) E. coli isolates with known resistance patterns were typed using pulsed-field gel electrophoresis (PFGE). Among the 25 detected PFGE types, ampicillin resistance was detected in 6, none of which was dominant over others among the dogs. The resistant types increased especially in the ampicillin group, whilst beta-lactamase inhibited their emergence. Selection of genetically unrelated resistant strains rather than emerging resistance among previously susceptible strains accounts for increasing resistance rates during ampicillin treatment.

The effects of feeding and withholding food on the canine small intestinal microbiota.

Prolonged lack of enteral feeding has a negative impact on gut physiology, potentially via microbiota modulation. The aims were to investigate the impact of fasting and post-prandial changes in canine jejunal microbiota. To study post-prandial effects, jejunal brushings were analyzed in 8 healthy fistulated dogs 15 min before feeding (baseline) and hourly for 8 h after feeding. To study effects of withholding food (WF), daily samples were collected for 15 days from 5 dogs. The first 5 days (PRE) dogs were fed regular diet. Food was withheld the next 5 days (days 6-10). For days 11-15 (POST), the original diet was reintroduced. Microbiota was characterized via denaturing gradient gel electrophoresis and 454-pyrosequencing of 16S rRNA genes. In the post-prandial study, no changes in microbiome structure were seen after feeding (ANOSIM, P = 0.28), but Betaproteobacteria (P = 0.04) and Bacteroidales decreased compared to baseline. Species richness decreased by 300 min (P = 0.04). During WF, microbiota structure differed from PRE and POST period (P = 0.001). During WF, species richness did not vary over time (P = 0.69). In conclusion, a prolonged period of food withholding results in altered jejunal microbiota. How these changes affect the microbiota metabolism warrants further studies.

Tylosin-responsive chronic diarrhea in dogs.

Fourteen dogs had shown chronic or intermittent diarrhea for more than 1 year. Diarrhea had been successfully treated with tylosin for at least 6 months but recurred when treatment was withdrawn on at least 2 occasions. Tylosin-responsive diarrhea (TRD) affects typically middle-aged, large-breed dogs and clinical signs indicate that TRD affects both the small and large intestine. Treatment with tylosin eliminated diarrhea in all dogs within 3 days and in most dogs within 24 hours. Tylosin administration controlled diarrhea in all dogs, but after it was discontinued, diarrhea reappeared in 12 (85.7%) of 14 dogs within 30 days. Prednisone given for 3 days did not completely resolve diarrhea. Probiotic Lactobacillus rhamnosus GG did not prevent the relapse of diarrhea in any of 9 dogs so treated. The etiology of TRD, a likely form of antibiotic-responsive diarrhea (ARD) is unclear. The following reasons for chronic diarrhea were excluded or found to be unlikely: parasites, exocrine pancreatic insufficiency, inflammatory bowel disease, small intestinal bacterial overgrowth, enteropathogenic bacteria (Salmonella spp., Campylobacter spp., Yersinia spp., or Lawsoni intracellularis), and Clostridium perfringens enterotoxin and Clostridium difficile A toxin. A possible etiologic factor is a specific enteropathogenic organism that is a common resident in the canine gastrointestinal tract and is sensitive to tylosin but difficult to eradicate. Additional studies are required to identify the specific cause of TRD.

Inhibition of ampicillin-induced emergence of resistance in intestinal coliforms by targeted recombinant beta-lactamase.

The aim of the present study was to determine whether oral targeted recombinant beta-lactamase (TRBL) administration could overcome the development of ampicillin-induced resistance in the gut microbiota. Eighteen laboratory beagles with permanent jejunal fistula were randomised to receive ampicillin + placebo, ampicillin + TRBL or placebo. A total of 982 coliform isolates, collected from jejunal and faecal samples before, during and after the treatment were tested against nine antimicrobials. The proportion of ampicillin resistance (multi-resistance) among coliform isolates increased from 20 to 36% in the ampicillin + placebo group but far less, 20-36%, in the ampicillin + TRBL group. These results indicate that TRBL may prevent the emergence of beta-lactam-associated resistance in coliforms in the gut.

Evaluation of pancreatic forceps biopsy by laparoscopy in healthy beagles.

The effects of laparoscopic biopsies were determined in four healthy laboratory beagles. Biopsies were taken from the pancreas of three dogs and from the peripancreatic fat of one dog. Clinical examinations and blood sampling for hematologic and biochemical tests were performed before laparoscopy and weekly throughout each dog's participation in the study (7 or 21 days). No clinical signs of pancreatitis were observed, and hematologic and biochemical parameters remained within normal limits in three dogs. One dog exhibited a transient increase in trypsinlike immunoreactivity, amylase, and lipase. Minor adhesions between the pancreas, small intestine, and peritoneum were observed macroscopically in this dog. Histologically, granulation tissue and a mild nonsuppurative inflammation in the pancreas were present. No abnormal changes were seen macroscopically or histologically in the other two dogs for which pancreatic biopsies were performed. Thus, laparoscopy appears to be safe, with neither permanent abnormalities in blood parameters nor changes in clinical health occurring during or after the procedure in healthy beagles.

Small intestinal permeability and serum folate and cobalamin absorption after surgical construction of permanent jejunal fistulas in laboratory beagle dogs.

Permanent jejunal fistulas enable easy, noninjurious, repeated and direct administration to and collection from the small intestines of conscious laboratory dogs. This study aimed at identifying potential alterations in the small intestinal morphology and function of this canine model after the surgery required to establish the fistulas. Assays of serum folate and cobalamin and (51)Cr-EDTA permeability tests were performed before and 4 wk after experimental jejunoplasties in 14 laboratory beagle dogs. Serum folate concentrations (mean ± SD) before (12.22 ± 1.80 µg/L) and after (14.14 ± 1.70 µg/L) jejunal surgery were within reference ranges for healthy dogs, although folate concentrations were higher after surgery. The cobalamin concentrations and the 6-h urinary excretion of (51)Cr-EDTA before (573.50 ± 150.04 ng/L and 6.75 ± 1.56%, respectively) and after (496.71 ± 164.22 ng/L and 6.41 ± 1.10%) were normal for healthy dogs, and no significant differences between pre- and postsurgical values were detected. The findings of the present study indicate that the small intestinal vitamin absorption and permeability of laboratory beagle dogs with jejunal fistulas remains unimpaired.

Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs.

The microbiota of the small intestine is poorly known because of difficulties in sampling. In this study, we examined whether the organisms cultured from the jejunum and feces resemble each other. Small-intestinal fluid samples were collected from 22 beagle dogs with a permanent jejunal fistula in parallel with fecal samples. In addition, corresponding samples from seven of the dogs were collected during a 4-week period (days 4, 10, 14, and 28) to examine the stability of the microbiota. In the jejunal samples, aerobic/facultative and anaerobic bacteria were equally represented, whereas anaerobes dominated in the fecal samples. Despite lower numbers of bacteria in the jejunum (range, 10(2) to 10(6) CFU/g) than in feces (range, 10(8) to 10(11) CFU/g), some microbial groups were more prevalent in the small intestine: staphylococci, 64% versus 36%; nonfermentative gram-negative rods, 27% versus 9%; and yeasts, 27% versus 5%, respectively. In contrast, part of the fecal dominant microbiota (bile-resistant Bacteroides spp., Clostridium hiranonis-like organisms, and lactobacilli) was practically absent in the jejunum. Many species were seldom isolated simultaneously from both sample types, regardless of their overall prevalence. In conclusion, the small intestine contains a few bacterial species at a time with vastly fluctuating counts, opposite to the results obtained for the colon, where the major bacterial groups remain relatively constant over time. Qualitative and quantitative differences between the corresponding jejunal and fecal samples indicate the inability of fecal samples to represent the microbiotas present in the upper gut.

Relationship between canine mucosal and serum immunoglobulin A (IgA) concentrations: serum IgA does not assess duodenal secretory IgA.

A double-sandwich enzyme immunoassay method was developed for determination of serum immunoglobulin A (S-IgA) and mucosal secretory immunoglobulin A (sIgA) in duodenal brush samples obtained via endoscopy and the relationship between enteric mucosal sIgA, salivary sIgA and S-IgA in dogs was examined. Twenty healthy dogs underwent routine endoscopy. A brush sample from the duodenal mucosa was obtained and washed in PBS, with a serum sample being taken concurrently. A saliva sample was collected from twelve of these dogs. S-IgA and sIgA with total protein concentrations in the duodenal washings and saliva samples were determined. A significant negative correlation (r = -0.64, P = 0.0059) was found between duodenal sIgA/protein ratios and S-IgA concentrations. Saliva sIgA/protein ratios did not correlate with sIgA/protein ratios of duodenal samples. The method described here allows for direct assessment of duodenal IgA; therefore indirect measures based on serum IgA or salivary IgA can be avoided. In addition, these indirect measures appear to be poor indicators of duodenal sIgA competence in dogs.

Enzymic degradation of a beta-lactam antibiotic, ampicillin, in the gut: a novel treatment modality.

Antibiotics can cause severe alterations in the gut microflora and promote diarrhoea and overgrowth of pathogenic bacteria. The present study investigated the potency of targeted recombinant beta-lactamase (TRBL) to degrade a beta-lactam antibiotic in the jejunum of fistula-operated beagles. We used different peroral doses of purified beta-lactamase (PenP) of Bacillus licheniformis in enteric-coated pellets together with intravenous ampicillin. Serum and jejunal samples were collected for ampicillin and beta-lactamase analysis. A dose-response effect of TRBL on ampicillin concentrations in the jejunal samples could be observed. The highest doses applied decreased the jejunal ampicillin concentrations to undetectable levels. In the serum samples, the ampicillin concentrations were not affected by the beta-lactamase dose used. Our results indicate that it may be possible to evolve a targeted treatment to degrade beta-lactam antibiotics intestinally and, thus, decrease antibiotic-induced adverse effects on the gut microflora.

Oral administration of beta-lactamase preserves colonization resistance of piperacillin-treated mice.

We hypothesized that orally administered, recombinant class A beta-lactamase would inactivate the portion of parenteral piperacillin excreted into the intestinal tract, preserving colonization resistance of mice against nosocomial pathogens. Subcutaneous piperacillin or piperacillin plus oral beta-lactamase were administered 24 and 12 h before orogastric inoculation of piperacillin-resistant pathogens. Oral administration of beta-lactamase reduced piperacillin-associated alteration of the indigenous microflora and prevented overgrowth of pathogens.

Orally administered targeted recombinant Beta-lactamase prevents ampicillin-induced selective pressure on the gut microbiota: a novel approach to reducing antimicrobial resistance.

Antibiotics that are excreted into the intestinal tract promote antibiotic resistance by exerting selective pressure on the gut microbiota. Using a beagle dog model, we show that an orally administered targeted recombinant beta-lactamase enzyme eliminates the portion of parenteral ampicillin that is excreted into the small intestine, preventing ampicillin-induced changes to the fecal microbiota without affecting ampicillin levels in serum. In dogs receiving ampicillin, significant disruption of the fecal microbiota and the emergence of ampicillin-resistant Escherichia coli and TEM genes were observed, whereas in dogs treated with ampicillin in combination with an oral beta-lactamase, these did not occur. These results suggest a new strategy for reducing antimicrobial resistance in humans.