Jaana Harmoinen

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.

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

ABSTRACT 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, 102 to 106 CFU/g) than in feces (range, 108 to 1011 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.

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.

Orally Administered Targeted Recombinant Beta-Lactamase Prevents Ampicillin-Induced Selective Pressure on the Gut Microbiota: a Novel Approach to Reducing Antimicrobial Resistance

ABSTRACT 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 β-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 β-lactamase, these did not occur. These results suggest a new strategy for reducing antimicrobial resistance in humans.

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.

Oral Administration of β-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.

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

BackgroundConsiderable 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 (%Gu2009+u2009C). In addition, 16u2009S rRNA gene amplicons were obtained from the most abundant %Gu2009+u2009C peaks and analysed by sequence analysis, producing a total of 720 non-redundant sequences (240 sequences per diet).ResultsThe 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.ConclusionsThis study detected Coriobacteriales in dog faeces, possibly due to the non-selective nature of the %Gu2009+u2009C 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.

Orally Administered Recombinant Metallo-β-Lactamase Preserves Colonization Resistance of Piperacillin-Tazobactam-Treated Mice

Antibiotics that are excreted into the intestinal tract may disrupt colonization resistance (i.e., the ability of the indigenous microflora to provide resistance against colonization by potentially pathogenic microorganisms) (1, 5). Because antibiotic activity within the intestinal lumenis is not needed for the treatment of most infections, we hypothesized that the enzymatic inactivation of the portion of antibiotic that is excreted into the intestinal tract would result in the preservation of colonization resistance during parenteral antibiotic therapy. We previously demonstrated that an orally administered recombinant class A β-lactamase preserved the colonization resistance of piperacillin-treated mice against nosocomial pathogens (4). Here, we show that a recombinant class B metallo-β-lactamase that is resistant to tazobactam inactivation preserves colonization resistance during treatment with the more widely used broad-spectrum antibiotic piperacillin-tazobactam. n nTargeted recombinant β-lactamase 2 (TRBL-2) containing amino acid residues 31 to 257 of the metalloenzyme of a clinical Bacillus cereus isolate (98ME 1552 from Helsinki University Hospital) was overproduced in Bacillus subtilis using a bacillar secretion vector (4). The hydrolysis rate of TRBL-2 for piperacillin with or without tazobactam was 275 μg/minute/μg of enzyme. Individually housed female CF-1 mice weighing 25 to 30 g (Harlan Sprague-Dawley, Indianapolis, Indiana) were used to examine the efficacy of TRBL-2 in the preservation of colonization resistance against vancomycin-resistant enterococci (VRE). At 24 h and again at 12 h prior to the orogastric inoculation of 104 CFU of VRE strain C68 (4), mice received either subcutaneous (0.2 ml) and orogastric (0.5 ml) phosphate-buffered saline (PBS), subcutaneous piperacillin-tazobactam (4 mg) and orogastric PBS, subcutaneous piperacillin-tazobactam (4 mg) and orogastric TRBL-2 (60 mg/kg of body weight), or subcutaneous piperacillin-tazobactam (4 mg) and orogastric TRBL-2 (60 mg/kg) that had been inactivated by boiling for 10 min. Stool VRE density was monitored as previously described (2). Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial rRNA genes was performed on stool samples collected 3 days after the inoculation of VRE in order to monitor changes in the indigenous microflora (3, 4). One-way analysis of variance was performed to compare VRE densities among treatment groups, with P values adjusted for multiple comparisons using the Scheffe correction. DGGE similarity indices were compared using Students t test. Computations were performed using Stata software (version 5.0; Stata, College Station, Texas). n nMice treated with piperacillin-tazobactam developed high-density VRE stool colonization; saline controls and mice treated with piperacillin-tazobactam in conjunction with TRBL-2 did not (P < 0.001) (Fig. u200b(Fig.1).1). The protective effect of TRBL-2 was eliminated by heat inactivation. DGGE analysis showed that piperacillin-tazobactam caused a significant disruption of the indigenous microflora but that piperacillin-tazobactam in conjunction with TRBL-2 caused a relatively minor alteration of the microflora (mean similarity indices of mice treated with piperacillin-tazobactam and mice treated with piperacillin-tazobactam and TRBL-2 in comparison to those of saline controls were 33% and 86%, respectively; P < 0.001) (Fig. u200b(Fig.22). n n n nFIG. 1. n nEfficacy of oral β-lactamase in preventing piperacillin-tazobactam-induced overgrowth of VRE. The densities (log10 CFU/g) of stool VRE are shown after the orogastric inoculation of 104 CFU of VRE on day 0. Prior to inoculation, none of the mice ... n n n n n nFIG. 2. n nDGGE analysis of stool microflora of individual mice. Lane 1, controls containing rRNA genes amplified from strains of Bacteroides thetaiotaomicron, Bacteroides uniformis, and Escherichia coli (top to bottom); lanes 2 to 4, saline control mice; lanes ... n n n nThese findings provide further evidence that oral β-lactamase treatment may be an effective means to preserve colonization resistance during therapy with broad-spectrum, parenteral β-lactam antibiotics. Additional studies are needed to determine the efficacy of this strategy as a means to limit the dissemination of nosocomial pathogens in humans.

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.

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.