Elias Westermarck

Interaction between probiotic lactic acid bacteria and canine enteric pathogens: a risk factor for intestinal Enterococcus faecium colonization?

Selected probiotic lactic acid bacteria (LAB) have been shown to elicit positive health effects particularly in humans. Competitive exclusion of pathogens is one of the most important beneficial health claims of probiotic bacteria. The effect of probiotic LAB on competitive exclusion of pathogens has been demonstrated in humans, chicken and pigs. In this study we evaluated the ability of certain LAB strains (Lactobacillus rhamnosus GG, Bifidobacterium lactis Bb12, Lactobacillus pentosus UK1A, L. pentosus SK2A, Enterococcus faecium M74 and E. faecium SF273) to inhibit the adhesion of selected canine and zoonotic pathogens (Staphylococcus intermedius, Salmonella Typhimurium ATCC 14028, Clostridium perfringens and Campylobacter jejuni) to immobilised mucus isolated from canine jejunal chyme in vitro. Adhesion of C. perfringens was reduced significantly by all tested LAB strains, between 53.7 and 79.1% of the control without LAB, the LAB of canine origin yielding the best reduction. The adhesion of S. Typhimurium and S. intermedius were not significantly altered by any of the LAB included in the study. Both enterococci tested significantly enhanced the adhesion of C. jejuni, to 134.6 and 205.5% of the control without LAB. E. faecium may thus favor the adhesion and colonization of C. jejuni in the dogs intestine, making it a potential carrier and possibly a source for human infection. Enhanced C. jejuni adhesion is a new potential risk factor of enterococci. Our results further emphasize the importance of safety guidelines to be established for the probiotics intended for animal use.

Absence of host specificity for in vitro adhesion of probiotic lactic acid bacteria to intestinal mucus

Adhesion of probiotic lactic acid bacteria (LAB) has been reported to be host species specific. Host specificity is regarded as a desirable property for probiotic bacteria and therefore recommended as one of the selection criteria. However, previous studies have indicated that LAB originating from one host adhere well also to the mucus of other species. The aim of the study was to investigate the host specificity of LAB adhesion in human, canine, possum, bird and fish mucus in vitro. An in vitro mucus adhesion model was utilized in this study using immobilized mucus from faeces or intestinal material of these hosts. The results indicate that the adhesion trait was not host specific but rather was characteristic to LAB species. In conclusion, mucus adhesion properties are more dependent on the LAB strain than on the host. This suggests that animal models in probiotic adhesion assays may be more applicable to other species than thought earlier. Positive health effects facilitated by adherent probiotics in humans may also denote the possibility of similar outcome in other species and vice versa.

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.

Comparison of diagnostic methods for detecting gastric Helicobacter-like organisms in dogs and cats.

Diagnostic methods for detecting gastric Helicobacter-like organisms (GHLOs) in dogs and cats were compared. Samples for brush cytology, the urease test and histological examination were collected post mortem from the fundus, corpus and antrum of 10 dogs (17 sample sites from each animal) and 10 cats (14 sample sites each). Samples of tissue from the fundus or corpus were taken for transmission electron microscopy and culture from three and eight dogs, respectively, and from six cats that gave a positive urease test with samples from these regions. In all dogs and in six of the 10 cats, GHLOs were detected by at least one of three methods (brush cytology, urease test or histological examination) in all regions. By brush cytology, GHLOs were demonstrated in all samples from the dogs and the positive cats. In cats, the urease test (60 min) gave a positive result in every sample site; in dogs it gave a positive result in 100% of the corpus samples, in 95% of the fundus samples and in 62% of the antral samples. Histological examination revealed GHLOs in all samples from the fundus and corpus of the dogs and of the positive cats; and in 74% and 91.7% of the antral samples of the dogs and cats, respectively. GHLOs were seen in all dogs and cats studied by transmission electron microscopy, and culture of gastric tissue was successful in 3/8 dogs and 1/6 cats. In this study, brush cytology was thus the most sensitive method for demonstrating GHLOs.

Exocrine pancreatic atrophy in German Shepherd Dogs and Rough-coated Collies: an end result of lymphocytic pancreatitis.

Previously published studies of the pathology of canine exocrine pancreatic insufficiency (EPI) have been based on morphological findings during the clinical phase of the disease, when atrophy of acinar parenchyma occurs. Recently, low serum trypsinlike immunoreactivity (TLI) concentration has been shown to precede clinical signs, making it possible to diagnose EPI prior to onset of the clinical disease. This study presents histological and ultrastructural findings of pancreatic biopsies from 11 German Shepherd Dogs and 2 Rough-coated Collies with subclinical EPI (SEPI). These findings were compared with those from dogs with clinical EPI (n = 11) and healthy control dogs (n = 5). Biopsied tissue from dogs with SEPI typically contained both normal and atrophied acinar parenchyma. The most significant finding was the marked lymphocytic infiltration, which was most prevalent at the border zone of affected and nonaffected parenchyma but had spread into the normal acinar tissue. Numerous intraacinar lymphocytes were found. Most of the lymphocytes were positive by immunostaining for CD3. In more advanced stages of destruction, the findings were characteristic of pancreatic acinar atrophy. In the atrophied parenchyma, the inflammatory reaction, if present, was less prominent. Ultrastructural changes were in accordance with those of the histological study showing infiltration of lymphocytes both in affected acini and in acini that revealed no obvious ultrastructural changes. Progressive degenerative changes of acinar cells were considered a nonspecific finding. Apoptotic death of acinar cells was occasionally found. The inflammatory reaction was clearly shown to precede the pancreatic acinar atrophy, and the findings suggested that lymphocytic pancreatitis leads to atrophy of the pancreas. The possibility that EPI is an immune-mediated disease in German Shepherd Dogs and Rough-coated Collies is discussed.

Streptococcus alactolyticus is the dominating culturable lactic acid bacterium species in canine jejunum and feces of four fistulated dogs.

Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4x10(6) to 1.5x10(7) CFU ml(-1) were obtained in jejunal chyme. In the fecal samples 7.0x10(7) and 2.0x10(8) CFU g(-1) were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16S and 23S rDNA restriction fragment length polymorphism patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both feces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected.