Intestinal Microbes and Digestive System Disease in Dogs

The intestinal microbiota comprises viruses, bacteria, fungi, and protozoa. In the past, the word microflora has been used to describe this complex ecosystem, but microbiota (from –bios, “living organisms”) is the more appropriate term. The microbiome is the collective genome of all these microbes. Most studies to date have focused on the bacterial microbiota, which is estimated to make up the vast majority of the intestinal microbiota.

An estimated 100 trillion microbial cells are present within the intestine, up to 10 times the number of mammalian cells in the whole body. Combined, microbial genes outnumber host genes by an estimated factor of 10.

This complex ecosystem of gut bacteria has a tremendous influence on host health. The interactions between bacteria and host are mediated through direct contact between microbes and the immune system and through various microbiota-derived metabolites. A physiologic microbiome modulates the immune system, protects against enteropathogens, and provides nutritional benefits to the host.

Conversely, changes in the intricate relationship between gut bacteria and host cells affect the host’s immune responses and metabolic status and may result in disease (Figure 1). Recent studies have described intestinal dysbiosis (ie, changes in intestinal microbiota composition and/or diversity) in various acute and chronic gastrointestinal (GI) disorders.¹ Additionally, initial data in human and animal models have linked chronic dysbiosis, such as that caused by antibiotic exposure, to extraintestinal disorders such as diabetes and obesity.^2,3 These findings highlight the importance of gut microbiota and dysbiosis in regulating host metabolism, with effects reaching far beyond the GI tract.

The dysbiosis patterns and metabolic signatures observed in acute and chronic GI diseases and the metabolic syndrome are only beginning to be described. Dysbiosis signatures and metabolic alterations are being evaluated for their diagnostic and therapeutic potential. This article provides an overview of the bacteria in the canine intestine and the role of dysbiosis in the etiology of GI diseases.

Figure 1. The interplay between the intestinal microbiota and the host immune system and metabolism. (A) In a healthy intestine, proper spatial arrangements exist between the highly abundant luminal microbiota, the epithelium, and immune cells (dendritic cells, lymphocytes, macrophages, neutrophils) that are present in low numbers. The microbiota provides immunomodulatory stimuli to the immune system and metabolizes and ferments complex carbohydrates into beneficial short-chain fatty acids (SCFA). These SCFA provide energy for endothelial cells, are anti-inflammatory, and regulate intestinal motility. The normal microbiota also converts primary bile acids to secondary bile acids, which are also anti-inflammatory, induce glucagon-like peptide 1 (which in turn increases insulin), and decrease, for example, sporulation of Clostridium difficile. (B) In a diseased state, regardless of the initiating cause, the decreased production of antimicrobial peptides and mucus leads to increased intestinal permeability and translocation of bacteria. Innate immune receptors, such as Toll-like receptors on macrophages and other immune cells, recognize specific pathogen-associated molecular patterns due to dysbiosis (eg, lipopolysaccharides in bacterial cell walls) and trigger inflammatory reactions. Macrophages phagocytize microbes, also triggering immune responses that can lead to oxidative stress. Oxidative stress, in turn, can cause intestinal dysbiosis.

The Intestinal Microbiota In Health

Identification and Components

Until recently, identification of intestinal bacteria was almost exclusively achieved using traditional bacterial culture. Fecal culture may still be useful for the detection of specific enteropathogens such as Salmonella or Campylobacter jejuni, as this approach allows for antibiotic susceptibility testing of clinical specimens, but the vast majority of intestinal bacteria are strict anaerobes and undetectable using standard cultivation methods. Therefore, routine bacterial culture does not allow in-depth characterization of complex intestinal bacterial communities (Figure 2). A number of molecular-based methods are now used for the characterization of the intestinal microbiota.⁴

Traditional bacterial culture as well molecular approaches have revealed differences in the type and amount of bacteria along the GI tract. Bacterial numbers in the duodenum of healthy dogs range from 10² to 10⁹ colony-forming units (cfu)/g.⁵ The colon harbors much higher numbers, with up to 10¹¹ cfu/g.⁶

Molecular tools have allowed identification of previously uncultivable, and hence unknown, bacteria. While the duodenum harbors a mixture of aerobic and facultative anaerobic bacteria, the colon is colonized almost exclusively by strict anaerobes.⁷ The bacterial groups Clostridiaceae, Bacteroidaceae, Prevotellaceae, and Fusobacteriaceae predominate in the large intestine (Figure 2). Of note is that every dog harbors a unique microbial community, with distinct differences in the proportions of these bacterial groups. Yet the bacterial gene content is conserved across individuals, suggesting that functional aspects of the microbiomes are similar across animals.⁸ Nevertheless, individual differences in microbial species may cause individualized responses to different diets, fiber sources, and probiotics.

The GI tract is also home to a diverse population of viruses and fungi. One study described up to 40 fungal species in canine fecal samples; most were various Candida spp.⁹ Based on these findings, it is expected that fungal organisms would be observed on routine fecal smears. Their exact contribution to health and disease remains unclear, as no significant differences in the types of fungi were reported when healthy dogs were compared with dogs with acute diarrhea.

Figure 2. Predominant bacterial taxa in fecal samples of healthy dogs. Each bar represents one healthy dog. Note that the bacterial families present vary widely, anaerobic bacteria predominate, and the ratios of these bacterial groups differ in each healthy dog. Routine bacterial culture misses these anaerobic bacteria. Fecal smears that evaluate ratios of gram-positive to gram-negative bacteria are also not useful to assess dysbiosis, as each dog harbors a unique ratio of these groups.

Roles

A balanced microbiome is crucial for maintaining host health. The normal microbiota performs the following functions:

• Modulates the immune system
• Keeps invading enteropathogens in check
• Provides nutrients to the host by metabolizing and fermenting various dietary components

Intestinal bacteria also contribute to the development of gut physiology. This has been demonstrated in studies with germ-free raised mice, which show altered epithelial architecture (eg, decreased number of lymphoid follicles) compared with mice that have been exposed to bacteria at birth.

Consistent crosstalk between the intestinal microbiota and host immune cells is mediated through a combination of microbial-derived metabolites (eg, short-chain fatty acids [SCFA], indole, secondary bile acids) as well as molecules on the surface of bacteria that activate receptors of the host innate immune system (eg, Toll-like receptors, dendritic cells).

Commensal bacteria, which prevent mucosal invasion of transient pathogens through competition for nutrients and epithelial adhesion sites, are an important part of the intestinal barrier. Furthermore, they establish a physiologically restrictive environment for nonresident bacterial species through secretion of antimicrobial compounds and modulation of luminal pH.

The major bacterial groups in the gut are strict or facultative anaerobes. The predominant bacterial families in the large intestine (Figure 2) ferment dietary carbohydrates (eg, starch, cellulose, pectin, inulin), resulting in the production of SCFA (eg, acetate, propionate, butyrate) and other metabolites. SCFA are an important source of energy and growth factors for intestinal epithelial cells and have a modulating effect on intestinal motility. Furthermore, SCFA are immunomodulatory. For example, butyrate induces immunoregulatory T cells and acetate beneficially modulates intestinal permeability.

An emerging area of research aims to better characterize the biologic functions of additional bacterial-derived metabolites that have been recently recognized as regulators of host health, such as indole and secondary bile acids. For example, dietary tryptophan is metabolized by bacteria into indole, which has been shown to decrease interleukin-8 expression, strengthen intestinal barrier function, and ameliorate nonsteroidal anti-inflammatory drug–induced enteropathy in mice.¹⁰ Some bacterial species in the colon convert primary bile acids into secondary bile acids (eg, lithocholic and deoxycholic acids). Therefore, secondary bile acids are present at much higher concentrations in the colon than primary bile acids. This high concentration of secondary bile acids in the colon is beneficial, as these acids are an important regulator of host homeostasis through activation of various receptors throughout the body. For example, the bile acid–specific membrane receptor TGR5 is expressed in the gallbladder, in bile duct epithelium, on monocytes and macrophages, and in muscle, kidney, pancreatic, and intestinal cells. The activation of these receptors, for which secondary bile acids have the highest affinity, downregulates the expression of proinflammatory cytokines and modulates insulin and glucose metabolism through activation of glucagon-like peptide 1.¹¹

Secondary bile acids also inhibit germination of Clostridium difficile spores, whereas an increase in primary bile acids (an effect of dysbiosis) promotes the germination of bacterial spores. Intestinal dysbiosis leads to a decrease in bile-acid converting bacterial species and, therefore, is associated with bile acid dysmetabolism (decrease in secondary and increase in primary bile acids) and potentially systemic effects on host metabolism.^12,13 Furthermore, an abnormal increase in primary bile acids may cause secretory diarrhea.

Dysbiosis

Intestinal dysbiosis is defined as differences in ratios of bacterial groups compared with those found in healthy dogs and is often accompanied by a reduction in species diversity. Intestinal dysbiosis has been reported in various acute and chronic GI disorders, but it can also be induced through use of broad-spectrum antibiotics.¹

The importance of the gut microbiota in host immune regulation and metabolism (Figure 1) means that a dysbiotic microbiome may have negative consequences for the host. However, the extent of clinical signs varies between individuals. For example, administration of metronidazole to healthy dogs induced major changes in the gut microbiota, with reductions in commensal anaerobic bacteria and a concurrent increase in Escherichia coli; these changes were accompanied by extensive changes in metabolomic pathways in the gut lumen (eg, increase in oxidative stress, reductions in secondary bile acids).¹⁴ Nine of 16 dogs developed loose stools while on antibiotics, but the remaining dogs exhibited no clinical signs despite having similar microbial and biochemical changes. This suggests that clinical signs depend on the interplay of multiple microbial and host factors, some of which (eg, underlying genetic susceptibility of the host, dietary and environmental triggers) remain to be elucidated. Nevertheless, antibiotic-induced dysbiosis is an example of how changes in microbial composition and metabolism can affect host health, as antibiotic-induced dysbiosis in early childhood or repeated pulse therapy has been recognized as a risk factor for development of allergies, obesity, and inflammatory bowel disease in humans.^3,15

The emerging epidemiologic data in humans and our evolving understanding of the immunomodulatory and metabolic properties of the gut microbiota suggest that proper diagnosis and correction of dysbiosis will be important goals in various diseases. A dysbiotic microbiome may cause harm through several mechanisms (Box 1), which are likely to be concurrent. Diarrhea can be due to bacterial enterotoxins that stimulate mucosal fluid secretions. Another recently recognized mechanism for diarrhea in humans is bile acid malabsorption due to the inability of the dysbiotic microbiota to convert primary to secondary bile acids.¹³ Initial studies suggest that such a mechanism may also occur in dogs and warrants further study.^12,16

Assessment of Dysbiosis

Because of the importance of the commensal microbiota to host homeostasis, it is important to diagnose dysbiosis. As noted, fecal bacterial culture cannot characterize the many anaerobes in the GI tract. It is estimated that only a very small percentage of intestinal bacteria are cultivable with standard laboratory techniques. The best way to fully characterize the microbiota is by high-throughput sequencing platforms that can provide an overview of the proportions of all bacterial groups within a sample; however, cost and long turnaround times limit the use of this method to research studies. Use of polymerase chain reaction (PCR) assays to target specific bacterial taxa that are consistently altered in dogs with chronic enteropathies (CE) can provide more rapid results.¹

The results of these multiple PCR assays can be combined and expressed as a mathematical ratio, the Dysbiosis Index (DI; Figure 3). A negative DI (<0) indicates a normal microbiota, whereas a positive DI (>0) indicates dysbiosis associated with CE.¹⁷ The DI can then be used to monitor the microbiota’s response to therapy for CE. Initial long-term follow-up studies in dogs with CE suggest that the microbiome requires several months to normalize, even when dogs respond within a few weeks with a decrease in clinical activity scores.¹⁸

A recent small study evaluating 3 dogs with CE used the DI to monitor fecal microbial changes in response to fecal microbial transplantation.¹⁹ All 3 dogs initially showed an immediate reduction in the DI, but after 3 weeks dysbiosis returned in 1 dog (increase in DI above 2), which showed no improvement in clinical signs. In the remaining 2 dogs, a partial improvement in clinical signs was observed, and their DI stayed below 0 for most of the 8-week follow-up period. This initial small dataset suggests a potential for monitoring the microbiota over time in patients with CE and after fecal microbial transplantation, but more studies are needed to determine the accuracy and clinical utility of microbiota analysis.

Analysis of fecal samples provides information only about changes in the luminal microbiota. The use of fluorescence in-situ hybridization (FISH) of intestinal biopsy specimens allows visualization of whether bacteria have translocated into the mucosal epithelium, as is observed in dogs with granulomatous colitis.²⁰ A positive result indicates the need for antimicrobial therapy to clear the translocated bacteria. FISH requires special analysis and is only available in a few reference laboratories.

Although assessing the fecal microbiota is useful for recognizing dysbiosis in the large intestine, fecal samples probably do not accurately reflect the situation in the small intestine. Although the fecal samples of many dogs with small intestinal disease show dysbiosis, a subset of dogs may have exclusively small intestinal dysbiosis. Measurements of serum cobalamin and folate concentrations remain the most useful markers of small intestinal dysbiosis. Serum cobalamin may be decreased and serum folate concentrations may be increased in dogs with small intestinal dysbiosis; alteration of both parameters is highly suggestive of the condition.

Recent studies have evaluated links between dysbiosis and changes in various biochemical pathways (eg, abnormal metabolism of bile acids, amino acids, and tryptophan) that affect the host immune system and metabolism.^18,21 Many novel metabolic biomarkers, such as concentrations of fecal bile acids, are being investigated for better assessment of the etiology and treatment of GI diseases, and these may soon become useful for routine practice.

Figure 3. The Dysbiosis Index (DI) is increased in dogs with chronic enteropathies (CE). The DI is a mathematical ratio that summarizes the abundances of various bacterial groups (eg, E coli, Faecalibacterium, Blautia, Fusobacterium) into a single number. A DI below 0 indicates a normal microbiota, and a DI above 0 indicates dysbiosis in the large intestine. The DI assay is commercially available through the Gastrointestinal Laboratory at Texas A&M University.

Therapeutic Considerations for Correction of Dysbiosis

The microbiota is an important player in host metabolism. Recent metabolomic studies have clearly linked dysbiosis with various diseases within and outside the GI tract. However, more work is needed to determine how to modulate the microbiome for best therapeutic success and to predict response to a specific therapy.

Diet and Antimicrobial Therapy

Dysbiosis is present in many dogs with CE and may be the cause of diarrhea in some patients, but dysbiosis may also be the consequence of GI inflammation in other patients. A gradient of various disease patterns across patients is likely, with host immune system and microbiome contributing to various degrees. Therefore, the presence of dysbiosis does not equate to an immediate need for antimicrobial therapy, as dogs with diet-responsive CE may also have a dysbiosis. Some animals with diarrhea respond favorably to antimicrobials, but antibiotics may induce diarrhea in others. Long-term antibiotic administration may induce dysbiosis patterns that could create a risk factor for various metabolic diseases, such as through induction of bile acid dysmetabolism.³ At this time, the best therapeutic approach to chronic GI disease remains empiric, with a sequential protocol of food trials, anti-inflammatory drugs, and/or antimicrobials.

Probiotics and Prebiotics

Because the microbiota is implicated in the pathophysiology of chronic GI disease, the addition of probiotic and prebiotic therapy may be appropriate. Probiotics are live microorganisms that, when administered in sufficient quantities, confer a health benefit to the host. Few studies have evaluated the benefits of probiotics in acute and chronic GI disease. Data suggest that probiotics have only a minor effect on the intestinal microbiota, but their beneficial effect in dogs with inflammatory bowel disease may be due in part to immune stimulation and/or enhancement of intestinal barrier function.²² Also, it appears that administration of higher doses and multiple strains leads to a higher probability that probiotic bacteria will be able to colonize the gut.

Prebiotics are fermentable and nonfermentable fibers that, after reaching the large intestine, are metabolized by intestinal bacteria to produce SCFA and other metabolites that may be immunomodulatory. Most commercial intestinal diets contain prebiotics.

Fecal Microbial Transplantation

Fecal microbial transplantation has garnered a lot of interest. Although it is a highly successful therapeutic approach in humans with recurrent C difficile, its use in CE of dogs requires further study because the pathophysiology between these diseases differs. Anecdotal evidence and small-scale studies suggest that fecal microbial transplantation may be promising in a subset of dogs with CE,¹⁹ but at this time proper patient selection is purely empiric and more studies are required.