11 Feb 2019
Kate Parkinson discusses new evidence on ways to manage this condition, aimed at general practitioners.
Kate Parkinson
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Image © huntun / Adobe Stock
All healthy organisms have a large bacterial community inhabiting their mucosal surfaces. This community is called the microbiota. Imbalance of these microorganisms is called dysbiosis. Interest in the microbiota has increased in the past decade. The faecal microbiome is easily accessible, but only 20% is culturable, so DNA sequencing is often used to identify bacterial DNA.
Acute and chronic gastrointestinal diseases, such as inflammatory bowel disease, are often associated with changes in the microbiota, and these changes may help diagnose and treat this condition. Antibiotics may alter the gastrointestinal microbiota, especially if given to neonates whose bacteria community has not fully developed. Probiotics may induce positive changes in the microbial community, but, in adults, most probiotics cause only minor changes lasting a few days.
Re-establishing a healthy and complex microbiota may be fundamental to treat many diseases. This may be achieved via faecal transplantation from healthy animals or auto-transplantation of healthy microorganisms isolated from a patient prior to illness. As complex interactions of host and microbiota may be involved in many diseases, treatments involving the microbiota may require a personalised approach to achieve their full potential.
All healthy organisms have trillions of microorganisms living on their mucosal surfaces (Werling and Barfield, 2018). In fact, there may be more than 10 times as many cells in your intestines than the rest of your body (Schmitz and Suchodolski, 2016) and more than 150 times more microbial DNA in your intestines than in your own cells (Werling and Barfield, 2018).
This vast and complex bacterial community is called the microbiota (Schmitz and Suchodolski, 2016). The entire genetic mass of microorganisms (bacteria, viruses, fungi, eukaryotes and archaea) is called the microbiome (Schmitz and Suchodolski, 2016).
The entire microbiota is critical to health (Chandler, 2017) and varies between individuals (Carding et al, 2015). A balanced and stable community of microorganisms is required for an effective immune response (Petersen and Round, 2014). These complex communities may be affected by many factors, including host genetics, diet, infection and medical intervention, especially antibiotics (Werling and Barfield, 2018). In fact, microbiota management may share more similarities with park governance than medicine, requiring a multi-pronged approach of habitat restoration, promotion of native species and targeted removal of invasive species (Chandler, 2017).
The microbiota constantly interacts with the environment and host genetics to maintain a stable, healthy state. Disruption of this stability causes disease (Spor et al, 2011), leading to loss of beneficial microorganisms, expansion of harmful microorganisms and loss of overall diversity (Petersen and Round, 2014). This is called dysbiosis.
Dysbiosis is associated with an abnormal immune response and is thought to be involved in immune-mediated diseases, such as inflammatory bowel disease (IBD), multiple sclerosis, asthma, diabetes and allergies (Petersen and Round, 2014). Patients affected by these conditions may have a different microbial community than healthy individuals; however, no consistent pattern of microbiota alterations has been identified.
Interest in the composition of the intestinal microbiota and the possibilities of its therapeutic modification has soared over the past decade (Schmitz and Suchodolski, 2016), with exponential growth in research papers on the gut microflora. Although much of this research has focused on the human microbiome, a growing interest in dysbiosis exists in the veterinary world.
Neonatal gut flora is first accumulated during intrauterine life (Gosalbes et al, 2012). After birth bacteria are shared between mother and child (Werling and Barfield, 2018), and breast milk delivers further commensal bacteria to the infant intestine (Jimenez et al, 2008). These bacteria are required to digest colostrum and are vital for postnatal immune system development (Werling and Barfield, 2018).
Early postnatal gut bacteria are highly changeable (Gensollen et al, 2016), but show limited variety. As neonates develop, the microbiota increases in diversity and progressively stabilises. Adult microbiota does not change dramatically, although bacterial number and diversity do increase gradually along the gastrointestinal tract (Schmitz and Suchodolski, 2016). This postnatal colonisation coincides with a period where the immune system is susceptible to microbial instruction, modulating early immune system development (Gensollen et al, 2016). Interference in this development may predispose patients to inflammatory or allergic disease later in life (Schmitz and Suchodolski, 2016).
In addition to receiving bacteria from their mother, neonates are exposed to microorganisms in their environment – in turn affecting their microbiota. For example, increased maternal exposure to microbes during pregnancy is associated with a decreased risk of atopic disease (Gosalbes et al, 2012).
Pigs raised indoors have less diversity and more pathogenic bacteria in their intestinal tract than pigs raised outdoors (Werling and Barfield, 2018). Genetically related individuals often share microbiota (Spor et al, 2011) and the intestinal microbiota of pet dogs may overlap with that of their human family (Werling and Barfield, 2018).
The faecal microbiota is easily accessible and frequently analysed, but does not necessarily reflect the intestinal microbial composition (Carding et al, 2015). Only 20% of the microbiota is culturable with conventional techniques (Petersen and Round, 2014), although culture medium enrichment may facilitate isolation (Brown et al, 2016). More diversity may be present in the mucosal layers than we can detect in faeces, even with advanced techniques such as high-throughput pyrosequencing.
Phyla, such as Firmicutes, Fusobacterium, Bacteroidetes, Proteobacteria and Actinobacteria, have all been identified in canine faecal microbiota (Schmitz and Suchodolski, 2016). Pathogenic species of bacteria include Escherichia coli, Clostridium perfringens and Salmonella, but these species may also be identified in healthy dogs’ intestines (Schmitz and Suchodolski, 2016). Even healthy individuals differ markedly in their microbial community.
Acute and chronic gastrointestinal diseases, such as IBD, are associated with microbiotic change (Chandler, 2017) and a breakdown in tolerance to gut bacteria (Round and Mazmanian, 2009). IBD patients have a decreased microbial population and reduction in the diversity and stability of the intestinal microbiota (Honneffer et al, 2014).
In IBD, commensal species are mistaken for pathogens (Honneffer et al, 2014) by the patient’s immune system, causing inflammation of the intestinal mucosa and, in some cases, beyond (Carding et al, 2015). Intestinal dysbiosis may cause the inflammation seen in IBD or be caused by a disturbed environment in the gastrointestinal tract.
As IBD is a multifactorial disease, it is likely the microbiota is only one of several factors influencing pathogenesis. Other factors include host genetics, immune system and environment (Vasquez-Baeza et al, 2016). This may be the cause of the overlap of dysbiosis seen in healthy and diseased dogs, which has also been reported in humans with IBD (AlShawaqfeh et al, 2017).
IBD is more common in certain breeds: for example, German shepherds, boxers, soft-coated wheaten terriers and Weimaraners (Allenspach and Jasani, 2014). German shepherds are particularly prone to chronic enteropathies, possessing changes in the receptors that recognise pathogens in the intestines and increased numbers of specific bacterial classes compared to other breeds (Allenspach et al, 2010).
On histopathology patients, with food allergies, idiopathic IBD and antibiotic-responsive diarrhoea all show lymphocytic, plasmacytic and/or eosinophilic infiltration. Due to this, IBD is difficult to diagnose by histopathology alone unless all other gastrointestinal disease is excluded (Allenspach and Jasani, 2014). A diagnostic workup often includes blood screening, urinalysis, abdominal imaging, diet trials and possibly biopsy.
Faecal samples are frequently unhelpful, as if a pathogenic organism is identified in faeces it is impossible to tell if this is the sole causing agent of the diarrhoea or a complicating factor of an underlying disease (Allenspach, 2007).
Blood tests are often likewise unrewarding, though hypocobalaminaemia and hypoalbuminaemia with high endoscopic score from duodenal biopsies are poor prognostic indicators (Allenspach, et al, 2007). An increase in mucosal echogenicity of the intestinal wall on ultrasound examination may help detect intestinal inflammation (Gaschen et al, 2008), and microbiota alteration may help us diagnose and treat this condition. As techniques advance, genetic analysis of biopsies and faeces may become commonplace.
Although antibiotics are often used in cases of chronic gastrointestinal disease (Schmitz and Suchodolski, 2016), we have little information on the effects of antibiotics on the canine microbiota (Suchodolski et al, 2009). Adult faecal microbiota is generally resilient to short-term antibiotic modification, but the effects on specific bacteria taxa may persist for several months (Suchodolski et al, 2009).
Treatment trials with antibiotics, such as metronidazole, tylosin and oxytetracycline, for four to six weeks seem to help some dogs with antibiotic responsive diarrhoea. Some patients will resolve permanently, but others will relapse and require long-term treatment (Allenspach and Jasani, 2014). Diseased dogs often have an altered microbial composition and it is possible antibiotics, such as tylosin, cause microbial changes that differ from those seen in healthy animals (Suchodolski et al, 2009).
Exposure to antibiotics in early life may result in permanent changes to the microbial composition in adults. In piglets and mice, antibiotic usage within the first week of life can affect growth (Yu et al, 2018; Zeineldin et al, 2018) and, in mice, behaviour (Leclercq et al, 2017). In addition, human children exposed to antibiotics within the first year of life have an increased risk of developing IBD, asthma and eczema (Gensollen et al, 2016).
Probiotics, prebiotics, or a combination of the two (synbiotics), are often used to modify intestinal microbiota (Schmitz and Suchodolski, 2016). Probiotics may enhance the growth of favourable microorganisms, increase bacterial adherence to the intestinal mucosa, displace pathogens from the intestinal wall, strengthen the gut-epithelial barrier, regulate various metabolites, and modulate the immune system to confer advantage on the host (Costello et al, 2012).
Probiotics have been used in human medicine since 2000 BC (Ozen and Dinleyici, 2015). Probiotic compounds have been shown to induce positive changes in the microbiota in the large intestine, are unlikely to cause harm and may contain other compounds, such as kaolin, that assist in swift resolution of a transient gastrointestinal upset. However, probiotic products do not have to prove their efficacy in applications, diseases or species, and little scientific evidence confirms the most effective bacterial strains and doses (Schmitz and Suchodolski, 2016).
Probiotics can expire, manufacturing processes may vary and individuals often respond differently. Most probiotics cause minor changes lasting only a few days, with a transient increase in administered species (Chandler, 2017). In adults with an established microbiota, the bacteria ingested are unlikely to colonise the gut and tend to be present, if at all, only for the period the probiotics are supplied, though some combination products may persist if given at high doses for long periods (Schmitz and Suchodolski, 2016).
Many studies have attempted to measure the effect of prebiotics, synbiotics or probiotics on the intestinal microbiota. However, some promising in vitro results could not be replicated in vivo during testing (Round and Mazmanian, 2009). Many studies are relatively small and take place in healthy dogs. Comparison is difficult, as wide ranges of products and doses are often administered. Despite these limitations, it is clear some bacterial strains show promise as probiotics (Schmitz and Suchodolski, 2016).
VSL#3, a high-dose multi-strain human probiotic used in IBD and Crohn’s disease, showed protective effects in patients with IBD; decreasing clinical and histological scores, decreasing T-cell infiltration and normalisation of dysbiosis after long-term therapy (Rossi et al, 2014).
A study (Rossi et al, 2018) demonstrated anti-proliferative and anti-inflammatory effects in high-dose multi-strain probiotics given for 60 days to canine patients with IBD and colonic polyps. Another study (Rossi et al, 2014) showed clinical improvement in dogs diagnosed with chronic enteropathies fed a hydrolysed diet plus probiotic supplement.
Over-the-counter probiotic products include Enterococcus, Lactobacillus, Bifidobacteria, yeasts, Bacillus and other fungi. VLS#3 includes four Lactobacillus strains, three bifidobacteria strains and one strain of Streptococcus thermophilus (Rossi et al, 2014). Several probiotic strains have been assessed by the European Food Safety Authority (EFSA). Of these, one strain of Enterococcus faecium has been approved for use in food-producing and small animals. The EFSA has not concluded the efficacy of other strains, including Bifidobacterium and Lactobacillus.
Although E faecium NCIMB 19415 (SF68) is the most commonly used strain of probiotic bacteria, not much is still understood. Little evidence is available about the correct dose and survival time of bacteria, or even if bacterial survival is required or if the ingestion of bacterial DNA is sufficient. Likewise, little is known about the best form of probiotic products. Are pills or powders, liquids, capsules, in-feed supplements or natural sources, such as yoghurt, more effective?
Some benefit exists in probiotic use in infectious acute intestinal disease, stress-induced diarrhoea, antibiotic-induced diarrhoea and idiopathic diarrhoea, though results vary. In cases of chronic diarrhoea secondary to IBD, probiotics are used to transiently influence the composition of microbiota and alleviate clinical signs, and some improvement has been noted in dogs treated with probiotic and diet compared to dogs treated with diet alone (Sauter et al, 2006).
Although probiotics are usually considered safe, one study in human patients showed an increased risk of death in patients with severe acute pancreatitis treated with probiotics. Some caution is advised before treating extremely sick patients with high-dose probiotics (Sherman et al, 2014).
The intestinal microbiota is vital for continued health, and dysbiosis can pose a serious problem. Rebiosis – re-establishing a healthy complex microbiota following dysbiosis – may be fundamental to treating some diseases (Petersen and Round, 2014).
Faecal transplants involve transferring intestinal microbiota from a healthy host to a patient affected by disease (Pereira et al, 2018). This method has proved effective in humans with C difficile infection – producing a high cure rate in human patients following a single faecal transplant (Petersen and Round, 2014). Canine studies are limited, though faecal transplants administered by enema to eight dogs with chronic diarrhoea thought to be caused by C perfringens appeared successful three months post-transplant (Schmitz and Suchodolski, 2016).
Faecal transplants may be carried out via enema (Pereira et al, 2018), by mixing faeces with ingested food, or by endoscopic or nasogastric administration. Some methods require anaesthesia of the canine recipient, which has the potential to change the microbiota. At present, although faecal transplantation methods show some success (Pereira et al, 2018), we do not know which method is the most effective. Faecal microbiota transplantation in parvovirus-infected puppies improved clinical recovery and decreased hospitalisation time (Pereira et al, 2018). Faecal transplants may be useful in some dogs failing other treatment options (Petersen and Round, 2014).
Although much research has little immediate application for the general practitioner, the potential of further research into the gastrointestinal microbiota, and its effect on the health and immune system of patients, is extremely interesting.
Advanced DNA sequencing can help us identify bacterial DNA found in faeces or intestinal biopsies. However, we can only identify species we already know, so it is possible many new species of bacteria are waiting to be identified.
Molecular-based assays may help us examine species and strain diversity, monitor the functionality of the microbiome throughout a patient’s life and gain an improved understanding of the pathophysiology of gastrointestinal diseases. This information could lead to new diagnostic and therapeutic approaches to dysbiosis, allowing us to manipulate the gut microbiota to maintain health and treat disease (Suchodolski, 2016).
The intestinal microbiome affects the immune system, so we may be able to reduce antibiotic usage by altering the microbiome (Werling and Barfield, 2018) to increase resistance to disease. Increased understanding of the microbiome may allow us to preserve the microbiota while treating illness.
At present, we still do not know the best method of administering healthy microbes to patients with dysbiosis. Faecal transplants are unreliable and may be distasteful to owners, while probiotics vary in effectiveness.
A safe, effective, easy, cheap and reliable delivery method of a proven combination of probiotic bacteria, perhaps a combination of strains freeze-dried and used as a probiotic pill (Leclercq et al, 2016), may increase the effectiveness of rebiosis.
In the future it may be possible to sample a patient’s own healthy microbiota prior to illness. The gut may then be seeded with bacterial starter colonies following potentially damaging treatments, such as chemotherapy.
The patient’s immune system may more readily recognise auto-transplanted bacteria, which fill pre-existing environmental niches and recolonise the intestine.
In conclusion, dysbiosis is a contentious issue, with many studies being relatively small and producing conflicting results. Although we know that the gastrointestinal microbiota can influence the immune system, we do not know exactly which bacteria are vital for a healthy microbiota and which are pathogenic.
Complex interactions of the microbiota and host may be involved in many immune-mediated diseases. Ultimately, treatments involving the microbiota may require a personalised approach to achieve full potential (Carding et al, 2015).
The author would like to thank Linda Matthewman BVSc, PhD, DACVIM, SAIM, MRCVS for access to her thesis (Matthewman, 2018) and assistance beyond the cause of duty, and Dirk Werling DrVetMed, PhD, MRCVS for help, interest, and assistance with this article.
