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SMALL THINGS CAN HAVE BIG INFLUENCES

The gastrointestinal microbiome and its effects

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Webinar notes Intestinal microbiome: “the total collection of microbial organisms within the gastrointestinal tract (GIT) of a host animal”. ƒƒPredominantly bacteria but also includes viruses, fungi and protozoa ƒƒCommensals and pathogens

It is essential for the normal development and functioning of the gastrointestinal tract and also general health.¹

Microbiome composition (and function) differs between species, breeds and even individuals within that species.1-5

This is determined by1-5: ƒƒEcological niche • And the dietary implications that this presents ƒƒEnvironment • E.g. seasonal food availability ƒƒGenetics • Demonstrated by variation between breeds and observed signatures of heritability

Do some genotypes encourage an intestinal microbiome that gives a clinically useful advantage? ƒƒThis is the basis for the development of Procanicare – dogs with renowned ‘iron stomachs’ were initially studied by Dr Beasley, when developing the product and deciding which Lactobacillus strains to include.

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Intestinal microbiome function Local effects

Digestion Digestion of non-digestible material, e.g. cellulose1: ƒƒAllows exploitation of ecological niches otherwise unsuitable for mammals ƒƒManagement of endogenous intestinal mucus

Short chain fatty acid (SCFA) production1: ƒƒSource of energy (for host and for microbiome itself) and many functions and effects across the body, e.g. • Direct nutrition to colonocytes • Instrumental in bile acid metabolism • Main source of energy for the bacteria of the microbiome • Essential for maintaining oxygen balance in the gut • Cholesterol metabolism and lipogenesis in peripheral tissues • Appetite regulation

Vitamin synthesis6: ƒƒDe novo synthesis and supply to host • Particularly B group vitamins

Protection Pathogen exclusion: ƒƒVia multiple mechanisms7,8 • Inhibition (production of antimicrobial peptides – AMPs) • Displacement • Competition

Maintain intestinal mucosal integrity1,8: ƒƒPrimarily via SCFA production • Direct nutrition, maintaining normal function • Alteration of tight junction integrity (higher levels of some SCFAs are associated with conferred protection against infection with enteropathogens) • Influence on mucin cells to promote mucus function (= physical barrier to pathogens)

Immunity and inflammation1,8: ƒƒMultiple effects via microbe metabolite production, e.g. • Act as signal molecules for altering numbers of inflammatory cell lines • Downregulating pro-inflammatory cytokines • Decreasing inflammatory gene expression in immune cells • Act as ligands for aryl hydrocarbon (AHR) receptors, regulating mucosal immune responses • Promote the production of AMPs

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Beyond the gut

Obesity The intestinal microbiome plays a role in the development and progression of obesity. Most studies of overweight and obese people show a dysbiosis characterised by lower diversity in their intestinal microbiome.1,9,10 ƒƒMany different metabolites and mechanisms are involved, affecting immune, energy and hormone regulation, e.g. • Higher production of SCFAs correlates with lower diet-induced obesity and with reduced insulin resistance • Butyrate and propionate seem to control gut hormones and reduce appetite and food intake in mice ƒƒStudies in identical twins have ruled out a genetic factor in these studies

Cognitive Accumulating data now indicates that the gut microbiota also communicates with the CNS - possibly through neural, endocrine and immune pathways - and thereby influences brain function and behaviour e.g.11-18 ƒƒThere is strong evidence that some species within the intestinal microbiome regulate the production and metabolism of serotonin, and its pre-curser amino acid tryptophan ƒƒSome microbiota have a direct anti-oxidant effect, reducing oxidative stress – a key factor for prognosis in many neurological disorders and following traumatic brain injuries ƒƒStudies in germ-free animals and in animals exposed to pathogenic bacterial infections, bacterial GI support products or antibiotic drugs, suggest a role for the gut microbiota in the regulation of mental health disorders, mood, cognition and pain ƒƒModulation of the gut microbiota may be a viable strategy for developing novel therapeutics for complex CNS disorders – so called psychobiotics • there is already evidence in humans that administering bacterial GI support products, with the intention of altering the intestinal microbiome, has positive effects on mental health conditions ƒƒThere is evidence that enteric microbiota play a role in early programming and later responses to acute and chronic stress ƒƒEvidence of an association between gut microbiome structure and dog aggression

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Neoplastic disease The intestinal microbiome changes during neoplastic disease.1,19 ƒƒNot clear if this is cause or effect (or both) • Some intestinal bacteria have been implicated in the development of some neoplastic diseases

Promising evidence of potential to reduce risk of neoplastic disease by modulating the intestinal microbiome, e.g.1 ƒƒBacterial GI support product formulations containing lactic acid bacteria have been shown to reduce the incidence of chemically mediated hepatocellular carcinoma and colon cancer in rats ƒƒButyrate has been shown to induce apoptosis of colon cancer cells

Atopic dermatitis Evidence from multiple studies to suggest that modulating the intestinal microbiome can significantly improve pruritus and visual skin lesions in atopic dogs.20,21 ƒƒIt is well recognised that administering bacterial GI support products to humans with atopic dermatitis has similar positive effects

Encouraging, as bacterial GI support products are widely accepted as safe for long term administration in dogs, with very low risk of side effects. ƒƒNot always the case for current therapeutics

Renal support One exciting pilot study has demonstrated that oral administration of various species of Lactobacillus increases glomerular filtration rate (GFR) and slows the decline of GFR over time in dogs with chronic kidney disease, compared to standard therapy.22 ƒƒThe same strains have demonstrated efficacy in humans for the prevention, treatment and maintenance of remission of pouchitis and ulcerative colitis, accelerate healing of gastric ulcers and reduce portal pressure in patients with cirrhosis. Recently, these strains have also been used in dogs with idiopathic inflammatory bowel disease (IBD) with promising results

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Pregnancy and early life 23-25 Changes (often dramatic) occur in intestinal microbiome during pregnancy naturally. ƒƒResult in unique inflammatory and immune changes, altered gut function and bacterial composition ƒƒOestrogen and progesterone impact the composition of the gut microbiome through their effect on bacterial metabolism and growth and virulence of pathogenic bacteria ƒƒHuman and animal studies have shown that microbial diversity in the gut at the start of pregnancy appears to be similar to that of non-pregnant women or animals. Yet, as the pregnancy advances, there are sometimes dramatic changes in the population. In humans, the abundance of gut bacteria associated with inflammatory states increased in nearly 70% of women

Maternal dysbiosis due to external causes during pregnancy may have detrimental effects on mother and foetus. ƒƒNegative dysbiosis during pregnancy – for example, as a result of antibiotic use - has been shown to increase the chances of chronic intestinal disorders for both mother and foetus and can even impact foetal brain development The intestinal microbiome of neonates starts to develop before birth and can be modulated during pregnancy, with the potential for long term effects for the neonate. ƒƒEvidence of maternal microorganisms being present in the meconium and cord blood suggest that the process of building an intestinal microbiome begins before birth. Evidence suggests the placenta and amniotic fluid are involved in this ƒƒIn humans, modulation of the maternal microbiome with bacterial GI support products during pregnancy has been shown to reduce the incidence of cutaneous allergic reactions in infants, with effects lasting into later life

Lactation is another source of bacteria for the development of the neonate’s intestinal microbiome. ƒƒMilk is being increasingly recognised as non-sterile, with many maternal microbiome bacteria species shown to be present in the breastmilk of humans

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The intestinal microbiome = an important aspect of health Preventative health approach is the best way to maintain its function. ƒƒA logical way to do this is via bacterial gut support products (most research where the intestinal microbiome was modulated in some way did so using these products)

Evidence suggests that this is worth doing from an early age – maybe even before birth.

Looking after the intestinal microbiome in practice Remember what can influence the intestinal microbiome – both positively and negatively – and adjust advice accordingly. ƒƒStress • provide advice to owners about supporting their dog’s intestinal microbiome in scenarios like changes of environment ƒƒDiet • Include advice that is sympathetic to the microbiome in dietary recommendations • E.g. switch diets gradually to try and reduce the risk of dysbiosis and consider the addition of bacterial GI support products • In a study comparing dogs fed a commercial extruded canine diet and those fed a raw diet, significant differences in their microbiome were observed, with the raw dogs having a higher incidence of Clostridium perfringens and E. coli than the commercial feed group26 ƒƒPhysical assaults to the intestinal microbiome, e.g. antibiotic administration • Can lead to severe dysbiosis that can take years to return to normal with no intervention27 • Consider making it standard protocol to prescribe bacterial GI support products after a course of antibiotics

Current and future therapeutic applications of the intestinal microbiome Bacterial GI support product administration does appear on a number of NHS clinical guidelines, including for pre-term babies. ƒƒAlthough not licensed in the UK for medicinal use

In the USA, the FDA does have jurisdiction over some bacterial GI support products, classifying them as ‘live biotherapeutics’ that are defined as “live microorganisms with an intended therapeutic effect in humans, including bacteria and yeast used in disease prevention or treatment, intended local or regional action”.28

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How to choose a bacterial GI support product Main difference is bacterial species included.

Safety is paramount. ƒƒThe Lactobacillus spp. found in Procanicare have been certified by the EFSA to present no pathogenic or antimicrobial resistance risk.29 They are typically sensitive to antibiotics and the genome of Lactobacillus species does not naturally receive foreign DNA, so development of resistance is extremely difficult

Most commercially available canine GI support products are of porcine, avian or human origin. ƒƒProcanicare™ is the first GI Support product available in the UK to be developed from the intestinal bacteria of healthy dogs and contains 3 canine strains of live Lactobacillus bacteria. This is important as it has been demonstrated that adhesion of gastrointestinal (GI) bacteria to epithelial cells of the gut is host specific30

The strains of Lactobacillus bacteria found in Procanicare have been shown to promote indigenous intestinal commensal populations and reduce numbers of potentially pathogenic bacteria. They do this via mechanisms unique to each strain, including inhibition, competitive exclusion and displacement.7,31

References: 1.Valdes AM, et al. Role of the gut microbiota in nutrition and health BMJ 2018; 361 :j2179. 2. Suzuki, TA. Links between Natural Variation in the Microbiome and Host Fitness in Wild Mammals. Integrative and Comparative Biology, Volume 57, Issue 4, October 2017, Pages 756–769. 3. Sharpton, TJ. Role of the Gut Microbiome in Vertebrate Evolution. American Society for Microbiology, mSystems Mar 2018, 3 (2) e00174-17. 4. Xiao, Y. et al. Comparative biogeography of the gut microbiome between Jinhua and Landrace pigs. Sci Rep 8, 5985 (2018) doi:10.1038/s41598-018-24289-z. 5. Koskella, B. et al. The microbiome beyond the horizon of ecological and evolutionary theory. Nat Ecol Evol 1, 1606–1615 (2017) doi:10.1038/s41559-017-0340-2. 6. LeBlanc JG, et al. Bacteria as vitamin suppliers to their host: a gut microbiota perspective. Current Opinion in Biotechnology, Volume 24, Issue 2, April 2013, Pages 160-168. 7. Grzeskowiak et al (2014) Pathogen exclusion properties of canine probiotics are influenced by the growth media and physical treatments simulating industrial processes. J Appl Microbia/. 116:1308-1314. 8. Rooks MG, Garrett WS. Gut microbiota, metabolites and host immunity. Nat Rev Immunol. 2016;16(6):341–352. 9. Turnbaugh PJ et al. A core gut microbiome in obese and lean twins. Nature. 2009 Jan 22;457(7228):480-4. 10. Ridaura VK, et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science. 2013;341(6150):1241214. 11. Cryan JF et al, Mind-altering microorganisms: the impact of the gut microbiota on brain and behavior. Nat Rev Neurosci. 2012 Oct;13(10):701-12. 12. Burokas A, et al. Microbiota regulation of the Mammalian gut-brain axis. Adv Appl Microbiol. 2015;91:1-62. 201. 13. Yano JM, et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015 Apr 9;161(2):264-76. 14. O’Mahony SM, et al. Serotonin, tryptophan metabolism and the brain-gut-microbiome axis. Behav Brain Res. 2015 Jan 15;277:32-48. 15. Borre YE, et al. The impact of microbiota on brain and behavior: mechanisms & therapeutic potential. Adv Exp Med Biol. 2014;817:373-403. 16. Dumitrescu, L, et al. Oxidative Stress and the Microbiota-Gut-Brain Axis. Oxidative Medicine and Cellular Longevity, Volume 2018, Article ID 2406594, 12 pages. 17. Mohammadi AA, et al. The effects of probiotics on mental health and hypothalamic-pituitary-adrenal axis: A randomized, double-blind, placebo-controlled trial in petrochemical workers. Nutr Neurosci. 2016 Nov;19(9):387-395. Epub 2015 Apr 16. 18. Kirchoff, NS, et al. The gut microbiome correlates with conspecific aggression in a small population of rescued dogs (Canis familiaris). PeerJ 7:e6103 (2019.) 19. Gavazza A, et al. Faecal microbiota in dogs with multicentric lymphoma. Vet Comp Oncol. 2018;16:E169–E175. 20. Hyejin Kim, et al. A Double-Blind, Placebo Controlled-Trial of a Probiotic Strain Lactobacillus sakei Probio-65 for the Prevention of Canine Atopic Dermatitis. J. Microbiol. Biotechnol. (2015), 25(11), 1966–1969. 21. Takafumi Osumi, et al. A double-blind, placebo- controlled evaluation of orally administered heat-killed Enterococcus faecalis FK-23 preparation in atopic dogs. Vet Dermatol 2019; 30: 127–e36. 22. Lippi I, et al. Effects of probiotic VSL#3 on glomerular filtration rate in dogs affected by chronic kidney disease: A pilot study. Can Vet J 2017;58:1301–1305. 23. Edwards SM, et al. The Maternal Gut Microbiome During Pregnancy. MCN Am J Matern Child Nurs. 2017;42(6):310–317. 24. Hongbin Liu, et al. Microbial and metabolic alterations in gut microbiota of sows during pregnancy and lactation. The FASEB Journal, 2019; fj.201801221RR. 25. Baldassarre ME, et al. Rationale of Probiotic Supplementation during Pregnancy and Neonatal Period. Nutrients 2018, 10, 1693. 26. Schmidt M, et al. (2018). The fecal microbiome and metabolome differs between dogs fed Bones and Raw Food (BARF) diets and dogs fed commercial diets. PLoS ONE 13 (8): e0201279. 27. Jakobsson, et al 2010. Short-Term Antibiotic Treatment Has Differing Long-Term Impacts on the Human Throat and Gut Microbiome. PLoS ONE, Volume 5, Issue 3, e9836.28. Hoffman FA. Development of Probiotics as Biologic Drugs. Clinical Infectious Diseases, Volume 46, Issue Supplement_2, February 2008, Pages S125–S127. 29. European Food Standards Agency (EFSA) Qualified Presumption of Safety (QPS) list, June 2019 https://www.efsa.europa.eu/en/efsajournal/pub/5753. 30. Kumar S, et al. Comparative assessment of canine-origin Lactobacillus johnsonii CPN23 and dairy origin Lactobacillus acidophillus NCDC 15 for nutrient digestibility, faecal fermentative metabolites and selected gut health indices in dogs. J Nutr Sci.2017;6:e38.31. Gomez-Gallego.C .et al (2016) A canine-specific probiotic product in treating acute or intermittent diarrhea in dogs: A double-blind placebo-controlled efficacy study. Vet Microbial. 197:122-128.

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