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New Approaches to Diagnosis and Interpretation of Intestinal Dysbiosis

New Approaches to Diagnosis and Interpretation of Intestinal Dysbiosis

Jan S. Suchodolski, DrMedVet, PhD, DACVM, AGAFAssociate Professor & Associate Director Research

Head of Microbiome Sciences, Gastrointestinal LaboratoryDepartment of Small Animal Clinical SciencesTexas A&M University, College Station, TX, USA

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https://research.pasteur.fr/en/team/microenvironment‐and‐immunity/

INTESTINAL MICROBIOME

1012 – 1014 bacterial cells inhabit the mammalian intestine

approx. 90% of cells in the body are bacteria

several hundred bacterial phylotypes

intestinal microbes contain 100‐fold more genes than the host genome

PREIDIS AND VERSALOVIC, GASTROENTEROLOGY 2009;136:2015–2031

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metabolic end‐products

metabolic activities of intestinal microbiota

effect on host health 

propionate, acetate, butyrate

carbohydrate fermentation

anti‐inflammatory, energy source of enterocytes, regulation of intestinal motility, 

amelioration of leaky gut barrier

Vitamin K2, B12, biotin, folate

vitamin synthesisimportant co‐factors for various metabolic 

pathways

indoledegradation of the 

amino acid tryptophan

increases epithelial‐cell tight‐junction resistance and attenuates indicators of 

inflammation

ceramide

induces degradation of sphingomyelin via 

alkaline sphingomyelinase

significant role in apoptosis and in the prevention of intestinal epithelial dysplasia 

and tumourigenesis

Cecum from germfree mouse 

Cecum from Wild typemouse 

The Role of the Microbiota in Gastrointestinal Health and Disease (Overstreet et al., InTech 2012)

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Inflammatory Bowel Disease (IBD)Metabolic syndromeCancerAsthmaDiabetesObesityStress related disorders

GUT MICROBIOTA IN DISEASE

ASSESSING MICROBIOTA

0%

20%

40%

60%

80%

100%Enterobacteriaceae

Succinivibrionaceae

Helicobacteraceae

Campylobacteraceae

Alcaligenaceae

Coriobacteriaceae

Bifidobacteriaceae

Turicibacteraceae

Streptococcaceae

Enterococcaceae

Bacillaceae

Fusobacteriaceae

Erysipelotrichaceae

Ruminococcaceae

Veillonellaceae

Peptostreptococcaceae

Peptococcaceae

Clostridiales;f__

Clostridiales;Other

Clostridiaceae

Lachnospiraceae

[Paraprevotellaceae]

Prevotellaceae

Porphyromonadaceae

Bacteroidaceae

Healthy dogs have a highly individual  microbiota

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0%

20%

40%

60%

80%

100%

Gram Positive

Gram Negative

Healthy dogs have a highly individual  microbiota

0%

20%

40%

60%

80%

100%

Organismal Systems|NervousSystem

Organismal Systems|ImmuneSystem

Organismal Systems|ExcretorySystem

OrganismalSystems|EnvironmentalAdaptationOrganismalSystems|Endocrine System

Organismal Systems|DigestiveSystem

OrganismalSystems|Circulatory System

HumanDiseases|NeurodegenerativeDiseasesHuman Diseases|MetabolicDiseases

Human Diseases|InfectiousDiseases

Human Diseases|ImmuneSystem Diseases

HumanDiseases|CardiovascularDiseasesHuman Diseases|Cancers

Environmental InformationProcessing|SignalingMolecules and InteractionCellular Processes|Transportand Catabolism

But predicted microbial function similar across healthy dogs

INTESTINAL MICROBIOTA IN DOGS

0%

20%

40%

60%

80%

100%

Duodenum Jejunum Ileum Colon

OTU

s

Bacteroidales

Enterobacteriales

Fusobacteriales

Lactobacillales

Clostridiales

Suchodolski et al, FEMS Microbiol Ecol, 2008

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DYSBIOSIS IN GI DISEASE

DUODENAL MICROBIOTA IN DOGS WITH IBD

0 20 40 60 80

Proteobacteria

Firmicutes

Bacteroidetes

Actinobacteria

Tenericutes

Fusobacteria

Cyanobacteria

Spirochaetes

Verrucomicrobia

% of sequences

Control IBD

Control dogs (n=6)Dogs with idiopathic IBD  (n=14)

* p<0.05

*

*

*

*

454‐pyrosequencing – 16S rRNA gene

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Suchodolski et al., 2012, PLOS ONEAcute diarrhea

PCR based Dysbiosis Index for 8 bacterial taxa

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Fecal dysbiosis in dogs with CE

IS DYSBIOSIS CAUSE OR EFFECT OF GI DISEASE?

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Winter et al., EMBO 2013

METABOLIC CONSEQUENCES OF GI INFLAMMATION AND DYSBIOSIS

Microbiota in GI health and disease

Intestinal microbiotain health

Intestinal microbiotain IBD

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Microbiota in GI health and disease

Intestinal microbiotain health

Intestinal microbiotain IBD

Healthy dog Dog with chronic enteropathy

metabolomicscomprehensive study of small molecules

present in biological samples

targeted metabolomics

measurement of specific metabolites (SCFA, vitamins)

untargeted metabolomics

global metabolic profile of many metabolites

understanding disease

pathogenesis

discovery of novel

biomarkers

noveltherapeutics approaches

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SHORT-CHAIN FATTY ACIDS (SCFAS)

• end-products of bacterial fermentation

• acetate, propionate, butyrate

• variety of beneficial effects anti-inflammatory

anti-diarrheic

regulation of GI motility

energy source for host

ABNORMALITIES IN MAJOR METABOLIC PATHWAYS IN DOGS WITH IBD BASED ON FECAL METABOLOMICS

Carbohydrate metabolism Glycolysis

Pentose phosphate pathway

TCA cycle

Lipid metabolism Primary bile acid synthesis

Secondary bile acid synthesis

Sterol absorption

Lipid Oxidation

• Amino acid metabolism– Lysine metabolism

– Aromatic amino acid metabolism

– Redox homeostasis

• Nucleotide metabolism

• Cofactors and vitamins

• Xenobiotics

Honneffer JB et al, DDW 2015

secondary BA inhibit germination of C. difficile spores 

primary BA promote 

germination

GALL BLADDER

LIVER

LUMEN OF INTESTINE

microbiota

Primary BA

Secondary BA

TGR5

TGR5

NUCLEUS

MITOCHONDRIA

FAT

MUSCLE

Secondary bile acids inhibit TNF‐α, IL‐1β and IL‐6 through 

activation of TGR5 

FXR

Feedback from the intestines regulates production of bile acids from the liver

INSULIN

GLUCOSE

TGR5

GLP‐1

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TGR5 BILE ACID RECEPTORS IN SMALL AND LARGE INTESTINE OF HEALTHY DOGS

GC= goblet cell; C= colonocyte; M= macrophage; EC= enteroendocrine cell; G= ganglion cell; En= endothelial cell.

BILE ACID DIARRHEA (BAD)

under recognized in humans (limited diagnostic testing) approx. 30% of patients with IBS, 40% of patients with Crohn’s Disease, 1%

of total population

type 1: bile acid malabsorption secondary to ileal inflammation

type 2: idiopathic primary bile acid diarrhea

type 3: secondary to small intestinal dysbiosis, radiation enteropathy, celiac disease, chronic pancreatitis

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Duboc et al., 2013

Fecal bile acids in dogs

p=0.0034

Blake Guard et al, ACVIM 2016

• insoluble chloride salt of a basic anion‐exchange resin

– neither digested or absorbed

– binds bile acids in gastrointestinal tract

– forms insoluble complex with bile acids

• cholestyramine used in patients with idiopathic bile acid diarrhea (Stotzer et al., Neuroenterol., 2013)

– reduced stool frequency

– improved stool consistency

– prolonged transit time

Bile acid sequestrant: cholestyramine

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515 patients with chronic diarrhea, 40% (207/515) diagnosed with BAD

107 of these patients were commenced on bile acid sequestrants (BAS)

54% (58/107) patients were followed up (median time since diagnosis 6 years)

median stool frequency decreased from seven stools per day to three (P= 0.0008) in those using BAS (38% were still using BAS at follow-up)

the 34% of patients not receiving treatment had no change in their daily bowel frequency

main reason for discontinuing treatment was poor tolerability of the BAS

conclusion: BAD is a chronic condition that best improves with BAS

EFFECTS OF ANTIBIOTICS

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Group 1 (Control) Diverse diets

No antimicrobial administration

Group 2 (Purina HA & Metronidazole) Purina HA hydrolyzed formula (dry diet)

2 week administration of metronidazole (15mg/kg PO q12h)

Group 3 (Metronidazole) Diverse diets

2 week administration of metronidazole (15mg/kg PO q12h)

Group 1 – Control group

Healthy dogs on various diets switched to Purina HA (hydrolyzed protein)

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Metronidazole14 days

Taxonomic changes over time for healthy dogs receiving metronidazole

Conversion from primary to secondary bile acids is disrupted by metronidazole

Metronidazoleadministration

* *

* p<0.05 compared to baseline

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ALTERED PATHWAYS DUE TO METRONIDAZOLE

Tyrosine metabolism

Glycine, serine and threonine metabolism

TCA cycle

Pyruvate metabolism

Tryptophan metabolism

Thiamine metabolism

Propanoate metabolism

Gylcolysis/Gluconeogenesis

Lysine degradation

Aminoacyl-tRNA biosyntesis

Galactose metabolism

Cysteine and methionine metabolism

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ACUTE HEMORRHAGIC DIARRHEA SYNDROME (AHDS)

endoscopic and histologic changes in mucosa stomach not affected => term “HGE” inadequate

new name => “acute hemorrhagic diarrhea syndrome”

acute necrosis in small and large intestinal mucosa

with predominantely neutrophilic inflammation

clinical presentation and progression severe diarrhea

rapid clinical improvement

good prognosis with aggressive fluid therapy

NETF - A NOVEL PORE-FORMING TOXIN

in Type A Clostridium perfringens associated with canine haemorrhagic gastroenteritis (Gohari and Prescott, 2015)

causes acute mucosal necrosis in small and large intestinewith predominantely neutrophilic inflammation

INDICATIONS FOR ANTIBIOTICS

rectal temperature > 39.5°C

tachycardia after rehydrationand pain management > 120/min

total WBC count < 4 or > 25 x 109/l

banded neutrophils > 1.5 x 109/l

inadequate response to fluid therapy after 4 hours

inadequate improvement of fecal quality after 4-5 days

• Unterer Stefan, ACVIM 2016

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24 dogs with AHDS – hospitalized• treated with fluid, anti‐emetics, analgesics• diet – small amounts, fat‐restricted, early• NO antibiotics

Day 0 significant different from all other days

Healthy dogs: n=47, with 0 positiveAcute diarrhea: n=15, with 0 positiveChronic diarrhea: n=36, with 0 positive

netF toxin on PCR:13/23 (56%)dogs positive

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Serum metabolite and microbiome profiles do not improve after therapy in canine IBD

CIBDAI

BL        1        2         3        4        5        6        7       8         9weeks

Healthy       Chronic enteropathy

SUMMARY - DYSBIOSIS

occurs commonly in GI disease but also inducible due to antibiotics, NSAIDs, etc.

loss of commensal microbiota and their metaboliteswhich are known to down-regulate immune responses

altered SCFA, secondary bile acids and other anti-inflammatory metabolites

bile acid malabsorption common component of chronic diarrhea

dysbiosis important component of many disorders may amplify intestinal inflammation may play role in other disorders (eg, Diabetes Mellitus)

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QUESTIONS?

jsuchodolski@cvm.tamu.edu

http://vetmed.tamu.edu/gilab