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9/8/2017
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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)