GI: microbiome and enteral nutrition

Question 1

Name 4 of the major bacteria phyla identified in the healthy gut of dogs and cats

Answer 1

Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria

Question 2

One of the major metabolic functions of the GI microbiome is to ferment non-digestible dietary residues into volatile fatty acids. Name the short chain volatile fatty acids

Answer 2

Acetate, propionate, butyrate

Question 3

What short chain fatty acid is the major energy source for colonocytes?

Answer 3

Butyrate

Question 4

Name 3 vitamins synthesized by the microbiome

Answer 4

Biotin, folate, vitamin K

Question 5

Name 3 minerals that the microbiome assists in the absorption of

Answer 5

Calcium, magnesium, iron

Question 6

Name 2 ways that the microbiome contributes to host mucosal immunity

Answer 6

1) Colonization by bacteria after birth influences T cell repertoires and Th1/Th2 cytokine profiles
2) Form a biofilm to prevent pathogenic bacterial colonization - displacement, competition for nutrients, production of antimicrobial substances (lactic acid, bacteriocins)

Question 7

Surface enterocytes sense danger signals within the intestinal lumen and respond by secreting what products?

Answer 7

Defensins, IgA, chemokines and cytokines

Question 8

What are M cells?

Answer 8

Specialized epithelial cells that overlie lymphoid follicles. Sample luminal antigens and deliver them to DCs for antigen presentation

Question 9

What are two pattern recognition receptor (PRR) systems used by the mucosal immune system to recognize pathogens? Where are they located?

Answer 9

Toll like receptors (TLRs) - transmembrane
Nucleotide-binding oligomerization domain (NOD1 and NOD2) - intracellular

Question 10

Immune responses to commensal bacteria in IBD may be driven by what TLR/antigen?

Answer 10

Flagellin activating TLR5

Question 11

Increased numbers of intestinal bacteria can cause malabsorption and diarrhea through what mechanisms?

Answer 11

1) Competition for nutrients - such as bacterial binding of cobalamin, which prevents absorption
2) Metabolism of nutrients into products that promote colonic secretion/diarrhea
3) Biochemical injury to the brush border, decreasing enzyme activity

Question 12

Selective IgA deficiency leading to bacterial overgrowth occurs in what breed?

Answer 12

German Shepherds

Question 13

What is the difference between microbiota and microbiome?

Answer 13

Microbiota = taxonomy (who is there)
Microbiome = the gene content and function (what are they doing)

Question 14

Why is bacterial culture less useful for evaluating the microbiome?

Answer 14

Majority of bacteria (especially in the colon) are anaerobes; a reduction in beneficial bacteria is often more relevant than overgrowth of one species - difficult to identify on culture

Question 15

What is DNA shotgun sequencing or metagenomics?

Answer 15

A technique that aims to sequence extracted DNA in a sample without prior PCR amplification on a high throughout sequencer - allows assessment of taxonomy and functional gene categories

Question 16

What are 3 advantages of DNA shotgun sequencing over 16S RNA sequencing?

Answer 16

• Better resolution at the bacterial species and strain level
• Can identify archaea, fungi, and DNA viruses
• Can provide more detailed info on virulence genes and antimicrobial resistance genes

Question 17

What are disadvantages of DNA shotgun sequencing over 16S RNA sequencing?

Answer 17

High cost, advanced bioinformatics needed to assemble the data

Question 18

What is the 16S rRNA gene?

Answer 18

Several bacteria variable gene regions that are flanked by conserved regions

Question 19

What is 16S rRNA gene sequencing?

Answer 19

DNA is extracted from a sample, and bacterial universal primers are used to amplify the conserved regions consisting of the 16S gene. PCR amplicons are sequenced using a high throughput sequencer

Question 20

What is alpha diversity?

Answer 20

The richness and diversity of a sample - how many taxa are in one sample

Question 21

What is beta diversity?

Answer 21

How similar one sample is to another sample based on the taxa present

Question 22

What are the advantages of 16S rRNA gene sequencing?

Answer 22

• Useful to detect overall differences in microbiota composition between groups; cannot detect the exact bacterial species (semi-quantitative)
• Relatively inexpensive

Question 23

What are the disadvantages of 16S rRNA gene sequencing?

Answer 23

• Changes in taxonomic databases and bioinformatics pipelines over time make comparing results across studies difficult
• Some taxa will differ significantly depending on the extraction/amplification technique used
• Gives you relative abundance of bacteria (not exact quantities)

Question 24

What method does the dysbiosis index use?

Answer 24

Quantitative PCR for specific taxa that have been identified as clinically relevant based on prior sequencing studies

Question 25

What percent of dogs and cats with chronic enteropathies have decreased abundance of C. hirononis?

Answer 25

50-60% of dogs, 30% of cats

Question 26

In one study, cats with small cell intestinal lymphoma had increased numbers of what bacteria attached to the mucosa in the ileum and colon compared to cats with IBD?

Answer 26

Fusobacterium sp.

Question 27

What is metabolomics?

Answer 27

Analysis of microbial-derived metabolites (SCFA, fecal bile acids, etc)

Question 28

Tryptophan is converted by bacteria into indole metabolites. What are their function in the GI tract?

Answer 28

Indoles can be anti-inflammatory (decrease IL-8 expression), induce mucin gene expression, and strengthen tight junctions

Question 29

Fecal concentrations of what short chain fatty acids were lower in dogs with chronic enteropathy than healthy controls?

Answer 29

Acetate and propionate

Question 30

What does richness refer to?

Answer 30

The overall number of unique operational taxonomic units (proxy for species) in a sample

Question 31

What does diversity refer to?

Answer 31

A measure of the variability of the microbiome, considering both the richness (number of unique species) and relative abundance of those species

Question 32

Puppies up to 5 to 6 weeks of age had what changes in their microbiome compared to older dogs?

Answer 32

Increased dysbiosis index, increased C. difficile, decreased C. hiranonis and secondary bile acids

Question 33

Administration of metronidazole for 14 days caused what changes to the microbiome in dogs? How long did it take for those changes to reverse?

Answer 33

Decreased richness, decreased Fusobacterium, increased dysbiosis index, increased fecal lactate and decreased secondary bile acids. Took 4 weeks to recover

Question 34

In a study of dogs with steroid responsive CE, how much lower were secondary fecal bile acids in affected dogs compared to control dogs? How did steroids affect it?

Answer 34

29% vs 88%; improved to 94% with steroid treatment

Question 35

In a study of dogs with steroid responsive CE, did the dogs' dysbiosis index improve with steroid treatment?

Answer 35

No - remained increased despite clinical resolution of disease. C hiranonis did improve though

Question 36

In a study of 773 shelter dogs, administration of a synbiotic reduced the occurrence of diarrhea in the first 14 days by what percent?

Answer 36

From 27% in the control group to 18% in the synbiotic group

Question 37

In a study of 773 shelter dogs, administration of a synbiotic reduced the occurrence of >2 consecutive days of diarrhea by what percent?

Answer 37

8% in the control, 4.6% in the synbiotic group

Question 38

In healthy cats given clindamycin, concurrent administration of a synbiotic had what beneficial effects?

Answer 38

Administration of synbiotics decreased hyporexia and vomiting

Question 39

For puppies with Parvovirus, administration of an FMT improved what measures of disease?

Answer 39

Faster resolution of diarrhea, shorter hospitalization (3 days vs 6 days), decreased mortality (21% vs 36%)

Question 40

What effects did tylosin (administered for 7 days) have on the microbiome?

Answer 40

Decreased bacterial diversity (some resolved, some didn't), increase in primary bile acids (lasted until day 63)

Question 41

What enzymatic process is required to convert primary to secondary bile acids?

Answer 41

7-alpha-dehydroxylation

Question 42

In healthy research dogs given enrofloxacin and metronidazole, what effect did giving a synbiotic have?

Answer 42

Reduced hyporexia, no difference in vomiting or diarrhea

Question 43

Based on multiple cross over studies, how long do the effects of synbiotics last?

Answer 43

At least 8-9 weeks (affected second study period too)

Question 44

Administration of Fortiflora to healthy dogs had what effect on cobalamin and folate?

Answer 44

Decrease in cobalamin with moderate hypocobalaminemia developing in 40%. No effect on folate

Question 45

When given to puppies for one year, Enterococcus faecium (Fortiflora), induced what changes?

Answer 45

Increased fecal and serum IgA levels, increased vaccine associated IgA and IgG levels

Question 46

Name the primary bile acids. What are they conjugated to?

Answer 46

Cholic acid, chenodeoxycholic acid - synthesized in the liver from cholesterol and conjugated to taurine or glycine

Question 47

Name the secondary bile acids

Answer 47

Deoxycholic acid, lithocholic acid

Question 48

What bacteria has been associated with AHDS?

Answer 48

Toxin-producing C. perfringens (potentially the netF toxin gene)

Question 49

Helicobacter species produce what enzyme that is used to buffer gastric acid?

Answer 49

Urease - converts urea to ammonia

Question 50

How does omeprazole administration change the gastric microbiota?

Answer 50

Decreases Helicobacter (from 98% of gastric bacteria to 92%), allowing for growth of Firmicutes/Fusobacteria. Increases total number of bacteria in the duodenum

Question 51

In patients with an esophagojejunal feeding tube placed fluoroscopically with wire guidance, post pyloric access was gained in what percent of cases? What was the mean duration of the procedure?

Answer 51

95% 63 minutes

Question 52

Dogs and cats receiving corticosteroids when a PEG tube is placed had what percent complication rate compared to controls?

Answer 52

43% vs 18% - included infection of the site, PEG tube dislodgment, and chewing of the tube around the stoma

Question 53

In a retrospective study evaluating E tube complications in 123 patients, what percent experienced a complication related to tube placement?

Answer 53

44% (same in dogs and cats) - 14-17% infection at the E tube site (mostly Enterococcus and E coli) - Regurgitation of food through the stoma noted in 7 dogs and 1 cat - 10-13% required re-suturing due to loose sutures - 5-6% dislodgment

Question 54

In a retrospective study evaluating E tube complications in 248 cats, what percent of cats had complications and what were they?

Answer 54

35.8% complication rate - Tube dislodgment 14% - Stoma site infections 12% - increased risk while on glucocorticoids or oncolytic agents