DISCLOSURES New approaches: FMT and probiotics
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5/19/2021
New approaches:
FMT and probiotics
Jan S. Suchodolski, DrMedVet, PhD, DACVM, AGAF
Professor & Associate Director Research
Head of Microbiome Sciences, Gastrointestinal Laboratory
Department of Small Animal Clinical Sciences
Texas A&M University, College Station, TX, USA
DISCLOSURES
Employee of Gastrointestinal Laboratory at Texas A&M University
provides diagnostic testing on service-for-fee basis
Consultant for Nestle Purina Petcare, IDEXX Laboratories, Exegi Pharma
Speaker fee past year: Royal Canin, Hill’s Pet Nutrition, Nutramax
Laboratories
1
5/19/2021
CASE REPORT
10 month old Boston Terrier
chronic diarrhea > 1 months duration
mixed origin, occasionally mucus in feces
ENTEROPATHOGEN PANEL
fecal centrifugal flotation (3 consecutive days)
IFA for Gardia and Cryptosporidium spp.
PCR
Clostridium perfringens genes:
toxin A, enterotoxin, netF toxin
Clostridium difficile
Campylobacter spp.
Salmonella spp.
2
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RESULTS
fecal flotation: Giardia cysts
IFA: Giardia & Cryptosporidium
PCR
Clostridium perfringens genes:
toxin A, enterotoxin, netF toxin
Clostridium difficile Toxin B gene
Campylobacter spp.
Salmonella spp.
CLOSTRIDIUM DIFFICILE
Prevalence of C. difficile in healthy companion animals
• cats and dogs 0 – 58%
• particularly young animals
• animals that visits human hospitals
dogs and humans have different ribotypes, but possible zoonotic risk!
Prevalence of C. difficile toxin A/B (CDT A/B) or C. difficile toxin b
gene (cdt b) in healthy companion animals
• dogs CDT A/B 0%; cdt b 3%
Marks et al., JVIM, 2011; Busch et al., Vet Rec, 2015
3
5/19/2021
ENTEROTOXIGENIC CLOSTRIDIUM DIFFICILE
PCR C. difficile toxin b ELISA for C. difficile toxin
gene (cdt b) A/B (CDT A/B)
a positive PCR should be confirmed with ELISA
for the expression of the toxin
abnormal butyrate/propionate
ratios lead to activation of abnormal bile acid conversion
virulence factors leads to overgrowth with
Salmonella, E. coli C. difficile, C. perfringens, E. coli
Dysbiosis
54/67 dogs (80%)
that were positive for
C. difficile lacked C.
hiranonis
(unpublished data)
4
5/19/2021
C. DIFFICILE IN HUMANS
important cause of hospital-associated and antibiotic-associated
diarrhea
estimated >250,000 cases in US occur in hospitals
many more develop in outpatient settings
diarrhea, often with dysentery, pseudomembranous
>20% of patients resistant to metronidazole or vancomycin
5
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Fecal Transplantation via retention enema is effective for
recurrent or refractory C. difficile‐associated diarrhea
89% of patients experienced clinical resolution within 24 hours of
FMT
some patients respond after 2nd FMT
no documented relapses or adverse events with mean follow‐up
at 216 days post‐FMT
• antibiotics promote C. difficile infection by inhibiting conversion of primary bile acids to
secondary bile acids
• normal secondary bile acid composition in the colon prevents germination of C.
difficile spores
• antibiotics allow increased levels of bile salts and primary bile acids in the colon, which
promote germination of C. difficile spores and growth of vegetative forms of bacteria
• FMT restores normal microbiota and the levels of secondary bile acids
6
5/19/2021
Fecal microbiota transplantation (FMT) as enema
• diarrhea stopped 2 days later
• negative for C. difficile on PCR and ELISA
Protocol:
• 60 g of faeces dissolved in 50 mL of
tap water, filtered through a medical
gauze.
• 30mL orally administered to the
recipient dog using a syringe
• 8-month-old French bulldog with 4-month history of intermittent large bowel
diarrhea
• C. difficile antigen, toxin A&B genes and proteins positive on days 1 and 25
• diarrhea considered as C. difficile-associated colitis
• dog was treated with oral FMT
• stool consistency and frequency and fecal blood and mucus became normal 2–3
days after oral FMT
• negative for C. difficile antigen, toxin A&B genes and proteins
7
Dysbiosis Index
0
2
4
6
8
-10
-8
-6
-4
-2
10
20 20
20 1
pr 20 7
e 18
p r 20d
e
p r 2d
e
1d
1
FMT
2
3
4
5
6
7
8
9
days post FMT
10
11
normal
dysbiosis
equivocal
log DNA
20
1
pr 20 7
e 18
p r 20d
e
p r 2d
e
1d
1
2
3
4
5
6
7
8
9
10
11
log DNA
Cricket – Miniature Schnauzer, FS, 11 years
0
1
2
3
4
5
20
1
pr 20 7
e 18
pr 20d
e
pr 2d
e
1d
1
2
3
4
5
FMT C. difficile
6
7
8
9
days post FMT
10
11
normal
8
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5/19/2021
FMT DONOR SCREENING
normal microbiota
Dysbiosis Index (IDEXX and TAMU GI Lab)
C. hiranonis ↓
Fusobacterium ↓
Blautia↓
normal C. hiranonis
indicates normal fecal bile acid conversion
5‐10% of healthy dogs do not have beneficial C.
hiranonis
screen for parasites
screen for enteropathogens – PCR panel
C. difficile, parvovirus, C. perfringens cpe and netF genes,
C. jejuni, Salmonella
Correlation with species diversity based on
full 16S rRNA gene sequencing r=0.647
p
5/19/2021
FMT DONOR SCREENING
microbiome generally stable, so no need for frequent
screening
alternatively select healthy dog
no signs of GI disease
no antibiotic administration for at least 6 months
STORAGE OF FECES
stable at 4C for up to 1 week
for longer storage in freezer, add glycerol
50 ml aliquots contain 10g of Stool, 30‐35 ml of saline, 5ml of glycerol
storage up to 6 months at ‐20C
105/19/2021
FMT protocol via enema based on Chaitman et al., ACVIM 2017
Blend approx. 5 gram of donor stool per kg BW with enough
0.9% NaCl to achieve a mushy consistency
Chaitman et al., ACVIM; 2017
115/19/2021
FMT protocol via enema based on Chaitman et al., ACVIM 2017
Draw up in catheter tip syringe, attach 12 French red rubber catheter
Push material into catheter until it comes out the tip to avoid air
transplantation
INDICATIONS FOR FMT
young animals with chronic diarrhea
animals with GI infection
in dogs with chronic CE, use FMT as adjunct treatment
dogs with CE require treatment for underlying GI inflammation
hypoallergenic diet as first step
125/19/2021
• 9 month old puppy presented from the pet shop with a resistant giardia
infection
• treated with panacur, metronidazole, sulfamethoxazole and trimethoprim over
3 months
• still soft stools – FMT performed
• clinical improvement
DOGS WITH ACUTE DIARRHEA
Chaitman et al, Frontiers in Vet Res 2020
135/19/2021
Protocol: 10 g of feces from a healthy dog diluted in
10 mL of saline given rectally 6-12 hours post-
admission
ANIMALS:
66 puppies with parvovirus
METHODS:
• standard treatment (STD) (n=33) with antimicrobials
• FMT (n=33) received FMT in addition
RESULTS:
• FMT had resolution of diarrhea and shorter hospitalization time (median 3 days vs. 6
days).
• Mortality in STD was 36% vs. 21% in FMT, but not statistical significant (P = .174)
AYLLA
June 2018
chronic vomiting and diarrhea
metronidazole
omeprazole
relapsed after 2 mo.
October 2018
endoscopy/colonoscopy revealed
mild lymphoplasmacytic gastritis, duodenitis,
ileitis, colitis with mild eosinophil infiltrates
improved with hydrolyzed protein diet but still
loose stool
145/19/2021
AYLLA
Nov 2019 – June 2020
owners brought Aylla to GI service for further work up
dewormed
changed to lamb meat as they weren’t accepting previous diet anymore
and it wasn’t helping with diarrhea
prednisone, cyclosporine with no improvement
despite diarrhea, good body score and eating well
owner’s didn’t want to do another endoscopy/colonoscopy
just managed life with diarrhea
AYLLA
Dec 2020
Aylla and Miya’s samples were submitted
DI as Miya could be Aylla’s FMT donor
both Aylla and Miya had results consistent
with dysbiosis
alternative donor was found
FMT was performed (5g/kg – diluted in
saline)
155/19/2021
C. hiranonis
8
Dysbiosis Index
6 normal
4
2
0
22 days post 2 FMT
28 days post
45 days post
Before FMT
3 dogs with chronic enteropathy
BL 1 2 3 4 5 6 7 8
weeks Gerbec Ziga, MS thesis Helsinki Univ
165/19/2021
Adjunct FMT in dogs with CE – partially responsive to standard therapy
Courtesy Dr. Linda Toresson, Evidensia, Sweden
• Preliminary data: retrospective
study of dogs with CE receiving
FMT as enema (protocol:
Chaitman 2021)
• 3x every 14 days
• 28% of dogs did not respond to
FMT (no improvement)
• remaining dogs showed
improvement in CIBDAI scores
• FMT (enema) as a last therapeutic option for cat before
euthanasia
• immediate improvement in regards to fecal texture, odor
and color
• due to relapse of diarrhea, a second FMT was performed 5
weeks later
• during a 3‐month period the cat started passing gradually
normal stools
• 11 months after FMT a prolonged clinical cure of the
diarrhea was achieved with the cat passing normal feces
175/19/2021
SUMMARY
FMT improves at least temporally clinical signs and microbiota in subset of dogs
correlates with secondary bile acid conversion in subset of dogs
donor screening should include testing for normal BA metabolism
FMT success may depend on underlying disease
if initial trigger gone (e.g., dysbiosis due to antibiotic use), then lasting improvement
of microbiota
luminal dysbiosis
in chronic enteropathies possibly repeated FMTs necessary as adjunct to standard
therapy
185/19/2021
Probiotics
Probiotics have potential for GI health, but we need to
understand the mechanisms
probiotic effect is strain specific
the administered dose is important
the quality of products is crucial for clinical success
195/19/2021
PROBIOTICS
Probiotics are defined as “live microorganisms, which
when administered in adequate amounts confer a
health benefit on the host”
Joint FAO/WHO Expert Consultation on Evaluation of Health and
Nutritional Properties of Probiotics
PREBIOTICS AND SYNBIOTICS
Prebiotics are non-digestible food ingredients
intended to stimulate growth of resident bacteria
typically carbohydrates
fructooligosaccharides (FOS), galactooligosaccharides (GOS)
beta-glucans
Synbiotics are a combination of probiotics and prebiotics
205/19/2021
MECHANISMS OF PROBIOTICS
exclusion of pathogens
secretion of anti-microbial peptides
anti-toxin effect on C. difficile
Saccharomyces boulardii
immunomodulatory effects Thomas et al., Gut Microbes 2010
IMPROVEMENT INTESTINAL BARRIER
• upregulation of tight junction
proteins
• increase mucin production –
improve epithelial mucus layer
long‐term administration in dogs
with CE useful as adjunct
treatment
215/19/2021
PROBIOTICS - FECAL COLONIZATION
M BS D1 D3 D5 D8 D17 D23 D26 D32 D38 M
rapid appearance (1‐2 days) and disappearance (1‐3 days)
of probiotic species
INDICATIONS FOR PROBIOTICS
stress‐related diarrhea (prevention or treatment)
boarding, traveling, working dogs
unspecific diarrhea, uncomplicated infectious diarrhea
prevention of antibiotic associated GI signs
chronic enteropathies
in mild to moderate CE in combination with diet
as adjunct to immunossuppresive therapy in severe cases
225/19/2021
DIARRHEA
improvement in fecal consistency
improvement in fecal scores in cats with chronic diarrhea (Proviable, Nutramax)
shortening of diarrhea by 1‐2 days in dogs with uncomplicated diarrhea
(Purina Fortiflora)
more rapid normalization of intestinal microbiota (Visbiome Vet)
dogs with acute hemorrhagic diarrhea syndrome
improvement in intestinal barrier in dogs with CE
ANTIBIOTIC‐ASSOCIATED GI SIGNS
occur commonly in dogs and cats
9/16 healthy dogs had worse fecal scores on metronidazole
85% of cats had worse fecal scores on amoxicillin‐clavulanate
100% of cats showed vomiting and diarrhea on clindamycin
235/19/2021
ANTIBIOTIC‐ASSOCIATED GI SIGNS
studies show that giving probiotics during periods of antibiotic
administration ameliorate some of the clinical signs
bacterial probiotics should be given a few hours apart from antibiotics
but started on the same day or even before first dose of antibiotics
EFFECT OF PROVIABLE
cats received 75 mg clindamycin with food once daily for 3 weeks
1 hour later by either 2 capsules of a synbiotic or a placebo
cats receiving synbiotic were more likely to complete treatment due to
decreased vomiting
100% vs. 50%, P=0.04
vomited less, higher food intake while receiving synbiotic
no significant effect on fecal scores
Stokes et al, JVIM 2017
245/19/2021
SACCHAROMYCES BOULARDII
yeast, 10x larger and different structured cell wall than bacterial cells
yeast cells recognized by different host receptors than bacterial cells causing
different antigenic responses
yeast cells are resistant
to low pH in the stomach, bile salts, intestinal enzymes
resistant to common antibiotics
SACCHAROMYCES BOULARDII
major clinical use in humans
meta‐analysis of 21 randomized controlled trials (4780 participants)
S. boulardii is effective in reducing the risk of antibiotic‐associated
diarrhea in children and adults
255/19/2021
QUALITY OF PROBIOTIC PRODUCTS
25 probiotics were purchased, labels were scrutinized, and bacterial
contents were enumerated
only 4/15 (27%) products that had specific claims of viable organisms met or
exceeded their label claim
only 2 of these also had an acceptable label, which properly described the
contents
veterinarians and owners should scrutinize commercial probiotics
Weese et al., 2011
PREBIOTICS
substances that induce the growth or activity of microorganisms (e.g.,
bacteria and fungi) that contribute to the well‐being of their host
soluble and insoluble fibers (Fructo‐oligosaccharides (FOS), mannan‐
oligosaccharides (MOS) etc.)
often included in high‐fiber diets and symbiotics
265/19/2021
PREBIOTICS
fiber is fermented by microbiota into different products
short-chain fatty acids (especially butyrate)
beneficial effects of colonocytes
fibers induce anti-inflammatory cytokines, and regulatory T
cells that can ameliorate an inflammatory response
PREBIOTICS
Psyllium - insoluble fiber source that also form soluble fiber (gel) in the GI
tract
has bile acid binding properties
dogs: 3 teaspoons/10 kilograms
cats: ¼ teaspoon to a meal
titrate up dependent on response
Excessive fiber may induce diarrhea
275/19/2021
QUALITY OF PROBIOTIC PRODUCTS
25 probiotics were purchased, labels were scrutinized, and bacterial
contents were enumerated
only 4/15 (27%) products that had specific claims of viable organisms met or
exceeded their label claim
only 2 of these also had an acceptable label, which properly described the
contents
veterinarians and owners should scrutinize commercial probiotics
Weese et al., 2011
QUESTIONS?
jsuchodolski@cvm.tamu.edu
http://vetmed.tamu.edu/gilab
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