Gasbarrini A. Microbiota, Antibiotici e Probiotici in Gastroenterologia. ASMaD 2016

Master in Gastroenterologia
Roma, 27 novembre 2016
La Gastroenterologia Clinica: from Deskside to Bedside
Microbiota, Antibiotici e
Probiotici in Gastroenterologia
Antonio Gasbarrini
Medicina Interna,
Gastroenterologia e Malatie del Fegato
Area Gastroenterologica
Polo Malattie dell’Apparato Digerente
Fondazione Policlinico Universitario Gemelli
Universita’ Cattolica, Roma

Balfour Sartor, Gastroenterology 2008
Gut Microbiota

Hollister EB et al. Gastroenterology 2014
Gastric acid
Biliary salts
Mucosal IgA
AN ANATOMO-MICROBIOLOGICAL BARRIER
Bacteria interactions
Mucus
Motility
CuriosityZein.net

Acquired
and
Innate
immunity
Vascular and lymphatic
systems
Neuroenteric system
Digestive enzymes
Mucosal
Barrier
Epithelial
barrier
Endocrine
system
Virus/phages Bacteria
Yeast
Microbiota has many components
Helminth
ParasiteArchea
Protozoa
Micro-eukaryotes

Acquired
and
Innate
immunity
systems
Neuroenteric system
Digestive enzymes
Mucosal
Barrier
Epithelial
barrier
Endocrine
system
Yeast
Gut Bacteriome
Helminth
ParasiteArchea
Protozoa
Micro-eukaryotes

Microbial Taxonomic Rank
DOMINIUM
REGNUM
PHYLUM
CLASSE
ORDO
FAMILIA
GENUS
SPECIES
SUBSPECIES
95%
99%
100%
Genes identity

Enterococcus
Dethlefsen et al., Nature, 2007 18;449(7164):811-8 Ley et al., Science, 2008, 20;320(5883):1647-51
Firmicutes
60 to 80 %
Firmicutes
60 to 80 %
Clostridium
coccoides (cluster
XIVa)
Clostridium
coccoides (cluster
XIVa)
Clostridium
leptum (cluster
IV)
Clostridium
leptum (cluster
IV)
LactobacillalesLactobacillales
Bacteroidetes
20 to 40 %
Bacteroidetes
20 to 40 %
Faecalibacterium
prausnitzii
Lactobacillus
Bacteroides
thetaiotaomicron
Streptococcus
thermophilus
Bifidobacterium
Escherichia coli
ActinobacteriaActinobacteria
ProteobacteriaProteobacteria
Phylogenetic diversity of human gut Bacteriome
Helicobacter pylori
2 major phyla: Firmicutes and Bacteroidetes (>70%)

Acquired
and
Innate
immunity
systems
Neuroenteric system
Digestive enzymes
Mucosal
Barrier
Epithelial
barrier
Endocrine
system
Yeast
Other components of the Microbiota
Helminth
ParasiteArchea
Protozoa
Micro-eukaryotes

The gut is home to >50 genera of fungi with Candida,
Saccharomyces and Cladosporium species being particularly
common
Commensal fungal populations are more
variable than those of bacteria and may be
influenced by fungi in the environment (less
abundant and less robust?)
Diet can affect the fungal microbiota: plant-
based diet ↑Candida species, animal-based diet
↑ Penicillium species
Dollive S, et al. Genome Biol 2014
Cui et al. Genome Med 2014
HUMAN GUT MYCOME
Fungal microbiome
Bacterial microbiome
MUCUS
Gut microbiome
Bacteria
Fungi
Huffnagle GB et al. Trends Microbiol 2014

HUMANE GUT VIROME
Berg Miller et al, Environ Microbiol 2011
• In the gut have been isolated >30.000 different viral
genotypes. Majority ( 78%) of sequences∼ did
not match any previously described virus
• Some are human, most are bacterial virus or
bacteriophages (caudovirales, corticoviridae,
• myoviridae, microviridae, siphoviridae..)
• Metabolic profiling revealed an enrichment
of sequences with putative functional roles in DNA,
protein and carbohydrate metabolism
• Phage have a main role in bacteriome adaptation to
perturbations (diet, antibiotics..)
• Pro phages outnumbered lytic phages: 2:1

INDIVIDUAL
HUMAN GUT
ENTEROTYPE

Correlation network analysis between relative abundance
of bacterial phylotypes, yeast and bacteriophage-matching
reads

EU= good BIOS= life
In a healthy Microbiota species
are in equilibrium: EUBIOSIS
How to define an
EUBIOTIC enterotype?

Kitamoto S et al. J Gastroenterol 2015
Microbiota “sensing”
Osmolarity
Bicarbonate
Oxygen pH
Fucose SCFAs
Bile
Viscosity
Attachment shear stress
Cell density
Unknown
Metabolic
sensing
Physico-chemical
sensing
Mechano
sensing
Quorum
sensing

Shenderov et al. Anaerobe 2011 Schauder et al. Genes & Development 2001
How Bacteria Talk to Each Other?
Highly specific as well as universal QUORUM
SENSING languages exist: METABIOTICS
Regardless of the type of signal used, QS allows
coordinated regulation of behavior
QS enables a group of bacteria to act in a
concerted manner, and thus acquire some of
the characteristics of multicellular
organisms, becoming similar to eukaryotes
Bacterial behaviors are regulated by QUORUM SENSING,
including symbiotic features, virulence, biofilm
formation, genes expression and epigenetic regulation,
apoptosis

 Some of the neurological diseases are associated with
an altered microbiota composition, such as autism
 In this study 3 chemically diverse quorum sensing
peptides were investigated for their brain influx and
efflux properties in an in vivo mouse model to determine
blood-brain transfer properties
Evelien Wynendaele et al., PLoS One 2015

3 chemically diverse QUOUM SENSING PEPTIDES:
BIP-2 (Quorumpeps ID 102, GLWEDLLYNINRYAHYIT)
PhrANTH2 (Quorumpeps ID 186, SKDYN)
PhrCACET1 (Quorumpeps ID 206, SYPGWSW)
1.BIP-2 (bacteriocin-inducing peptide 2): synthesized by
Streptococcus pneumonia (commensal of the human
nasopharynx)
2.PhrANTH2: produced by Bacillus anthracis
3.PhrCACET1: formed by Clostridium acetobutylicum
Clostridium spp are predominant in the gut of
autistic children
Evelien Wynendaele et al., PLoS One 2015

Wynendaele et al., PLoS One 2015
 PhrCACET1 showed
the highest brain
influx: it very efficiently
crossed the BBB with a
measured clearance by
the brain that was
higher than that of
dermorphin (positive
control), followed by
BIP-2
 PhrANTH2 shows no
significant brain influx,
similar to BSA
(negative control)

…specific effects in each GI tract!
GUT MICROBIOTA AND HOST
HEALTH
Barrier effect
Immunocompetence/Tolerance
Synthesis
Metabolism
Drug metabolism
Behavior conditioning

Small Bowel
Barrier effect
Synthesis
Metabolism
Drug metabolism
HEALTH

Innate immune response
•Epithelial cells
•Myeloid cells
•Innate lymphoid cells
Adaptive immune response
•IgA
•TH17 cells
•Treg cells
Microbiota and immune system
relationship

Microbiota and epithelial cells
Thaiss et al, Nature 2016

Microbiota and innate lymphoid cells

Microbiota and myeloid cells

Microbiota and B and T cells
stimulation (IgA production)
Honda and Littmann Nature 2016

Microbiota induces TH17 cells
Honda and Littmann Nature 2016

Microbiota stimulation of Treg cells
SCFA derived from fementation of complex dietary
carbohydrates (high-fiber diet) by colonic microbiota
SCFA downregulate proinflammatory responses at the site of
antigenic insult and also influence bone marrow-derived APCs
Honda and Littmann Nature
2016

Microbiota educates the
immune system to tolerance
Kamada e Nunez Gastroenterology 2014

Colon
Barrier effect
Synthesis
Metabolism
Drug metabolism
HEALTH

NUTRIENT SENSOR PATHWAY
Tilg et al, Clin Gastroenterol. 2010 Sep;44 Suppl 1:S16-8

Failure of MICROBIOTA equilibrium
Quali-quantitative alterations of oral,
esophageal, gastric, small bowel and/or
colonic microbiota
DYSBIOSIS
Digestive and extradigestive diseases
EUBIOSIS

Dysbiosis is caused by several life events
Ottmann N et al. Front Cell Infect Microb 2012

Breastfeeding/
formula feeding
Fecal microbiota
(mother)
Koenig JE et al, PNAS 2010
During the weaning phase (first 2-3 years of age)
a Native CORE microbiota populates the
gut (early programming with life long-effects )
Mode of delivery (vaginal microbiota)
Other (e.g. antibiotcs)
Environment
(mother/father/parents/
babysitter/siblings/pets..)

Backhed et al. Cell Host & Microbe, 2014
..an early programming with long-term effects

Huffnagle GB et al. Trends Microbiol 2014
EARLY LIFE DYSBIOTIC EVENTS
Fungal microbiome
Bacterial microbiome

• Existence of a critical window in early life, when the gut
microbiota can influence the development of persisting
metabolic traits
• Recipients of penicillin altered microbiota had decreased
expression of intestinal immune-response genes, similar to
their donors Immunologic and metabolic changes are
not caused by direct effects of antibiotics but rather by
derived changes in the gut microbiota
• Currently there is no direct evidence for a causal relationship
in humans
Jess T., N Engl J Med. 2014

Cox – Cell 2014
• Mice receiving penicillin
during weaning gained
total mass and fat mass
in adult age
• Mice receiving penicillin-
altered microbiota
(transfer of the cecal
microbiota from 18 w-old
penicillin-treated mice to
3 w-old Germ Free mice)
gained total mass and fat
mass at a significantly
faster rate
n early life and obesity

Hollister EB et al. Gastroenterology 2014
Gastric acid
Biliary salts
Mucosal IgA
GUT BARRIER INTEGRITY
Bacteria interactions
Mucus
Motility

Gut Barrier disfunction
Intestinal permeability: Leaky gut

Intestinal hyper-permeability
Leaky gut
•Physiological
•Pathological

DYSBIOSIS
OUTGROWTHOVERGROWTH
DOWNGROWTH
LEAKY GUT

DYSBIOSIS
REDUCTION OF ABUNDANCE OF SPECIES
LEAKY GUT

Microbiota new knowledge caused
the falling of the Single Germ Theory
• With the Microbiota revolution differences in
proportions of various bacteria in different
disease state are important rather than the
appearance of a single microrganism
• To understand disease pathogenesis the
emphasis has to be on the balance of different
microbes rather than a single pathological
microrganism

All Digestive Diseases have been
associated to a LEAKY GUT
• Gastrointestinal infections
• Irritable Bowel Syndrome
• Small Intestine Bacterial Overgrowth
• Diverticulosis
• Inflammatory Bowel Diseases
• Gastro-intestinal Cancers
• Food Intolerance/Allergy
• Celiac disease
• Liver diseases
• Pancreatic diseases
• Obesity, Diabetes and Metabolic Syndrome

Vogt SL et al. Anaerobes 2015
Clostridium difficile infection

• Small Intestine Bacterial Overgrowth
• Diverticulosis
• Celiac disease
• Liver diseases
Digestive Diseases associated to
DYSBIOSIS/LEAKY GUT

SMALL INTESTINE BACTERIAL OVERGROWTH
is associated with IBS symptoms (bloating,
diarrhea/constipation, pain) and food intolerance
Lin, JAMA 2004Fermentation and gas production

Husebye E, Chem 2005
SIBO RISK FACTORS:
1. GASTRIC ACID REDUCTION

SIBO
prevalence
increases
with age
< 50 years
50-74 years
> 75 years
0%
64%
10%
Riordan SM, Am J Gastro 1997
SIBO RISK FACTORS:
2. AGEING

IBS pts are DYSBIOTIC
1. Reduction of bacterial abundance
Human intestinal tract chip
phylogenetic microarray that
enables the parallel profiling
and semi-quantitative
analysis of >1000
representative intestinal
phylotypes
Microbiota of IBS and
healthy subjects are
significantly different (P
=0.0005) with a
reduction of abundance
of species
HC
IBS
Rajilic-Stojanovic. Gastroenterology 2011;141(5):1792-801

Rajilic-Stojanovic - Gastroenterology 2011
45 phylogenetic groups differed significantly between IBS and controls
*Especially B gallicum and B pseudocatenulatum
*
IBS pts are DYSBIOTIC
2. Different bacterial variety

Digestive diseases associated to
DYSBIOSIS/LEAKY GUT
• Intestinal Bacterial Overgrowth
• Diverticulosis
• Celiac disease
• Liver diseases

Junjie Qin et al. Nature 2010;464(7285):59-65
IBD pts are DYSBIOTIC
1. Reduction of bacterial abundance

Daniel N. Frank et al., PNAS 2007;104(34):13780-5
2. Different bacterial variety

Norman – Cell 2015
The enteric virome
richness increases in
CD and UC
Decreases in bacterial
diversity and richness
in IBD do not explain
virome changes
Virome changes in CD
and UC are disease-
specific
3. Alterations in enteric Virome

 Increased abundance of Basidiomycota
and an equivalent decrease in Ascomycota in
IBD (particularly in flare)
 Among decreased Ascomycota in IBD flare,
Malassezia sympodialis was identified
 In IBD, Saccharomyces genus (particularly
S. cerevisiae) exhibited the strongest signals.
S. Cerevisiae decreases both in absolute
number and regarding the proportion,
particularly in flare
4. Alterations in enteric Mycobiome
Sokol H, GUT 2016

Sanchez. Appl Environ Microbiol 2013
Untreated CD VS GFD-CD VS Controls
Proteobacteria (11 vs 2%)
Enterobacteriaceae
(15 vs 5%)
Staphylococcaceae
(22 vs 10%)
Firmicutes (73 vs 92%)
Bacterial Microbiota dysbiosis
in Celiac Disease

 Breastfed/vaginally delivered infants with first-degree CD relative
 HLA-DQ2 VS non-HLA-DQ2/8 carriers
 16S rRNA gene Pyrosequencing + qRT PCR
DQ2 vs Non DQ2/8 - Genus level
Bifidobacterium
Unclassified Bifidobacteriaceae
Corynebacterium; Gemella
Clostridium sensu stricto,
Unclassified Clostridiaceae
Unclassified Enterobacteriaceae
Raoultella
Olivares. Gut 2015;64(3):406-17

De Palma. Br J Nutr 2009;102(8):1154-60
30 days of GFD in healthy people
Bifidobacterium
C. lituseburense
F. prausnitzii
Bifidobacterium
Lactobacillus
Enterobacteriaceae
E.coli
FISH
qPCR
Gluten Free Diet causes dysbiosis
in not Celiacs subjects

Is Gut Microbiota involved
in Obesity and Metabolic
disorders?

GUT MICROBIOTA INFLUENCE
ON ENERGY STORAGE
• Wild Type (WT) mice have 42%
more total body fat and 47%
more gonadal fat than germ-
free (GF) mice
• Colonisation of GF mice with
microbiota from WT produces a
60% increase in body fat mass,
associated with increased
insulin resistance
Backhed et al – PNAS 2004

Microbiota transmit adiposity phenotype
Ridaura et al. Science 2013
Walker AW et al. Science 2013
TRANSFERRED INTO THE
INTESTINES
OF GERM-FREE MICE
(Ob) twin + mice = adiposity
(Ln) twin + mice =  adiposity
Fecal microbiota from 4 human female twin pairs discordant for obesity
COHOUSING
(Ob) twin transplanted mice +
(Ln) twin transplanted mice =
(Ob) mice became LEAN
(Ln) mice remain LEAN
TRANSMISSIBILITY
OF INTESTINAL MICROBES
AND ADIPOSITY PHENOTYPE
ARE TIGHTLY LINKED

How Gut Microbiota is
involved in Obesity and
Metabolic disorders?

Acetate promotes metabolic syndrome through
activation of the parasympathetic nervous system
Perry – Nature 2016
• Increased production of acetate due to a
gut microbiota–nutrient interaction in
HFD-fed rodents
• Activation of the parasympathetic
nervous system
• Increased glucose-stimulated
insulin secretion
• Increased ghrelin secretion
• Hyperphagia
• Obesity

Bile acids - Microbiota-induced obesity
requires farnesoid X receptor
Parseus - Gut 2016
• Farnesoid X receptor (FXR) is a bile acid nuclear receptor
• GF versus wild-type and Fxr−/− mice fed with HFD for 10 weeks
• FXR and gut microbiota regulate
development of diet-induced obesity
• Gut microbiota increases
hepatic steatosis and
expression of genes
involved in lipoprotein
uptake through FXR

• The obesity phenotype is transferable by transferring the
caecal microbiota from HFD-fed Fxr−/− and wild-type mice
into GF mice
Bile acids - Microbiota-induced obesity
requires farnesoid X receptor
Parseus - Gut 2016

Which changes in Gut
Microbiota composition in
Obesity and Metabolic
disorders?

Obesity is associated
with:
•Reduced bacterial
diversity
•Phylum-level changes
•Altered representation
of bacterial genes and
metabolic pathways
Turnbaugh – Nature 2009
obesecontrol
Gut microbiota in obese humans
BACTEROIDETES/
FIRMICUTES
Adiposity index

Changes in gut microbial ecology
• Low bacterial richness (Low gene count)
• Microbiotal phenotype
• Higher rate of systemic inflammation
Bacterial alteration
Reduction in F. prausnitzii, A. muciniphila, Alistipes…
Proinflammatory bacteria dominate (Ruminococcus gnavus.)
Consequences
• Reduction in butyrate production and increased mucus degradation
• Increased oxidative stress and metainflammation
Tilg and Moschen , Gut 2014
obesecontrol
Gut microbiota in obese humans

Akkermansia muciniphila
Microbiota fingerprint of obesity?
Everard – PNAS 2013
Akkermansia muciniphila
is a mucin-degrading
bacteria that resides in the
mucus layer
Lower abundance of A.
muciniphila in leptin-
deficient obese than in lean
mice
100-fold decrease of A.
muciniphila in high-fat-fed
mice

Schneerberg et al - Sci Rep 2015
A. muciniphila inversely correlates with onset of
inflammation, altered adipose tissue metabolism and
metabolic disorders during obesity in mice exposed to HFD
• A. muciniphila, Bifidobacterium
spp. and Lactobacillus spp.
were significantly decreased
after HFD, although this
decrease was transient for
Bifidobacteria and Lactobacilli
• Abundance of Akkermansia muciniphila
decreased gradually to finally reach a level
~ 10,000 times lower than the initial one

Dao et al – Gut 2015
A. muciniphila is associated with a healthier metabolic
status and better clinical outcomes after Caloric
Restriction in overweight/obese adults
6-w caloric restriction period followed by a 6-w weight stabilisation diet
in 49 overweight and obese adults
• Baseline A. muciniphila inversely
related to fasting glucose, waist-to-
hip ratio and subcutaneous
adipocyte diameter.
• Individuals with higher A.
muciniphila abundance at baseline
displayed greater improvement in
insulin sensitivity markers and other
clinical parameters after CR

Metformine and diabetes
A microbiota-dependent pathway?
Meta-analysis of metagenomic data from 199 T2D patients, from whom information on antidiabetic
treatment was available, and 554 non-diabetic controls, comprising Swedish, Danish and Chinese
individuals
Metformin changes gut microbiota in T2D patients
Forslund et al – Nature 2015
Metformin-treated T2D pts
Intestinibacter spp abundance
Escherichia spp abundance

Metformine and diabetes
A microbiota-dependent pathway?
Meta-analysis of metagenomic data from 199 T2D patients, from whom information on antidiabetic
treatment was available, and 554 non-diabetic controls, comprising Swedish, Danish, and Chinese
individuals
• Bacterial gene function modules for butyrate
and propionate production increase as
metformine serum levels do
• Identification of T2D
patients treated with
metformin based on
their gut microbial
composition
Forslund et al – Nature 2015
Metformin changes gut microbiota in T2D patients

Beside diet composition
(HFD..) and caloric amount,
other Microbiota influencers
could have a role in Obesity
and Metabolic disorders

4 groups of C57BL/6 mice:
•Low Fat/Sedentary
•Low Fat/Exercise
•High Fat/Sedentary
•Hfigh Fat/Exercise
•HFD resulted in significantly
greater body weight and adiposity
as well as decreased glucose
tolerance that were prevented by
Exercise
Evans – Plos One 2014
Microbiota influencers in Met Disorders
Exercise
Exercise prevents weight gain and
alters microbiota in HFD-induced
obesity

• At week 12,
exercise changed
the levels of phyla
of bacteria:
Bacteroidetes
Firmicutes
Proteoacteria
Evans – Plos One 2014
Exercise

 Non caloric artificial sweeteners (NAS: SACHARIN, SUCRALOSE,
ASPARTAME) drive development of glucose intolerance through
induction of compositional and functional alterations of gut
microbiota
 NAS-mediated effects can be abrogated by antibiotic treatment
 NAS-mediated effects are fully transferrable to germ free mice upon
transplantation of microbiota from NAS consuming mice or of
microbiota anaerobically incubated in presence of NAS
Suez et al, Nature 2014
CALLING FOR A REASSESSMENT OF
MASSIVE SWEETENERS USAGE
Non caloric artificial sweeteners

Poroiko, Sci Rep 2016
Chronic sleep disruption
Chronic Sleep Disruption Alters Gut Microbiota, Induces
Systemic and Adipose Tissue Inflammation and Insulin
Resistance in Mice
Mice were exposed to Sleep Fragmentation (SF) for 4 w and then allowed to
recover for 2 w
• Sleep Fragmentation increases food intake, visceral fat and
insulin resistance

Poroiko – Sci Rep 2016
Chronic sleep disruption
Chronic Sleep Disruption Alters Gut Microbiota
Mice were exposed to Sleep Fragmentation (SF) for 4 w and then allowed to recover
for 2 w
Firmicutes
Lachnospiraceae
Ruminococcaceae
Bacteroidetes
Bifidobacteriaceae
Lactobacillaceae
Reversible gut microbiota
changes after SF

GI Cancers associated to DYSBIOSIS
• Oral cavity
• Esophagus
• Stomach
• Small Bowel
• Colon
• Liver
• Bile trat
• Pancreas
H. pylori
Gut microbiota

Microbiota in anticancer immunotherapy
Antibodies targeting CTLA-4 have been successfully
used as cancer immunotherapy. Their effect depends on
the presence of microbiota.
Vétizou et al. Science 2015
Tumors in antibiotic-treated or germ-free mice did
not respond to CTLA blockade.

The antitumor effects of CTLA-4 blockade
depend on distinct Bacteroides species.
In mice and patients,
T cell responses
specific for B.
thetaiotaomicron or
B. fragilis were
associated with the
efficacy of CTLA-4
blockade.
Microbiota in anticancer immunotherapy
Vétizou et al. Science 2015

ETOH NAFLD
HBV/HCV
HCC
Auto
immunity
Encephalopathy
Iron.. Portal
hypertension
HRS
Ascites/SBP
DYSBIOSIS
+
LEAKY GUT

DISEASE
PROGRESSION
DISEASE
PROGRESSION
DISEASE
PROGRESSION
Ge PS et al. NEJM 2016
TODAY…
GUT MICROBIOTA HAS A PROGNOSTIC VALUE
IN LIVER CIRRHOSIS

Gut microbiota Mucosal immune system
Muco-epithelial barrier
Vascular pathway Neuroendocrine/
Neuroenteric
Systems
How to mantain an
Eubiotic gut barrier?

How to modulate Gut Microbiota?
Diet and Nutritional Support
Diet composition (meat, cheese, fibers, high glicemic index,
saturated fatty acids, ethanol, sweeteners…)
Caloric amount, minerals, vitamins..
Removal of predisposing conditions
Treat diabetes, endocrine, other motility disorders..
Surgery or prokinetics when indicated
Stop PPI or other antiacid, NSAIDs, antibiotic,
immunosoppressant, antidepressant….
Intervention
Fecal Microbial Transplantation
Biotherapy (prebiotics, probiotis, symbiotics, postbiotics)
Antibiotics

FMT: therapeutical applications
• C Difficile* and other antibiotic resistant GI
infection
• IBD and IBS
• Other inflammatory/autoimmune conditions
• NAFLD and other liver diseases
• Diabetes, Metabolic Syndrome, Obesity
• GI cancer
• Oncohematology
• Neurological and psichiatric disorders
*Approved

 Short vanco+FMT vs Short
vanco+bowel prep vs Standard vanco
 Study stop after an interim analysis
 Resolution of CDAD
 Mild diarrhea and abdominal
cramping in the FMT group on the
infusion day
• FMT group (n=16): 81%1 FMT, 94% >1 FMT
• Vancomycin group (n=13): 31%
• Bowel prep (n=13): 23%
Van Nood et al – NEJM 2013
RCT: FMT nasoduodenal tube

 Short vanco+FMT vs Standard vanco
 Study stop after a 1-year interim
analysis
 Resolution of CDAD
 5/7 pts with severe disease (PMC):
progressive disappearance of PMC and
resolution of CDAD after multiple FMT
 No significant adverse events
RCT: FMT colonoscopy
• FMT group (n=20): 90%
• Vancomycin group (n=19): 26%
Cammarota et al – APT 2015

FMT for recurrent C. Diff entered
in the European Guidelines
 FMT is strongly
suggested in
combination with
antibiotics for multiple
recurrent CDI
SoR: A
QoE: 1
Debast et al – Clin Microbiol Infect 2014

Youngster – JAMA 2014
 20 pts with rCDI received 15 FMT capsules by healthy
volunteers on 2 consecutive days and were followed up for
symptom resolution and adverse events for up to 6 months
 Resolution of diarrhea in 14 patients (70%; 95%CI, 47%-
85%) after a single capsule-based FMT
 All 6 non-responders were re-treated; 4 had resolution of
diarrhea, resulting in an overall 90% rate of clinical
resolution of diarrhea (18/20)
 No serious adverse events attributed to FMT

Moayeddi et al Gastroenterology 2015, 16-5085 (15) 451-5
FMT induces remission of UC
 Parallel study of UC patients
 FMT vs placebo
 50 ml via Enema from anonymous donors
 FMT once weekly for 6 weeks
 Primary end points: remission (Mayo score <2) and
endoscopic Mayo score of 0 at week 7
 Trial stopped for futility
 24% of FMT reached remission vs 5% of controls
 No differences in adverse events
 7 out of 9 pts in remission received FMT from a
single donor THE MAGIC POOP!

Diet composition (fibers/high glicemic index/saturated fatty
acids…)
Intervention
Antibiotics
Biotherapy (prebiotics, probiotics, symbiotics, postbiotics)

Lactobacillus spp
• casei spp (Rhamnosus, DN..)
• reuteri
• acidophilus
• shirota
• delbrueckii, sp. Bulgaricus
• brevis
• plantarum
Bifidobacterium spp
• bifidum
• infantum
• longum
• thermophilum
• lactis
Which PROBIOTIC?
Cocci gram-positive
• Streptococcus thermophilus
• Enterococcus faecium
• Streptococcus intermedieus
• Streptococcus alfa-emoliticus
Bacillus gram-negative
• Escherichia coli Nissle (1917)
Guarino A. Bruzzese E. 2001; FAO/WHO, 2001
Bacillus gram-positive
• Bacillus clausii
Yeast
• Saccharomyces boulardii

Needs for a Subspecie
(Strain)-specific Microbial
Therapy
Different action for each Probiotic:
Knowledge of micro-organism functions and host
genetic modulation by different Species/Strain is
crucial

How to choose the right
Probiotic?
Different action for each Probiotic:
Knowledge of micro-organism functions and host
genetic modulation by different Species/Strain is
crucial

HOW TO CHOOSE A
PROBIOTIC?
Single strain Multistrains
Alive Dead
Bacteria Yeasts
Mechanism of action

Needs to know:
mechanisms of Probiotic/Host interaction
Immunological benefits:
Macrophage activation to increase antigen presentation to B cells and IgA
production
Modulate cytokine profiles
 Induce hyporesponsiveness to food antigens
Non-immunological benefits:
 Digest food and compete for nutrients with pathogens
 Alter local pH to create an unfavorable local environment for pathogens
 Produce bacteriocins to inhibit pathogens
 Scavenge superoxide radicals
 Stimulate epithelial mucin production//enhance intestinal barrier function
 Compete for adhesion with pathogens
 Modify pathogen-derived toxins WGO 2011

LEVEL OF EVIDENCE FOR PROBIOTICS
IN GASTROINTESTINAL DISORDERS
Reduction of Antibiotic-associated Diarrhea
Prevention and treatment of Infectious Diarrhea
Adjuvant for H. pylori and C. difficile treatment
Treatment of Necrotizing enterocolitis
Treatment of Sugar Intolerance
Prevention and treatment of Pouchitis
Maintenance of remission of IBD
Treatment of IBS
A
Practice guidelines on Probiotics usage
World Gastroenterology Organization (2011)

Probiotics has to be
choosen according to level
of evidence
Strain-specific Microbial
Therapy

LACTOBACILLUS CASEI sp RHAMNOSUS
Maintenance of remission in Ulcerative Colitis
Treatment of IBS

LACTOBACILLUS REUTERII
Maintenance of remission in Ulcerative Colitis
Treatment of IBS

ESCHERICHIA COLI sp NISSLE 1917
Adjuvant for H. pylori treatment
Maintenance of remission of Ulcerative Colitis
Treatment of IBS

BACILLUS COAGULANS GBI-30, 6086
Adjuvant for H. pylori treatment
Treatment and maintenance of remission of Ulcerative
Colitis
Treatment of IBS

Multistrains combination VSL 3
Treatment and maintenance of remission of Ulcerative
Colitis
Treatment of IBS

SACCHAROMYCES CEREVISAE sp BOULARDII
Adjuvant for H. pylori and C. Difficile treatment
Treatment of Traveller’s diarrhea
Maintenance of remission in IBD
Treatment of IBS

Mechanisms of action of each strain
Optimal dose
Duration of treatment
Selection of strains and/or strains
combinatios
Safety? Stability?
Probiotics: many unsolved
questions

..waiting NEXT GENERATION
PROBIOTICS
1.Faecalibacterium Prausnitzii
2.Akkermansia Muciniphila
3.Eubacterium halii
4.…

Diet composition (meat, cheese, fibers, high glicemic index,
saturated fatty acids, ethanol, sweeteners…)
Intervention
Fecal Microbial Transplantation
Biotherapy (prebiotics, probiotics, symbiotics, postbiotics)
Antibiotics

Targets of antibiotic therapy
1. Eradicate specific pathogenic bacteria
• H. Pylori, Yersinia, Shigella, Toxic E. Coli....
2. Modulate mutualistic gut microbiota
• Colonic diverticulosis
• Liver cirrhosis complications (encephalopathy, PBS)
• Intestine Bacterial Overgrowth
• IBS/IBD
•…..

Antibiotics
Gut-specificSystemic

Rifaximin and liver disease
-Rifaximin with or without lactulose is able to reduce the risk of hepatic
encephalopathy (HE) recurrence and the rate of HE-related hospitalizations.
Rifaximin has also been reported to improve operational abilities and input
integration capacity in patients with minimal HE
-Rifaximin reduces the occurrence of spontaneous bacterial peritonitis
(SBP) increasing transplant free survival
-Rifaximin reduces endotoxemia and the hepatic venous pressure
gradient (HVPG), as well as the incidence of complications related to portal
hypertension, such as variceal bleeding and thrombocytopenia
Bass NM et al. N Engl J Med. 2010
Neff GW wt al. J Clin Gastroenterol 2012
Sharma BC et al. Am H Gastroenterol 2013
Maharshi S et al. Gut 2014
Sharma K et al. Saudi J Gastorenterol 2014
Bajaj JS et al Gastroenterology 2011
Hanouneh MA et al. J Clin Gastroenterol 2012
Vlachogiannakos J et al. J Gastroenterol Hepatol 2012
Kalambokis GN et al. Hepatology 2012
Kalambokis GN et al. Liv Int 2012
Bajaj JS et al. PLoS One 2013

Rifaximin and GI diseases
-Rifaximin is effective in preventing traveler’s dyarrhea,
treat Small Intestine Bacterial Overgrowth, obtaining
symptoms relief in patients with uncomplicated
diverticular disease and in those with irritable bowel
syndrome (IBS) without constipation
-Rifaximin induces remission in patients with active
Crohn’s disease
-Rifaximin reduces stool frequency, rectal bleeding and
sigmoidoscopic score and helps in achieving remission in
patients with ulcerative colitis
Guslandi M et al. Inflamm Bowel Dis 2006
Gionchetti P et al. Digest Dis Sci 1999
Papi C et al. Aliment Pharmacol Ther 1995
Colecchia A et al. World J Gastroenterol 2007
Gasbarrini et al, Alim Pharm Ther 2005
Pimentel M et al. N Engl J Med 2011
Prantera C et al. Aliment Pharmacol Ther 2006
Shafran I et al. Curr Med Res Opin 2005
Shafran I et al. Am J Gastroenterol 2008
Prantera C. Gastroenterology 2012

•Alteration of virulence
•Inhibition of bacterial adherence to gut mucosa
•Reduced bacteria internalization
•Anti-inflammatory activity
•Gut microbiota modulation
HOW RIFAXIMIN WORKS?
A «NON CONVENTIONAL» ANTIBIOTIC

Decrease in
Veillonellaceae
and increase in
Eubacteriaceae
abundance

EUBIOTIC EFFECT OF RIFAXIMIN
Design of the study:
observational prospective
Population under study:
•Ulcerative Colitis (U),
•Crohn’s disease (C)
•Irritable bowel syndrome (I)
•Diverticular disease (D)
•Liver cirrhosis with hepatic encephalopathy (HE)
undergoing 1200 mg/day of Rifaximin x 10 days
Gasbarrini et al, Dig Dis 2016

Time a vs Time b P.Value adj.P.Val
f__Rikenellaceae 0,0001 0,002 <
f__Streptococcaceae 0,002 0,026 <
f__Lactobacillaceae 0,005 0,039 >
Time a vs Time c P.Value adj.P.Val
f__Rikenellaceae 0,0002 0,002 >
f__Enterobacteriaceae 0,007 0,057 >
f__Streptococcaceae 0,011 0,06 <
Time b vs Time c P.Value adj.P.Val
f__Streptococcaceae 0,0003 0,007 >
f__Rikenellaceae 0,003 0,028 >
f__Enterobacteriaceae 0,008 0,048 >
Time a (baseline) vs time b (stop Rifa)
Time a (baseline) vs time c (1 month after Rifa)
Time b (stop Rifa) vs time c (1 month after Rifa)
Gasbarrini et al, Dig Dis 2016
EUBIOTIC EFFECT OF RIFAXIMIN

Soldi, Gasbarrini et al. Clin Exp Gastroenterol 2015
• 15 non-C IBS patients
• Treated with Rifaximin 550 mg t.i.d. for 14
days
Fecal sample collection was performed at baseline
(T0), at the end of 14 days of treatment, and at the
end of the 6-week follow-up period (T56)
In IBS patients, a general stability of the
investigated groups across the different time
points was observed
EUBIOTIC EFFECT OF RIFAXIMIN IN IBS

• Faecalibacterium prausnitzii (from 5.6% at T0 to 8.5% at T14)
• Roseburia inulinivorans (from 2.4% at T0 to 1.9% at T56)
• Streptococcus salivarius (from 1% at T0 to 0.4% at T14
• Blautia luti (from 1.6% at T0 to 0.7% at T14)
Soldi, Gasbarrini et al. Clin Exp Gastroenterol 2015
EUBIOTIC EFFECT OF RIFAXIMIN IN IBS

Painting the landascape
of GUT barrier and role of
microbiota
Take Home Message

Loosely
adherent
mucus layer
Firmly
adherent
mucus layer
Bad
bacteria
Bile
acids
Lumen
Recettori
ionici
Wate
r
Stomach Duodenu
m and
Jejunum
Ileum Colon
Adhesions molecules
Immune
cells
Food
antigens
Endothelium
And fibroblasts
Nerve and miocytes
Non-Immune
cells
Food
antigens
Good
bacteria

Lumen
Immune
cells
Endothelium
And fibroblasts
Nerve and miocytes
Non-Immune
cells
SEVERE LEAKY GUT AND DYSBIOSIS
GASBARRINI A, UNPUBLISHED

Dysbiosis DIET
PREBIOTICS
ANTIBIOTICS
MICROBIOTA
TRANSPLANTATION
POSTBIOTICS
EUBIOTIC (RIFAXIMIN)

Gasbarrini A. Microbiota, Antibiotici e Probiotici in Gastroenterologia. ASMaD 2016

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Editor's Notes