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UNIVERSITA’ CATTOLICA DEL SACRO CUORE
Facoltà di Medicina e Chirurgia “A. Gemelli”
UOC DI MEDICINA INTERNA E GASTROENTEROLOGIA
direttore Prof. A Gasbarrini
“La disbiosi indotta da antibiotici a breve
e a lungo termine:
dalla patogenesi al trattamento”
Franco Scaldaferri,
MD, PhD
Department of internal Medicine, Gastroenterology Division
IBD Unit – “Policlinico “A. Gemelli” Hospital
Catholic University of Sacred Hearth
Rome, Italy
[email protected]
THE HUMAN INTESTINAL MICROBIOTA
ENDOCRINE/METABOLIC
INTESTINAL
BARRIER
IMMUNE
Human Gut Microbiome
Metabolomics
>25.000
Chemicals
Proteomics
>58.000 Enzymes
Genomics
>3.000.000 Genes
Kau et al, Nature 2011
Qin et al, Nature 2011
Composition of gut microbiota
Gram-positives (60-80%)
Gram-negatives (20-40%)
Anaerobic (60-80%)
Zoetendal 2006, Bottazzi 2009
Gut microbiota and enterotypes
NATURE, VOL473, 12MAY2011
Microbiota as
“individual fingerprint”
 A core microbiota may account for up to 30% of the
microbiota
 70% of species phylotypes are subject specific: EACH
HEALTHY SUBJECT POSSESSES A SPECIFIC AND
DYNAMIC SUBSET OF HUNDREDS OF SPECIE
THE INDIVIDUAL
MICROBIOTA SHOWS AN
ASTONISHING LEVEL OF
INTER-INDIVIDUAL
VARIABILITY
MICROBIAL FINGERPRINT
Courtesy of Dr. Silvia Turroni, Bologna
Microbiota: phyla
FIRMICUTES
up to 40-65%
ACTINOBACTERIA
up to 5%
bifidobacteria
PROTEOBACTERIA
up to 8%
VERRUCOMICROBIA
up to 1%
BACTEROIDETES
up to 25-60%
D. Berry, W. Reinisch / Best Practice & Research Clinical Gastroenterology 27 (2013) 47–58
Prevalent bacterial phyla present in
IBD, DD, IBS and CT (overall values)
Controls
k__Bacteria;p__Bacteroidetes
Ulcerative Colitis
k__Bacteria;p__Firmicutes
k__Bacteria;p__Proteobacteria
k__Bacteria;p__Fusobacteria
Diverticular Disease
k__Bacteria;p__Elusimicrobia
Crohn’s Disease
Irritable Bowel
Syndrome
CIRRHOSIS
Scaldaferri F et al, ECCO 2015, FISMAD 2015, DDW2015
Diseases associated to an unbalanced
GUT Microbiota
cirrhosis
inflammatory bowel disease
IBS
diverticular disease
controls
0
50
100
150
200
250
300
350
chao index (complexity)
Species richness in Cirrhosis, IBD, DD, IBS and CT (Chao Score)
Scaldaferri F et al, ECCO 2015, FISMAD 2015, DDW2015
400
Microbiota and antibiotics
 Antibiotics act on single germ level
Goldman: Goldman's Cecil Medicine, 24th ed., 2011
Impact of antibiotics on GUT
MICROBIOTA
After the onset of treatment, increase in resistant bacteria (purple rods) due to:
• resistant bacteria (green rods), present in low levels, which increase in number
• horizontal gene transfer or mutation events (white arrow).
Some bacteria may be protected from antibiotic exposure in the mucin layer
(yellow shading). A temporary decrease in diversity can also be seen.
Cecilia Jernberg et al, Microbiology (2010), 156, 3216–3223
Impact of antibiotics on GUT
MICROBIOTA
• Antibiotic concentration in blood ?
• Antibiotic concentration within gut lumen?
• Antibiotic concentration within gut mucus?
• Subject variability?
Generation and Spread of
Antibiotic Resistance
Antibiotic resistance (ABX) is
generated by mutations that
can be induced by several
driving forces.
A) competition of bacteria
(inter- or intra-species)
B) Exogenous antibiotic
pressure through medical or
industrial practices
C) Antibiotic resistance
genes (ARGs) through
horizontal gene transfer, in
other words conjugation,
transduction, or transformation.
Trends in Molecular Medicine 2016 22, 458-478DOI: (10.1016/j.molmed.2016.04.003)
Microbiota and antibiotics
 Antibiotics act on single germ level
 Antibiotics act on gut microbiota composition
16S ribosomal RNA gene sequence-based microbiota analysis
quantitative polymerase chain reaction to evaluate the establishment of the intestinal
microbiota
preterm infants (n = 27) compared
with full-term babies (n = 13).
Arboleya et al, J Pediatr 2015;166:538-44)
Antibiotics potently affect microbiota
composition
Aggregate microbiota at family level, of samples collected at the different
time points from term infants whose mother received a single dose of IAP
with ampicillin (n = 3) and those whose mothers did not receive IAP (n =
10).
Arboleya et al, J Pediatr 2015;166:538-44)
Antibiotics potently affect microbiota
composition
Vangay P et al, Cell Host & Microbe 17, May 13, 2015 a2015 Elsevier Inc.
Antibiotics potently affect microbiota
composition
Vangay P et al, Cell Host & Microbe 17, May 13, 2015 a2015 Elsevier Inc.
Definition of Dysbiosis
“Dysbiosis”
“Eubiosis”
Reduced microbiota biodiversity
and increased instability
Increase in Proteobacteria
(pathogenic bacteria)
Reduction of anti- inflammatory
bacteria
SPECIFIC ALTERATIONS FOR SPECIFIC DISEASE
….
Microbiota and antibiotics
 Antibiotics act on single germ level
 Antibiotics act on gut microbiota
 Antibiotics impact on gut microbiota functions
within the GI tract
Clostrium difficile colitis:
the prototypycal “dysbiosios”
C difficile is a gram-positive, anaerobic, sporeforming bacterium, highly prevalent in
infants as well as among adults with
frequent health care– associated contact
and patients in chronic-care facilities
The bacterium became virulent by the
production of 2 toxins (TcdA and TcdB)
Symptoms of active infection range from mild
diarrhea to inflammatory colitis which can
cause death or recurrent colitis (20%)
CDI costs more than $3 billion annual hospital
costs
Britton R and Young, Gastroenterology 2014;146:1547–1553,
Bibbò S et al, J Immunol Res. 2014;2014:462740. doi: 10.1155/2014/462740. Epub 2014 Jun 5.
Clostrium difficile colitis:
the prototypycal “dysbiosios”
Microbiota alterations:
Reduced biodiversity
Increase in Proteobacteria
Loss of Lachnospiraceae
Britton R and Young, Gastroenterology 2014;146:1547–1553,
Bibbò S et al, J Immunol Res. 2014;2014:462740. doi: 10.1155/2014/462740. Epub 2014 Jun 5.
Antibiotic associated diarrhea
•
Antibiotic associated diarrhea (AAD)ranges from
mild diarrhea to inflammatory colitis to
pseudomembranous colitis
•
AAD may occur while taking antibiotics, or
develop as much as 8 weeks afterwards.
•
Duration of AAD ranges from 1 day to several
months, being recurrent in up to 20-50% of
patients
Mc Farland VL, Future Microbiology. 3.5 (Oct. 2008)
Antibiotic associated diarrhea
Pathogenesis
• Disruption of normal intestinal microbiota is the
key step that causes susceptibility: DYSBIOSIS
• Overgrowth of opportunistic pathogens,
like Clostridium difficile , Clostridium perfringens
, Klebsiella oxytoca and Staphylococcus aureus or
Candida
• Worse outcomes for High-risk antibiotics or in
poor host conditions (immunecompromised or
hospitalized patients)
Mc Farland VL, Future Microbiology. 3.5 (Oct. 2008)
Incidence of AAD in trials
Mc Farland VL, Future Microbiology. 3.5 (Oct. 2008)
Incidence of Clostridium Difficile
Mc Farland VL, Future Microbiology. 3.5 (Oct. 2008)
Microbiota and antibiotics
 Antibiotics act on single germ level
 Antibiotics act on gut microbiota
 Antibiotics impact on gut microbiota functions
within the GI tract
 Antibiotics impact on gut microbiota functions
outside the GI tract
this is a Swedish population-based case–control study
2,933 individuals with celiac disease (CD) - Marsh stage 3 (villous atrophy) enrolled from
the histopathology registry
Aim was to study association between celiac disease and antibiotics (Swedish Prescribed
Drug Register )
Data from July 1st 2005 (launch of the Prescribed Drug Register) through January 29th
2008 (end of the study period)
Of the 2,933 individuals with
CD,
27.0% had received at least
one course of antibiotics
during the study period
before biopsy
as compared with 21.1% in
the controls,
corresponding to an odds
ratio for subsequent CD of
1.40 (95% CI = 1.27-1.53)
Mårild et al. BMC Gastroenterology 2013, 13:109
This is a nested case-control study from the Health Improvement Network database (United
Kingdom), from June 1994, through January 2013
Children aged 1 to 15 years with newly diagnosed psoriasis (n = 845) were
compared with age- and sex-matched controls (n = 8450) randomly chosen at the
time of psoriasis diagnosis from general practices
JAMA Dermatol. 2016;152(2):191-199. Published online November 11, 2015.
JAMA Dermatol. 2016;152(2):191-199. Published online November 11, 2015.
After adjusting for matching, country, socioeconomic deprivation,outpatient visits,
and infections within the past 2 years,
antibiotic exposure in the last 2 years was weakly associated with incident
psoriasis (adjusted odds ratio [aOR], 1.2; 95% CI, 1.0-1.5).
The associations for infections of skin (aOR, 1.5; 95% CI, 1.2-1.7) and other sites
(aOR, 1.3; 95% CI, 1.1-1.6) were similar.
Untreated nonskin infections (aOR, 1.5; 95% CI, 1.3-1.8) but not antibiotic-treated
nonskin infections (aOR, 1.1; 95% CI, 0.9-1.4) were associated with psoriasis.
JAMA Dermatol. 2016;152(2):191-199. Published online November 11, 2015.
Cohort study spanning 2001-2013 using electronic health records from primary care
practices affiliated with the Children’s Hospital of Philadelphia
All children with annual visits at ages 0 to 59 months, were enrolled.
The cohort comprised 64 580 children.
JAMA Pediatr. 2014;168(11):1063-1069. doi:10.1001/jamapediatrics.2014.1539
Published online September 29, 2014.
69 of children were exposed to
antibiotics before age 24months,
with a mean (SD) of 2.3 (1.5)
episodes per child.
JAMA Pediatr. 2014;168(11):1063-1069. doi:10.1001/jamapediatrics.2014.1539
Published online September 29, 2014.
Steroid use, male sex, urban practice,
public insurance, Hispanic ethnicity,
and diagnosed asthma or wheezing were also
predictors of obesity
common infectious diagnoses and anti-reflux
medications were not.
Cumulative exposure to antibiotics was associated with later obesity (rate
ratio [RR], 1.11; 95% CI, 1.02-1.21 for 4 episodes); this effect was
stronger for broad-spectrum antibiotics (RR, 1.16; 95% CI, 1.06-1.29).
JAMA Pediatr. 2014;168(11):1063-1069. doi:10.1001/jamapediatrics.2014.1539
Published online September 29, 2014.
FMT “BLOOMING”
FMT “BLOOMING”
INCLUSION CRITERIA
TREATMENT
 vancomycin
 at least 18 years of age
(500 mg orally x 4/day x 4/5 days),
 a relapse of C. difficile infection
after at least one course of
adequate antibiotic therapy
(≥10
vancomycin
days
≥125 mg x 4
or
metronidazole 500 mg x 3/day).
of
/day
 C. difficile infection defined as
**
diarrhea
(≥3 watery stools/day for 2 days or ≥8 loose stools in
24
hours)
** positive C. difficile toxin.
followed
by
bowel lavage with 4 liters of
macrogol solution (Klean-Prep)
and
then
infusion
of
FECAL
MICROBIOTA
by nasoduodenal tube
 standard vancomycin regimen
(500 mg orally four times per
day
for
14
days);
or a standard vancomycin
regimen with bowel lavage on
day 4 or 5.
FMT “BLOOMING”
FMT TREATMENT
Feces were collected by the donor on the day of infusion and by 6 hours:
• Feces were diluted with 500 ml of sterile saline (0.9%)
• They stirred, strained and poured in a sterile bottle
• Feces were then infused through a nasoduodenal tube
(2 to 3 minutes per 50 ml).
Few patients received a second infusion
CLINICAL OUTCOMES
• The primary end point was cure without relapse within 10 weeks after the
initiation of therapy.
FMT “BLOOMING”
Because most patients in control groups had
a relapse, the data and safety monitoring
board performed the interim efficacy analysis
and advised an early termination of the trial
for excess of positive results
(46 total patients recruited)
Microbiota transplantation:
the most powerfull
“microbial therapy”
Vijay Shankar et al,
Species and genus level resolution analysis of gut microbiota in Clostridium difficile patients following fecal
microbiota transplantation.
Microbiome. 2014; 2: 13.
Microbiota transplantation:
the most powerfull
“microbial therapy”
Vijay Shankar et al,
Species and genus level resolution analysis of gut microbiota in Clostridium difficile patients following fecal
microbiota transplantation.
Microbiome. 2014; 2: 13.
FMT
by colonoscopy

Short vanco+FMT vs Standard vanco
 Study stopped after interim analysis
 Resolution of CDAD
•
•
FMT group (n=20): 90%
Vancomycin group (n=19): 26%
 5/7 pts with severe disease (PMC):
progressive disappearance of PMC and
resolution of CDAD after multiple FMT
 No significant adverse events
Cammarota et al – APT 2015
Fecal Microbiota Transplantation
- FMT -
In pediatrics??
Pediatric FMT: rCDI



7 reports, 19 patients (youngest: 16 month-old)
Overall 89.6% cure rate - no adverse events reported
Similar methodology to adult FMT
Walia – Curr Opin Pediatrics
2014
Clinical Trials for
fecal-M transplantation
www.clinicaltrials.gov on June 2016
..on going Clinical Trials
microbiota
Barrier
functions

OBESITY and
metabolic
syndrome
Immune
functions

Irritable
bowel
syndrome (IBS)
Metabolic
functions

Inflammatory bowel
disease (IBD)

Other microbiotarelated disorders
other
functions
FMT
Therapeutic pyramid
for dysbiosis
FMT
antibiotics
Probiotics and
Prebiotics
Fecal microbiota transplantation (FMT),
defined as
“ the introduction of a fecal suspension
from a healthy donor into the
gastrointestinal tract of a diseased
individual”
is perhaps the most potent modulator of
GUT MICROBIOTA COMPOSITION
and also the most powerful weapon against
DYSBIOSIS
DIET, sport…
TREATMENT
OF DYSBIOSIS
53
Probiotics: definitions
“Microorganisms that,
when administrered in adequate amounts,
confer health benefit to the host,
expecially by improving
intestinal microbial balance”
. FAO/WHO. Joint FAO/WHO Working Group Report
on Drafting Guidelines for the evaluation of Probiotics
in Food (FAO/WHO, London, Canada, 2002).
FAO/WHO. Probiotics in food. Health and nutritional
properties and guidelines for evaluation. En “FAO
Food and Nutrition
Paper 85”, 2006, ISBN 92-5-105513-0. Available at: ftp:
//ftp.fao.org/docrep/fao/009/a0512e/a0512e00.pdf .
Bacillus Clausii
Antibiotic induced diarrhea
55
Gareau, M. G. et al. Nat. Rev. Gastroenterol. Hepatol. 7, 503–514 (2010)
Mechanisms of actions
56
Gareau, M. G. et al. Nat. Rev. Gastroenterol. Hepatol. 7, 503–514 (2010)
MODULATE GUT MICROBIOTA
COMPOSITION???
57
Results from a preclinical study
“Clausii in colitis:
role and mechanisms of action of Bacillus
clausii in experimental colitis”
Franco Scaldaferri1, Cristina Graziani1, Valentina Petito1-3, Loris Riccardo Lopetuso1, Gianluca
Quaranta2, Luca Masucci2, Francesco Franceschi1, Andrea Poscia3, Vincenzo Arena3, Domenico
Scannone3, Gianluca Ianiro1, Giovanni Cammarota1, Alessandro Sgambato5 and Antonio Gasbarrini1
1 ISTITUTO DI PATOLOGIA SPECIALE MEDICA, Università Cattolica del Sacro Cuore, L.go Gemelli 8, 00168 Rome, Italy
2 ISTITUTO DI MICROBIOLOGIA, Università Cattolica del Sacro Cuore, L.go Gemelli 8, 00168 Rome, Italy
3 ISTITUTO DI IGIENE, Università Cattolica del Sacro Cuore, L.go Gemelli 8, 00168 Rome, Italy
3 ISTITUTO ANATOMIA PATOLOGICA, Università Cattolica del Sacro Cuore, L.go Gemelli 8, 00168 Rome, Italy
4 ISTITUTO DI PATOLOGIA GENERALE, Università Cattolica del Sacro Cuore, L.go Gemelli 8, 00168 Rome, Italy
SCALDAFERRI F ET AL, UEGW 2016, SIMI 2016, EHMSG 2016
DSS model of colitis
START
day 1
day 5
stop DSS
and 1st sacrifice
ACUTE
PHASE
END of
experiments
day 14
RECOVERY
PHASE
• Each experiment: 30 mice C57BL6 (10 per group)
• DSS 2.5% given for 5 days (mild colitis)
• Bacillus Clausii (dose 1= 80 millions /dose 2=20 millions spores)
given by oral gavage during the 5 days of DSS treatment
SCALDAFERRI F ET AL, UEGW 2016, SIMI 2016, EHMSG 2016
Bacillus clausii improves experimental colitis
in mice in dose dependent fashion
Higher dose (dose 1= 80 mill of bac clausii per day for 5 days)
of BACILLUS CLAUSII was associated to better control of colitis
SCALDAFERRI F ET AL, UEGW 2016, SIMI 2016, EHMSG 2016
Bacillus clausii modulates gut microbiota in
mild DSS murine colitis, increasing aerobes
microbial counts in colitic mice.
AEROBES COLTURE
COLITIC MICE: microbial (aerobes ) load CFU/g
a)
12000
DSS + placebo
DSS + dose 1
ANAEROBES COLTURE
b)
DSS + dose 2
6000
8000
6000
4000
2000
3000
2000
0
T1
T2
HEALTHY MICE: microbial (aerobes) load CFU/g
12000
placebo
dose 1
dose 2
10000
8000
T0
d)
6000
4000
2000
T1
T2
HEALTHY MICE: microbial (anaerobes) load CFU/g
6000
placebo
dose 1
dose 2
5000
milions of CFU/g
milions of CFU/g
DSS + dose 2
1000
T0
healthy
mice
DSS + dose 1
4000
0
c)
DSS + placebo
5000
milions of CFU/g
milions of CFU/g
10000
colitic mice
(DSS)
COLITIC MICE: microbial (anaerobes) load CFU/g
4000
3000
2000
1000
0
T0
T1
T2
0
T0
T1
SCALDAFERRI F ET AL, UEGW 2016, SIMI 2016, EHMSG 2016
T2
Bacillus clausii modulates
gut microbiota
Bacillus clausii was
associated to a reduction
of
 Staphilococcus aureus
 Clostridium innocuum
 Enterobacteriacae
like Klebsiella oxytoca,
Enterococcus
casselliflavus and
gallinarum.
SCALDAFERRI F ET AL, UEGW 2016, SIMI 2016, EHMSG 2016
• The gene, called aadD2, encoding a putative 246-amino acid protein, shared
47% identity with ant-4-Ia from Staphylococcus aureus, which encodes an
aminoglycoside 4’-O-nucleotidyltransferase, RESPONSIBLE FOR
RESISTENCE WAS IDENTIFIED IN BACILLUS CLAUSII
• The aadD2 gene was chromosomally located in all strains and was not
transferable by conjugation. These data indicate that chromosomal aadD2 is
specific to B. clausii.
Antibiotic resistance
Chromosomal resistance:
vertically trasmissible by bacterial division
No risk of antibiotic-resistance transfer
Courvalin P, DLD 2006
Antibiotic resistance
Plasmid resistance:
horizontally trasmissible by bacterial conjugation
High risk of antibiotic-resistance transfer
Courvalin P, DLD 2006
Probiotics and
antibiotics
Favours to Favours to
Probiotics Placebo
Hempell S et al, JAMA. 2012;307(18):1959-1969
67
Review Team
Francisco Guarner (Chair, Spain) Aamir G. Khan (Pakistan) James Garisch (South Africa)
Rami
Eliakim
(Israel)
Alfred
Gangl
(Austria)
Alan Thomson (Canada) Justus Krabshuis (France) Ton Lemair (The Netherlands)
Invited outside experts
Pedro Kaufmann (Uruguay) Juan Andres de Paula (Argentina) Richard Fedorak (Canada)
Fergus Shanahan (Ireland) Mary Ellen Sanders (USA) Hania Szajewska (Poland) B.S.
Ramakrishna (India) Tarkan Karakan (Turkey) Nayoung Kim (South Korea)
WGO: World Gastroenterology Organisation
WGO, 2011
68
Evidence-based adult indications
for probiotics
Disorder
Prevention of
antibiotic
associated
diarrhea in
adults
Probiotic strain
Evidence level
E. faecium LAB
SF68
108 cfu, bid
1b
S. boulardii, ceppo
di S. cerevisiae
1 g o 4 x109 cfu
die
1b
L. rhamnosus GG
1010- 1011 cfu bid
1b
L. casei DN-114 001
in latte fermentato
1010 cfu bid
Bacillus clausii
2x109 spore tid
1b
5x1010 cfu uid o
bid
1b
L. acidophilus
CL1285 +
L. casei LBC80R
WGO: Organizzazione Mondiale di Gastroenterologia
Cfu: unità formanti colonie
Recommended dose
1b
WGO, 2011
Prebiotics
“Non digestible food ingredients that beneficially
affects the host by selectively stimulating the growth
and/or the activity of one or a limited number of
bacteria in the colon and thus improve host health”
•Inulin
•Lactulose
•Fructo-oligosaccharides (FOS)
•Galacto-oligosaccharides (GOS)
•Other oligosaccaharides (xylo-, soyo-,
gluco-, gentio-, isomalto-oligosaccharides…)
Gibson, J Nutr 1995
Mechanism of action
of prebiotics
Gibson, DDT 2003
“Dysbiosis”
Increased intestinal
permeability
Epithelial cells
Diet and food
disregulation
72
73
Take home message
 Antibiotics act not just at single germ level,
but also on the entire gut microbiota
 Antibiotics impact on gut microbiota functions
within the GI tract and outside the GI tract
 Microbiota modulation is a reality for
intestinal disorders (FMT in C Diff Colitis,
pre and pro-biotics) and perhaps for extra
intestinal disorders in the future
74
April 2017
75