Post on 14-Feb-2019
Antonio Gasbarrini
Microbiota e Cirrosi
Microbiota intestinale e malattia cirrotica complicata
Medicina Interna e GastroenterologiaPoliclinico Universitario Gemelli
Universita’ Cattolica, Roma
Monotematica AISF 2014 Cirrosi Epatica: Napoli 10 anni dopo
Napoli, 9-11 otobre 2014
Advisory board: Sanofi, Alfa Wassermann, Abbvie, MSD, Angelini, Zambon,, Jansen, Boheringer, Admirall, Gilead, Bayer, IBI
Speaker’s bureau: Sanofi, Alfa Wasserman, Jansen, Boheringer, Angelini, Zambon, MSD, Gilead, Abbvie, Metagenics, Malesci, Admirall, Shire
Conflict of interest
Systems biology (systeomics):1.Computational and mathematical modeling of complex biological systems
2.Biology-based inter-disciplinary field of study that focus on complex interactions within biological systems (ie metabolic networks or cell signalling networks)
Human Microbiota: new actor, new knowledges
VirusBacteriophages
BacteriaYeast
Acquiredand
Innate immunity
MucosalBarrier
Epithelial barrier
Vascular and lymphatic systems
Neuroenteric systemDigestive enzymes
Endocrinesystem
HelminthParasiteArchea
Protozoa
GUT BARRIER
GUT MYCOBIOME
HBV-CIRRHOTICS:Candida (33.78%), uncultured fungi (12.53%), Aspergillus (7.99%), Simplicillium (5.65%), Chaetomium (2.46%), Galactomyces (2.33%), Rhizopus (1.96%), Wallemia (1.10%), Fusarium (1.10%), Iodophanus (0.12%), Penicillium (0.49%), Saccharomyces (25.18%), uncultured Pezizomycotina (0.86%), uncultured Pucciniomycotina (1.10%), uncultured Agaricomycotina (0.74%), Aureobasidium (0.61%), Hyphozyma (0.49%), Asterotremella(0.49%), Cryptococcus (0.49%)..
• The fungal biota of the gut, plays an important role in health and diseases interplaying with other biomes of the gut
HEALTHY: Wallemia, Trichocomaceae, Saccharomycetaceae, Rhodotorula, Pleosporaceae, Metschnikowiaceae, Cystofilobasidiaceae, Ascomycota, Amphisphaeriaceae, Agaricaceae Dollive S, et al. Genome Biol 2012
Chen Y, et al. Diagn Micr Infec Dis 2011
GUT VIROME
Berg Miller et al, Environ Microbiol 2011
• Collection of all viruses of the gut• Harmful viruses are a minority compared to benign viruses• Bacterial virus are called bacteriophages
Random pyrosequencing of virus-enriched metagenomes have been isolated from bovine rumen. In the bovine rumen have been isolated up to 28.000 different viral genotypes; the majority (∼78%) of sequences did not match any previously described virus
Pro phages outnumbered lytic phages approximately 2:1
Metabolic profiling revealed an enrichment of sequences with putative functional roles in DNA and protein metabolism, but a low proportion of sequences assigned to carbohydrate and amino acid metabolism
GUT BACTERIOME
>9 phyla95% genes identity
>1000 species99% genes identity
>15000 strains100% genes identity
800-900 gr, >3.300.000 genes
Microbiome MetabolomeLeser et al, Environ Microbiol 2011 Qin et al, Nature 2011
BACTERIAL TAXONOMIC RANK
DOMINIO
REGNO
PHYLUM
CLASSE
ORDINE
FAMIGLIA
GENERE
SPECIE
CEPPO (STRAIN)
95%
99% 100%
Genes identity
MOLECULAR BACTERIOLOGY:most abundant PHYLA in the GUT (>60%)
Eckburg et al, Science 2005
BACTEROIDETES
FIRMICUTES
HUMAN GUTENTEROTYPE
Microbiota composition is affected by life events
Ottmann N et al. Front Cell Infect Microb 2012
Failure of MICROBIOTA control’s mechanisms
Quali-quantitative alterations of oral, esophageal, gastric, small bowel and/or
colonic microbiota
DYSBIOSIS
Digestive and extradigestive diseases
EUBIOSIS
DYSBIOSIS
LEAKY GUT
Digestive diseases associated to DYSBIOSIS/LEAKY GUT
• Gastrointestinal infections• Irritable Bowel Syndrome • Intestinal Bacterial Overgrowth• Colonic diverticulosis• Inflammatory Bowel Diseases• Gastro-intestinal Cancers• Food Intolerance/Allergy• Liver diseases• Pancreatic diseases• Obesity, Diabetes and Metabolic Syndrome• …
Ruolo del Microbiota intestinale nelle principali complicanze della Cirrosi
Ascite/PBS
Encefalopatia
Sindrome epato-renale
Trombosi portale
Emorragia digestiva
Epatocarcinoma
Quale e’ il microbiota del paziente cirrotico?
Qin, Nature 2014
A novel gut gene catalogue (98 patients with liver cirrhosis and 83 controls)
Comparison with MetaHIT, Human Microbiome Project, T2D catalogues
2.69 million genes, 36.1% are novel
4 currently available major human microbiome gene sets
Cirrhotics and controls separated by principal component analysis
Genes grouped into 66 clusters, denoted metagenomic species (MGS)
28 MGS enriched in patients and 38 MGS in controls
Qin, Nature 2014
Bacteroides was significantly decreased in the liver cirrhosis group
Veillonella, Streptococcus, Clostridium and Prevotella were enriched in the liver cirrhosis group
Eubacterium and Alistipes were dominant in the healthy controls
Severity of liver disease was positivelycorrelated with the abundance of a number of Metagenomics Species (MGS) enriched in patientsand negatively correlated with those of the MGS enriched in controls
Disease statusof the patients with the highest load of these bacteria was significantly worse than that of the patients with the lowest load
54% of the patient-enriched, taxonomically assigned species are of buccal origin
14 day course of 40mg/day of omeprazole is associated with significant shift in gut microbiota composition in patients and controls
Relative Streptococcaceae abundance, normally abundant in saliva, significantly increased post-omeprazole in controls (1% vs. 5%) and cirrhosis (0% vs 9%) and was correlated with serum gastrin levels (r=0.4, p=0.005)
Significant changes in linkages of bacteria with metabolites (hippurate/DMA/lactate) were found post-omeprazole, compared to pre-PPI in cirrhosis patients.
Short course of a PPI significantly shifts urinary NMR metabolic profile and stool microbiota function and composition linked to the
gastric acid suppression and overgrowth of bacteria normally present in the oral cavity
On the basis of only 15 biomarkers, a highly accurate Patient Discrimination Index (PDI) is created and validated on an independent cohort
Biomarkers specific to cirrhosis are revealed by a comparison with those for T2M and IBD
Liver cirrhosis, T2D and IBD displayed a relatively unique profile, even if some markers were shared
Patient Discriminatory Index could be used to identify patients with liver cirrhosis
Discriminatory power of the PDI was validated using an independent group
Potential use of gut microbiota information for identifying patients with liver cirrhosis and severity of liver disease
Alterazioni di permeabilita’/disbiosi sono associate alla severita’ della
cirrosi?
Intestinal Permeability and CHILD status
0102030405060708090
100
IP
Child AChild BChild C
IP impairment followed progressing Child status: Child A 22
%; Child B 39 %; Child C 75 %
%*
**
Scarpellini E, Gasbarrini A. Am J Gastroenterol 2010
Aim: To study changes in microbiome over cirrhosis severity, its stability over time and its longitudinal alterations with decompensation
Methods:Stool microbiota quantified using multi-tagged
pyrosequencingCirrhosis dysbiosis ratio (CDR): autochthonous/non-
autochthonous taxa (low number indicating dysbiosis)The microbiome was compared between controls and
cirrhotic sub-groupsLongitudinal assessment after decompensation, outcome and
infectionBajaj 2014 J Hepatol
Bajaj 2014 J Hepatol
Red: inpatient cirrhotics
Green: outpatient cirrhotics
Blue: controls
Bajaj 2014 J Hepatol
32
MICROBIOTA ANALISYS: 454 Technology• In DNA synthesis, a dNTP is attached to the 3’ end of the growing DNA
strand. The two phosphates on the end are released as pyrophosphate (PPi).
• Luciferase is the enzyme that causes fireflies to glow. It uses luciferin and ATP as substrates, converting luciferin to oxyluciferin and releasing visible light.
• The amount of light released is proportional to the number of nucleotides added to the new DNA strand.
• The plate is coupled to a fiber optic chip. A CCD camera records the light flashes from each well.
33
Our results: phyla
Cirrhosis BacteroidetesFirmicutesProteobacteriaFusobacteriaUnassignedVerrucomicrobiaActinobacteriaSpirochaetesElusimicrobiaTM7SynergistetesTenericutesCyanobacteriaLentisphaerae
Controls
34
Our results: genus
Cirrhosis g__Bacteroides
g__Prevotella
g__Lactobacillus
f__Rikenellaceae
g__Faecalibacterium
f__Ruminococcaceae
g__Acidaminococcus
f__Enterobacteriaceae
g__Parabacteroides
f__Lachnospiraceae
Controls
35
Our results: phyla
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Controls Child A Child B
% of total
TM7
Fusobacteria
Synergistetes
Actinobacteria
Unassigned
Elusimicrobia
Spirochaetes
Verrucomicrobia
Proteobacteria
Firmicutes
Bacteroidetes
CHILD stratificied Cirrhosis vs controls (analysis of gut microbiota phyla)
36
Our results: genus
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Controls Child A Child B
% of total
g__Acidaminococcus
g__Enterococcus
f__Enterobacteriaceae
g__Streptococcus
f__Lachnospiraceae
g__Parabacteroides
g__Oscillospira
g__Veillonella
f__Rikenellaceae
f__Ruminococcaceae
g__Faecalibacterium
g__Lactobacillus
g__Bacteroides
g__Prevotella
CHILD stratificied Cirrhosis vs controls (analysis of gut microbiota genus)
37
Our results(1)
A) CTRL vs Hepatopatic patients(5 healthy subject, 6 patients)
at baseline
• Child A
• Child B
Rifaximin treatment (1200 mg/day x 10 days) and fecal microbiota
characterization
Hepatic Encephalopathy before and after rifaximin
38
Our results: phyla
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%% of total
p__Proteobacteria
p__Firmicutes
p__Bacteroidetes
p__Actinobacteria
Unassigned
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
% of total
39
Our results: genus
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
% of totalp__Proteobacteria;c__Haemophilusp__Proteobacteria;c____Klebsiellap__Proteobacteria;c__Enterobacteriaceae;g__p__Proteobacteria;c____Campylobacterp__Proteobacteria;c___Neisseriap__Proteobacteria;c__Betaproteobacteria;o___Sutterellap__Firmicutes;c__Erysipelotrichi;o___[Eubacterium]p__Firmicutes;c__Erysipelotrichi;o___Coprobacillusp__Firmicutes;c__Erysipelotrichi;o___Clostridiump__Firmicutes;c__Erysipelotrichi;o_g__Bulleidiap__Firmicutes;c__Erysipelotrichi_Erysipelotrichaceae;g__p__Firmicutes;c__Clostridia;o___Veillonellap__Firmicutes;c__Clostridia;o___Selenomonasp__Firmicutes;c__Clostridia;o___Phascolarctobacteriump__Firmicutes;c__Clostridia;o___Megasphaerap__Firmicutes;c__Clostridia;o___Megamonasp__Firmicutes;c__Clostridia;o___Dialisterp__Firmicutes;c__Clostridia;o___Acidaminococcusp__Firmicutes;c__Clostridia;o____Ruminococcusp__Firmicutes;c__Clostridia;o___Oscillospirap__Firmicutes;c__Clostridia;o___Faecalibacteriump__Firmicutes;c__Clostridia;o___Anaerotruncusp__Firmicutes;c__Clostridia;o__Ruminococcaceae;g__p__Firmicutes;c__Clostridia;o__Peptostreptococcaceae;g__p__Firmicutes;c__Clostridia;o___[Ruminococcus]p__Firmicutes;c__Clostridia;__Lachnospirap__Firmicutes;c__Clostridia;__Doreap__Firmicutes;c__Clostridia;o__Blautiap__Firmicutes;c__Clostridia_Lachnospiraceae;g__p__Firmicutes;c__Clostridia;Otherp__Firmicutes;c__Clostridia;_Clostridiump__Firmicutes;c__Clostridia;Clostridiaceae;g__p__Firmicutes;c__Clostridia;;Otherp__Firmicutes;c__Clostridia;o_;g__p__Firmicutes;c__Clostridia;o_;Otherp__Firmicutes;c___Streptococcusp__Firmicutes;c____Lactococcusp__Firmicutes;c____Lactobacillusp__Firmicutes;c___Granulicatellap__Firmicutes;c___Aerococcaceae;g__p__Firmicutes;c____Gemellap__Firmicutes;c_Gemellaceae;g__p__Bacteroidetes;__Parabacteroidesp__Bacteroidetes;__Bacteroidesp__Actinobacteria;__Eggerthella
A ti b t i A ti
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
% of total
Ruolo del Microbiota intestinale nelle complicanze della Cirrosi
Ascite
Encefalopatia
Trombosi portale
Emorragia digestiva
Epatocarcinoma
25 cirrhotics undergoing paracentesis provided ascites
Samples treated with propidium monoazide to exclude non-viable bacterial DNA
Bacterial load was quantified by 16S rRNA Q-PCR with species identity and relative abundance was determined by 16S rRNA gene pyrosequencing
Correlation of molecular microbiology data with clinical measures and diagnostic microbiology was performed
Not viable anaerobic bacteria and viable aerobic bacteria are present in the ascites of a majority of patients with cirrhosis including those with no clinical signs of infection
Ascitic microbiota composition significantly correlates with clinical measures
Microbiota similarity between patients correlated with asciticpolymorphonuclear leukocyte count and Child-Pugh Score
Entry of bacteria into ascites is unlikely to be limited to translocation from the gut, raising questions about the processes that underlie the development of SBP
Samples analyzed are negative for bacterial growth based on standard microbiological tests
Viable bacterial signal was obtained in 84% of ascites samples, both by Q-PCR and pyrosequencing
Approximately 190,000 ribosomal pyrosequences were obtained, representing 236 species, including both gut and non gut-associated species
Substantial variation in the species detected was observed between patients
152 hospitalized patients with advanced liver cirrhosis underwent a diagnostic paracentesis from March 2012 to April 2013 for SBP
Data about type of SBP prophylaxis, prior use of rifaximin, concomitant complications of cirrhosis, laboratory results and bacteriological findings are collected
Patients were divided in three groups: no antibiotic prophylaxis, prophylaxis with rifaximin or with systemically absorbed antibiotic prophylaxis
Escherichia coli and enterococci were dominant in 72% of patients without any prophylaxis and were not detected in the Rifaximin group
Klebsiella species were recovered from 75% of ascites samples in the Rifaximin group; the bacterial species causing SBP were changed by Rifaximin
Ruolo del Microbiota intestinale nelle complicanze della Cirrosi
Ascite
Encefalopatia
Trombosi portale
Emorragia digestiva
Epatocarcinoma
GUT microbiota: key player in hepatic Encephalopathy
Moriwaki H et al J Gastr Hepatol 2009
Abundance of Streptococcus salivarius and Veillonellacee was significantly higher in cirrhotic patients with MHE than in those without ( P = 0.030)Change in the amount of this bacteria was positively correlated with ammonia accumulation ( R = 0.58, P = 0.003) in cirrhotic patients with MHE but not in those withoutUrease producer
Gupta A et al, J Hepatology 2010
26% of cirrhotics with MHE had IntestinalBacterial Overgrowth (IBO)
IBO prevalence: CHILD A: 31%CHILD B-C: 70%
OCTT significantly prolonged in SIBO+ (175 vs 127 min, p <0.0001)
Lactulose, probiotics, prebiotics, antibiotics in hepatic Encephalopathy
Als-Nielsen B et al BMJ 2003
Lactulose in hepatic Encephalopathy
There was a significant improvement in cognition and endotoxemia afterrifaximinThe networks centered on Enterobacteriaceae, Porphyromonadaceae andBacteroidaceae indicated a shift from pathogenic to beneficial metabolitelinkages and better cognition
20 MHE pts underwent cognitive testing and functional MRI, diffusiontensor imaging and MR spectroscopy before and after rifaximin therapy
Therapy with Rifaximin is associated with improved cognition, workingmemory performance and inhibitory control in MHE pts
Improvement in white matter microstructural integrity as depicted by Diffusion tensor imaging
Higher effective connectivity in white matter region reflecting improved psychophysiological
interaction (functional MRI)
Ahluwalia et al, march 2014 Metab Brain Dis
Eltawil K et al. WJG 2012
Rifaximin is superior to any other therapies to treat HE
Sharma 2013 Am J Gastro
Significant decrease in mortality inrifaximin plus lactulose
More deaths in lactulose group dueto sepsis
Shorter hospital stay for rifaximinplus lactulose
Recurrence of HE: 22.1% (31/140)rifaximin vs. 45.9% (73 of 159) placebo pts
Incidence of recurrent HE: reduced by58%
Hospitalization due to HE: reduced by 50%(13.6% rifaximin pts vs. 22.6% placebo pts)
No major adverse events were noted in therifaximin group. Mortality rate was the samein the two groups.
Bass et al. N Eng J Med 2010
Secondary end point:RIFAXIMIN SIGNIFICANTLY
REDUCED THE RISK OF HOSPITALIZATION
INVOLVING HEPATIC ENCEPHALOPATHYhazard ratio 0.50
relative risk reduction 50%
Primary end point:RIFAXIMIN MAINTAINED
REMISSION FROM HEPATICENCEPHALOPATHY MORE EFFECTIVELY THAN DID
PLACEBO FOR A 6-MONTH PERIODS
hazard ratio 0.42relative risk reduction 58%
24-month, open-label maintenance study of rifaximin (550 mg, twice daily) in patients with HE who participated in the previous RCT of rifaximin (NEJM 2010) or new patients enrolled
Median exposure to rifaximin was 427.0 days (range, 2–1427 d) Profile and rate of adverse events with long-term rifaximin treatment were similar
to those of the original RCT No increase in the rate of infections, including with Clostridium difficile, or
development of bacterial antibiotic resistance; rates of hospitalizations with long-term rifaximin administration remained low
Long-term treatment (≥24 mo) with Rifaximin provides a
continued reduction in the rate of HE-related and all-cause
hospitalization, without increased rate of adverse events
Mullen et al, Clinical Gastro and Hepatol 2014
n of speeding tickets n of illegal turns
= n of collisions
RIFAXIMIN showed an higher rate of improvement in total driving errors, specifically speeding tickets and navigation of illegal turns on a driving simulator compared with those on
placebo. This was accompanied by improved cognitive performance and psychosocial aspects of quality of life
Bajaj JS et al, Gastroenterology 2011
219 Patients (multicentric RCT) with cirrhosis in remission from HE and with documented history of recurrent HE episodes
Rifaximin 550 mg twice daily (N = 101) or placebo (N = 118) for 6 months
Microbiota in cirrhosis complications
Take Home Message
Loosely adherent
mucus layer
Firmly adherent mucus layer
Bad bacteria
Bile acids
Lumen
Recettori ionici
Water
Stomach Duodenum and
Jejunum
Ileum Colon
Adhesions molecules
Immune cells
Food antigens
EndotheliumAnd fibroblasts
Nerve and miocytes
Non‐Immune cells
Food antigens
Good bacteria
Lumen
Immune cells
EndotheliumAnd fibroblasts
Nerve and miocytes
Non‐Immune cells
SEVERE LIVER DISEASES
GASBARRINI A, UNPUBLISHED
Dysbiosis and leaky gut characterizes natural history of liver diseases
Type of dysbiosis is related to ethiological cause of liver diseases
Dysbiosis affects liver diseases pathogenesis
Dysbiosis has a main role in development of cirrhosis’ complications
TAKE HOME MESSAGES
ETOH NAFLDHBV/HCV
HCC
Auto immunity
Encephalopathy
Iron.. Portal hypertension
DYSBIOSIS+
LEAKY GUT
HRS
Ascites/PBS
HBV/HCVLiver damage
Dysbiosis
Portal hypertension/Increased permeability
PAMPs absorption
Endotoxemia
Inflammation
Fibrosis/ Complications
Ist HIT
IInd HIT
High Fat Diet
Increased permeability
PAMPs absorption
Endotoxemia
Inflammation
Steatohepatitis/ Metabolic disorders
Ist HIT
IInd HIT
NAFLD
Dysbiosis + Liver steatosis
ETOHLiver damage + GI damage
Dysbiosis
Portal hypertension/Increased permeability
PAMPs absorption
Endotoxemia
Inflammation
Hepatitis/ Complications
Ist HIT
IInd HIT
Gut microbiota remodulation affects the management of cirrhosis’ complications
Efficacy and safety of systemic antibiotics for treatment and prevention of SBP and of Rifaximin for treatment and prevention of overt and minimal HE has been extensively established in various RCT and meta-analysis
Potential of dysbiosis diagnosis and treatment in liver diseases goes much beyond PBS and encephalopathy treatment
TAKE HOME MESSAGES
TRUC mice, deficient for Tbet and Rag
Colitic phenotype could be transmitted vertically to the
progeny of the affected parents and horizontally to
unrelated animals
Microbiota transmits colitic phenotype
Garrett, Cell 2007
De Gottardi A, J Hepatology 2011
Microbiota transmits hepatosteatosic phenotype
Transplantation of germ-free mice with a microbiota from obese/steatosic mice stimulates triglyceride
synthesis and glycogenesis in the liver
Delzenne et al., Nat Rev Endocrinol 2011Abu-Shanab et al., Nat Rev Gast Hep 2011
Microbiota transmits adiposity phenotype
Ridaura et al. Science 2013Walker AW et al. Science 2013
TRANSFERRED INTO THE INTESTINESOF 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