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