Gianluca Ianiro

Gastroenterology AreaFondazione Policlinico Universitario “A. Gemelli”, Università Cattolica del Sacro Cuore, Rome, ItalyUEG Young Talent Group

Gut microbiotae apparato digerente

Email: gianluca.ianiro@hotmail.it

@gianluca1aniro

MICROBIOTAREVOLUTION

• Gut microbiota

• Muco-epithelial barrier

• Immune system

• Vascular pathway

• Endocrine system

• Neuroenteric system

• Digestive enzymes

Not only gut microbiota The functional interplay of the gut barrier

The “Tiki-Taka” model

• Il microbiota intestinale è l’insieme dei batteri, virus, funghi, parassiti e altri microbi, che vivono nel nostro intestino

• Il microbiota intestinale stabilisce con l’uomo un rapporto di simbiosi(“super-organismo”)

10:1Rapporto cellule microbiche-cellule umane nel nostro organismo

3.300.000 Vs 22.000N° geni del microbiota umano vs N° geni del nostro organismoMicrobioma come “parte variabile” del nostro genoma

>10.000N° specie microbiche identificate nel nostro organismo

1 kgPeso del microbiotaintestinale umano

80-90%Differenza in termini di genoma microbico fra due individui

0.01%Differenza in termini di genoma umano fra due individui

Human microbiota composition

Enterococcus

Dethlefsen et al., Nature, 2007 - Ley et al., Science, 2008 - Tap et al., Environ Microbiol, 2009,

Firmicutes60 to 80 %

Clostridium coccoides (cluster

XIVa)

Clostridium leptum (cluster IV)

Lactobacillales

Bacteroidetes20 to 40 %

Faecalibacteriumprausnitzii

Lactobacillus

Bacteroidesthetaiotaomicron

Streptococcus thermophilus

Bifidobacterium

Escherichia coli

Actinobacteria

Proteobacteria

Helicobacter pylori

GUT BACTERIOME

Functions of gut microbiota on host health

• Barrier effect

• Immunocompetence/Tolerance

• Synthesis

• Metabolism

• Drug metabolism

• Behavior conditioning

Lifelong immunostimulation by enteric commensal and pathogenic bacteria

Maynard CL et al. Nature 2012

Gut microbiota isan excellent anaerobicenergetic bioreactor

•It consumes, stores and redestributes energy•It allows us to extract calories from otherwise indigestiblecarbohydrates

EU= good; BIOS= life

What is EUBIOSIS?

Eubiosis is the healthyrelationship among commensal

bacteria of the gutCOMPOSITION

• Diversity• Richness• Relative Abundance

FUNCTION

• Microbiota’s effect on hosthealth

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 is a consequence of life events

Ottmann N et al. Front Cell Infect Microb 2012

Weaning

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..)

Verdu – Nat Rev Gastro Hepatol 2015

Early determinants of GUT Microbiota composition

Cox – Cell 2014Jess T.,N Engl J Med.

• Transfer of cecal microbiota from 18-week-old controls and penicillin-treated mice to 3-week-old germ-free mice

• Mice receiving penicillin-altered microbiota gained total mass and fat mass at a significantly faster rate than controls

• Mice whose mothers were treated with penicillin before the birth of the pups and throughout the weaning process had increased total and fat mass, increased ectopic fat deposition, increased hepatic expression of genes involved in adipogenesis,

• The body composition of adult male mice who had received penicillin after weaning was similar to that of controls

Microbiota, antibiotics & obesity

• Administration of low doses of penicillin or oxytetracycline has been shownto lead to weight gain in mice, but high doses lead to weight loss

• Early life is the key period for microbe-mediated programming of hostmetabolism Cox et al – Cell 2014

Cox et al – Nature Rev Endocrinol 2015

Blaser – Science 2016

• The decline in microbiota diversity in the US happened simultaneously with the early introduction of sanitation

• In late modernization countries: later but faster diversity loss, because of the effects of the accelerated pace of modernization in recent years on human microbiota biodiversity loss in developing countries.

Dysbiosis is a consequence of life events

Ottmann N et al. Front Cell Infect Microb 2012

Adult

Microbiota influencers• Diet

– Composition (calories, fat, vegetable, meat..)– Cooking– Natural food additives (safrolo..) – Artificial chemical food additives:

• Preservatives (benzoic acid, sodium benzoate, nitrite/nitrate, sulfur dioxide/sulfite..)

• Sweeteners, emulsifiers and stabilizers, flavors, thickeners, antifoaming, anticaking, bulking, antioxidants..)

• Others (titanium dioxide..)• Exercise• Sleep• Stress• Drugs

The animal-based diet increases the abundance

of bile-tolerant microorganisms

(Alistipes, Bilophila, and Bacteroides) and

decreases the levels of Firmicutes that

metabolize dietary plant polysaccharides

(Roseburia, Eubacterium rectale, and

Ruminococcus bromii )

The human gut microbiome can rapidly switch between herbivorous andcarnivorous functional profiles. It may reflect past selective pressures duringhuman evolution.

The short-term modification of diet alters microbial community structure and microbial gene expression

David – Nature 2014

De Palma– Br J Nutr 2009

30 days of gluten-free diet in healthy people

BifidobacteriumC. lituseburense F. prausnitzii

BifidobacteriumLactobacillus

Enterobacteriaceae E.coli

FISH

qPCR

Microbiota & GFD: healthy people

Butyrate-producing Clostridiumcluster XIVa (6.62-fold)

Akkermansia muciniphila (19.3 fold)

Ruminococcus torquesHalmos – Gut 2015

• Cross-over randomised controlled trial - 21 pts with IBS and 6 controls

• 21d low-FODMAP diet vs typical Australian diet, followed by a 21d washout period before crossing over to the alternate diet

Low-FODMAP diet influence on large bowel microbiota

Reduction of potential prebiotic and fermentative effects might adversely affect the colonic microenvironment

Low-FODMAP vs Australian diet

Fermentable, Oligo-, Di-, Mono-saccharides And Polyols

Non caloric artificial sweeteners (NAS: SACHARIN, SUCRALOSE,ASPARTAME) drive development of glucose intolerance throughinduction of compositional and functional alterations of gutmicrobiota

NAS-mediated effects can be abrogated by antibiotic treatment

NAS-mediated effects are fully transferrable to germ free mice upontransplantation of microbiota from NAS consuming mice or ofmicrobiota anaerobically incubated in presence of NAS

Suez et al, Nature 2014

CALLING FOR A REASSESSMENT OF MASSIVE SWEETENERS USAGE

Microbiota influencersNon caloric artificial sweeteners

4 groups of C57BL/6 mice:• Low fat (LF) sedentary (Sed)• LF/Exercise (Ex)• High Fat/Sed• HF/Ex

Evans – Plos One 2014

• Diet and activity altered the relative level of Bacteroidetesand Firmicutes

• Beta Diversity is Elevated by HF Diet and Exercise

Poroiko – Sci Rep 2016

Microbiota influencersChronic sleep disruption

Chronic Sleep Disruption Alters Gut MicrobiotaMice were exposed to Sleep Fragmentation (SF) for 4 w and then allowed to recover for 2 w

FirmicutesLachnospiraceae Ruminococcaceae

BacteroidetesBifidobacteriaceaeLactobacillaceae

Reversible gut microbiota changes after SF

Sommer – Nat Rev Microbiol 2013

GUT MICROBIOTA IN DISEASEFall of the “Single germ” theory

• Our concept of bacterial-induced diseases has beendominated by the SINGLE GERM THEORY

• With Microbiota, instead, differences in proportions of variousbacteria in different disease state are important rather thanthe 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

• Gut Microbiota is dynamic and responsive to environmentalfactors due to a past selective pressure during human evolution

GI and LIVER diseases associated to GUT MICROBIOTA IMPAIRMENT

• Gastrointestinal infections• Irritable Bowel Syndrome • Intestinal Bacterial Overgrowth• Food Intolerance/Allergy• Celiac disease• Inflammatory Bowel Diseases• GI Cancer• Obesity and MetS• Liver diseases• …

GUT MICROBIOTA INFLUENCE ON ENERGY STORAGE

• Wild Type (WT) mice have42% more total body fat thangerm-free (GF) mice

• Colonisation of GF mice withmicrobiota from WT produces a60% increase in body fat mass,associated with increasedinsulin resistance

Backhed et al – PNAS 2004

Obesity is associatedwith:• 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 alterationReduction 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

Gut microbiota in obese humans

Akkermansia muciniphilaMicrobiota 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 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

Akkermansia muciniphilaMicrobiota fingerprint of obesity?

GUT MICROBIOTA in T2D• Case-control metagenome-wide association study (MGWAS)• Faecal DNA samples from 345 Chinese T2D patients and nondiabetic controls

Qin – Nature 2012

Butyrate-producing bacteria(E. rectale, F. prausnitzii, etc.)

Opportunistic pathogens (C. hathewayi, C. ramosum, E. coli)

Mucin degrading species Sulphate-reducing species

T2D vs Controls

Metformin and diabetesA microbiota-dependent pathway?

• Metformin treatment significantly improved the glycaemic profile of HFD-fed mice

• HFD-fed mice treated with metformin showed a higher abundance of Akkermansia than HFD-fed control mice

• The number of mucin-producing goblet cells was significantly increased by metformin treatment (p<0.0001)

C57BL/6 mice were fed either a normal diet or a HFD and treated with metformin for 6 weeks - 454 pyrosequencing.

Increase in Akkermansia muciniphila induced by metformin improves glucose homeostasis in diet-induced obese T2M mice

Shin et al – Gut 2013

Metformine and diabetesA 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

Gut microbiota modulationDiet & nutritional support Caloric amount, minerals, vitamins

Diet composition (fibers/high glicemic index/saturated fatty acids…)

Removal of predisposing conditions Treat diabetes, endocrine, other motility disorders..

Surgery or prokinetics when indicated

Therapeutic interventions Antibiotics

Prebiotics, probiotics, postbiotics, symbiotics

Fecal Microbiota Transplantation

Abbassy J JAMA 2017

The “Magic” of Bariatric

Surgery

Resolution of diabetes after bariatric surgery was noted on

many occasions

Rubino F Nature 2016

In 1925, a report in The Lancet described a 'side effect' of a gastrointestinal operation to

treat a peptic ulcer. This was the almost overnight resolution of an excess of sugar in the urine (glycosuria) — the chief symptom of

diabetes at the time.

The two surgical procedures induced similar and durable

changes on the gut microbiomethat were not dependent on

body mass index and resultedin altered levels of fecal and

circulating metabolitescompared with obese controls

The Gammaproteobacteria class were higher while the levels of three species in the Firmicutes phylum (Clostridium

difficile, Clostridium hiranonis, and Gemella sanguinis) were lower.

At the genus level, several facultative anaerobes in the Proteobacteria (e.g.,

Escherichia, Klebsiella, and Pseudomonas) were present at

increased relative abundance in RYGB.

Roux-en-Y gastric bypass (RYGB) versusvertical banded gastroplasty (VGB) versus OBS

By colonizing germ-free mice with stools from the patients, the surgically alteredmicrobiota promoted reduced fat deposition in recipient mice. These mice also had alower respiratory quotient, indicating decreased utilization of carbohydrates as fuel.

Microbiota in anticancer immunotherapy

Vétizou et al. Science 2015; Eggenfort et al – NEJM 2016; Ianiro et al – NEJM 2017

• Ipilimumab, a human monoclonal antibody against cytotoxic T-lymphocyte antigen 4, is known to be an effective treatment against

• The anti-cancer effect of ipilimumab is influenced by the microbiota composition of the host through modulation of interleukin 12–dependent TH1 immune responses

• Mostly involved species were Bacteroides fragilis and Bacteroides thetaiotaomicron

• Ipilimumab was not effective in reducing tumour growth whenadministered to germ-free mice, or to mice treated with antibiotics, but its anti-tumour properties were reattained afterthese mice received microbiota transplantation from human feces rich in Bacteroides.

A new scenario of individualized medicine for cancer patients relies in gut microbiota

1958

2013

Eiseman – Surgery 1958; Surawicz – AJG 2013; Smith - Nature 2014

Fecal microbiota transplantation

First reported application of FMTfor pseudomembranous colitis

FMT included in Americanguidelines for the management ofC. difficile

FMT for Cdiff: ready for prime timeo van Nood et al – 2013

o Kassam et al – 2013

o Cammarota et al - 2014

• RCT – FMT by nasojejunal tube vs vancomycin• 94% vs 31% Cdiff resolution rate

• Systematic review of 36 studies, 536 pts• 87% pooled Cdiff resolution rate

• Syst rev/metanalysis of 11 studies, 273 pts• 89.7% Cdiff resolution rate

o Cammarota et al – 2015

o Drekonja et al – 2015

o Lee et al – 2016

o Kelly et al - 2016

• RCT – FMT by colonoscopy vs vancomycin• 90% vs 26% Cdiff resolution rate

• Systematic review of 35 studies, 516 pts• 85% pooled Cdiff resolution rate

• Noninferiority RCT – Fresh vs frozen FMT by enema• 85.1% vs 83.5% Cdiff resolution rate

• RCT – autologous vs donor FMT by colonoscopy• 62.5% vs 90.9% Cdiff resolution rate

FMT in C. difficile guidelines

Surawicz et al – AJG 2013; Debast et al – Clin Microbiol Infect 2014

o American College of Gastroenterology

o European Society of Clinical Microbiology & Infect Dis

• FMT at 3° recurrence after vancomycin

• FMT + antibiotics for multiply recurrentdisease

FMT for recurrent Clostridium difficile infection

Statement: FMT is recommended as a highly effective and safe treatment option for both mild and severe rCDI. Its implementation in clinical practice is recommended

Quality of evidence: high Strength of recommendation: strong

European Consensus Conference on Faecal Microbiota Transplantation in Clinical PracticeGiovanni Cammarota,1 Gianluca Ianiro,1 Herbert Tilg,2 Mirjana Rajilić-Stojanović,3 Patrizia Kump,4 Reetta Satokari,5 Harry Sokol,6 Perttu Arkkila,7 Cristina Pintus,8 Ailsa Hart,9 Jonathan Segal,9 Marina Aloi,10 Luca Masucci,11 Antonio Molinaro,12

Franco Scaldaferri,1 Giovanni Gasbarrini,1 Antonio Lopez-Sanroman,13 Alexander Link,14 Pieter de Groot,15 Willem de Vos,5,16 Christoph Högenauer,4 Peter Malfertheiner,14 Eero Mattila,17 Tomica Milosavljević,18 Max Nieuwdorp,12,15,19

Maurizio Sanguinetti,11 Magnus Simren,20 Antonio Gasbarrini,1 The European FMT Working Group

Cammarota et al – Gut – 2017

FMT for refractory Clostridium difficile infection

Statement: FMT can be considered as a treatment option for refractory CDI

Quality of evidence: high Strength of recommendation: strong

FMT for the first episode of Clostridium difficile infection

Statement: There is insufficient evidence to recommend FMT as a treatment for the first episode of CDI.Additional studies are needed to determine if FMT could have an advantage over antibiotics for this indication

Quality of evidence: low Strength of recommendation: weak

18 treatment-naive MetS males

Random assignment to either an allogenic (from lean male donors; n=9) orautologous FMT (n=9)

Vrieze – Gastroenterology

Improvement in peripheral insulinsensitivity with allogenic FMT

Significant increase of gut microbiota diversity

16 bacterial groups increased significantly,including those related to the butyrate-producerRoseburia intestinalis (2.5-fold increase)

Conclusions

• As gut microbiota appears to be strictly associated with the developmentof many human disorders, its modulation should be considered as a maintreatment target

• Several pathogenetics and methodological gaps are still to be solved, such as the role of non-bacterial components, and the way to analyzemicrobiota findings

• Therapeutic modulation of microbiota, including diet, anti-pre-probiotics, and FMT, should come within the therapeutic armamentarium of each GI expert

Save the dates!

EAGEN/EHMSG FMT Course UEG Long-term Training Support

Basic Science Course

Gut microbiota: Relevance, analysis, modulation

22-23 November 2017 July 6-8, 2017

Fondazione Policlinico Universitario “A. Gemelli”, Rome, Italy

© UEG. 2017