Presentazione a cura del Professor Cesare Cremon - M.A.S.T.E.R. ECM in Gastroenterologia: Focus on: Microbiota e dintorni - Fondazione Santa Lucia - Roma

1. Cesare Cremon
Policlinico S. Orsola – Malpighi
Azienda Ospedaliero - Universitaria di Bologna
FOCUS ON:
MICROBIOTA E DINTORNI
Disturbi digestivi funzionali
e microbiota
(1088 – 2018)

2. Microbiological revolution: What have we learned?
• Number of microbiota genes > 100 time of human genome
• >9 phyla (95% genes identity), >1000 species (99% genes identity), >15000 strains
(100% genes identity)
• “A silent organ” with high metabolic activity (biomass ≈ liver)
Gill SR, et al. Science 2006;312:1355–1359
Lee YK, and Mazmanian SK. Science 2010;330:1768-73
Arumugam M et al., Nature 2011;473:174–180
Agar plate
culture
• Long time to culture
• 60-80% unculturable
• Molecular biology
• 16S rRNA

3. Functional and phylogenetic profiles of human gut microbiome
Arumugam M et al., Nature 2011;473:174–180
Phylum
Class
Order
Family
Genus
Species

4. Intestinal microbiota
physiological and pathological conditions
Pathological conditions
• GI infections
• Antibiotic-associated colitis
• IBD
• Food intolerance/allergy
• FGIDs/IBS
• SIBO
• Liver encephalopathy
• CRC
• Behaviour, Psychology
• …
Lynch SV, Pedersen O. N Engl J Med 2016;375:2369-2379

5. Hypothesis of pathogenesis caused
by dysbiotic gut microbiota
Lynch SV, Pedersen O. N Engl J Med 2016;375:2369-2379

6. Microbiota and FGIDs: what is the link?
 New diagnosis of IBS/FD after infectious gastroenteritis
– Spiller R, Garsed K. Gastroenterology 2009;136:1979-88
– Klem F et al., Gastroenterology 2017;152:1042-54
 Qualitative and quantitative changes in intestinal microbiota composition of FGIDs
– Simrén M, Barbara G et al., Gut 2013;62:159-76
– Barbara G et al., Gastroenterology 2016;150;1305-18
 Non-absorbable antibiotics and probiotics are effective in FGIDs
– Pimentel M, et al., N Engl J Med 2011;364:22-32; Lembo A et al., Gastroenterology 2016;151:1113-21
– Tan VPY et al., Aliment Pharmacol Ther 2017;45:767–76
– Moayyedi P, et al., Gut 2010;59:325-32; Ford AC et al., Am J Gastroenterol 2014;109:1547-61

7. 1811 subjects infected (acute symptoms)
 1684 pupils (93%) – children 3-10 yrs
 127 teachers or other school employees (7%) – adults 19-59 yrs
19 ottobre 1994
36 schools
in the Bologna area
Salmonella outbreak in Bologna
(October 19, 1994)
Tuna sauce contaminated
(Salmonella enteritidis D)
Barbara et al., Aliment Pharmacol Ther 2000;14:1127-31

8. Non-exposed
Exposed
*P = 0.004
OR = 1.92
(95% C.I.= 1.23-2.98)
*
PrevalenceofIBS(%)
44/189
(23.3%)
75/204
(36.8%)
Early life salmonellosis and IBS in adulthood
Salmonella outbreak in Bologna (1994-2010)
Cremon C. et al., Gastroenterology 2014;17:69-77

9. IBS after infectious enteritis: a meta-analysis
Klem F et al., Gastroenterology 2017;152:1042-54

10. Futagami S et al., Aliment Pharmacol Ther 2015;41:177–88

11. Changes in intestinal microbiota of FGIDs/IBS

12. Different composition of fecal microbiota in IBS vs healthy
• 62 primary care IBS (Rome II), 46 HC
• Deep molecular analysis
• 2-fold  Firmicutes/Bacteroidetes ratio
• Multivariate analysis identifies 2 distinct
enterotypes
• IBS symptoms correlated with 18
phylogenetic groups (R = 0.29-0.43)
Rajilić-Stojanović et al., Gastroenterology 2011;141:1792-801
Multivariate analysis (RDA)
C
V
Axis1 (50%)
Axis2(20.3%)1.5-1.5
-1.5 1.5
HC
IBS

13. Irritable bowel syndrome subtype defined by
species-specific alterations in faecal microbiota
Jerrery IB et al., Gut 2012,61:997-1006
High Firmicutes:Bacteroidetes ratio IBS (red)
Normal Firmicutes:Bacteroidetes ratio IBS (blu)
HAD depression score in normal-like IBS group
Sequenced 30.000 16S rRNA gene V4 region
amplicons per subjects

14. Gut microbiota composition and host-microbe cross-talk
Jalanka-Tuovinen J et al., Gut 2014;63:1737-45

15. Casén C et al., Aliment Pharmacol Ther 2015;42:71-83
Pre-dominant bacteria contributing to dysbiosis in IBS
Firmicutes (Bacilli)
Proteobacteria
Actinobacteria
Shigella/Escherichia
Ruminococcus gravus

16. Intestinal microbiota and IBS severity
Tap J et al., Gastroenterology 2017;152:111–123

17. Short chain fatty acids in IBS
and correlation with clinical/biological parameters
Gargari G, Cremon C et al., Submitted 2018

18. Intestinal ecosystem:
Differences between IBS-C and FC
HC
FC
IBS-C
Weighted UniFra Beta-diversity
Lachnospiraceae Coriobacteriaceae
Erysipelotrichaceae Bacteroidaceae
Phylogeneticabundance
(familylevel)
Phylogeneticabundance
(familylevel)
Cremon C et al., UEG Journal;5S:A122

20. Strategies for modifying gut microbiota
Simrén M et al., Gut 2013;62:159-76
Fecal microbial
transfer

21. • Two identically designed, phase 3, multicentre,
double blind, placebo-controlled studies
(TARGET 1 and TARGET 2) in 1260 patients
with IBS
Placebo Rifaximin
Primary End-point
0
10
20
30
40
50
%Responders
* *P<0.001
Rifaximin for patients with IBS without constipation
Pimentel et al., N Engl J Med 2011;364:22-32

22. IBS-D Rifaximin Re-Treatment Study (TARGET 3)
Lembo A et al., Gastroenterology 2016;151:1113-21

23. Rifaximin for the treatment of functional dyspepsia
Tan VPY et al., Aliment Pharmacol Ther 2017;45:767–76

24. Probiotics
FAO/WHO definition
“live microorganisms that, when
administered in adequate amounts, confer a
health benefit on the host”
Hill C et al., Nat Rev Gastroenterol Hepatol 2014;11:506-14
Latin preposition pro
("for")
Greek adjective βιωτικός
("fit for life, lively")
Élie Metchnikoff,
1908 Nobel Prize for Medicine

25. Ohland CL. Et al. Am J Physiol Gastrointest Liver Physiol 2010 ;298:G807-19
 Modulate gastrointestinal motility
 Reduce visceral hypersensitivity
 Reduce low grade mucosal immune
activation
 Improve epithelial permeability
 Enhance gut-brain communication
 Restore intestinal dysbiosis (?)
Major claims of probiotics in IBS
Several of these claims based on convincing experimental evidence from preclinical studies

26. Symptom Probiotics Agreement % Grade of
evidence
Overall
IBS
Bifidobacterium bifidum MIMBb75, B. Longum subsp.
infantis 35624, Escherichia coli DSM17252,
investigative combinations, marketed combinations
100 High
IBS-C B. animalis subsp. lactis DN-173 010 80 Low
IBS-D B. longum subsp. infantis 35624, marketed
combinations
100 Moderate
Hungin APS et al,. Aliment Pharmacol Ther 2013; 38: 864–86
Systematic review: probiotics in the management of
gastrointestinal symptoms in clinical practice:
an evidence-based international guide
Statement
Specific probiotics help relieve overall symptom burden in some patients with IBS
However…
No claims approved by European Food & Health Safety Authority (EFSA)
No products containing probiotics approved by Food and Drug Administration (FDA)

27. ClinicalTrials.gov Identifier: NCT02371499
2 wks
-
Screening
4 wks
-
First treatment
Randomization
1:1
End of
Study
Placebo
L. casei DG® b.i.d
End of
Treatment
4 wks
-
Cross-over
4 wks
-
Second treatment
4 wks
-
Follow-up
Wash-out
Period
Placebo
L. casei DG® b.i.d
Stool samples Stool samples Stool samples Stool samples

28. Treatment effect on modulation of
IBS symptoms, fecal microbiota, and short chain fatty acids%Responders
Abdominal pain/discomfort score
P=0.338
D = 7.5%
n=12/40
n=15/40
Wilcoxon test
MEDIAN
ABUNDANCE
(mg/kg)
L. casei DG®
treatment p.value before after
Acetate 0.021* 36.63 43.06
Propionate 0.289 15.18 16.73
Butyrate 0.047* 5.99 10.73
Isobutyrate 0.133 1.11 1.22
Isovalerate 0.428 1.14 0.95
Valerate 0.080 1.82 2.14
Placebo treatment p.value before after
Acetate 0.388 47.83 33.08
Propionate 0.622 16.37 17.13
Butyrate 0.746 10.52 8.47
Isobutyrate 0.387 1.55 1.64
Isovalerate 0.360 1.04 1.28
Valerate 0.572 2.45 1.90
Cremon C et al., UEG Journal 2018, in press

29. Quigley EM, Lembo AJ. Gastroenterology 2017;152:936-938