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EFFICACIA DEI PROBIOTICI NELLE MALATTIE DIGESTIVE
Antonio Gasbarrini, Gianluca Ianiro
Gastroenterologia Policlinico Universitario Gemelli
Università CaBolica del Sacro Cuore -‐ Roma
Come le conoscenze aBuali su Gut Microbiota influenzano la gesIone dei probioIci?
Microbiota
GUT BARRIER
Acquired and
Innate immunity
Mucosal Barrier
Epithelial barrier
Vascular and lymphatic systems
Neuroenteric system Digestive enzymes
Endocrine system
Virus Bacteriophages
Bacteria Yeast
Not only Bacteria..
Acquired and
Innate immunity
Mucosal Barrier
Epithelial barrier
Vascular and lymphatic systems
Neuroenteric system Digestive enzymes
Endocrine system
Helminth Parasite Archea
Protozoa
GUT MYCOME • Yeasts are commensal to the gut at low concentrations
• In the healthy gut dominant species are: Wallemia, Trichocomaceae, Rhodotorula, Saccharomycetaceae, Pleosporaceae, Agaricaceae, Metschnikowiaceae, Cystofilobasidiaceae, Ascomycota, Amphisphaeriaceae...
• Yeast’s functions in the gut it is not clear
• Yeasts overgrowth/dysbiosis (related to host genetic and physiology, lifestyle, antibiotic usage, immune system disfunction) could be pathogenic fo the gut
Dollive S, et al. Genome Biol 2012 Cui et al. Genome Medicine 2013 Thewes S, Mol Microbiol 2007
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 phyla 95% genes identity
>1000 species
99% genes identity
>15000 strains 100% genes identity
900-1200 gr, >17.000.000 genes
Microbiome Metabolome
BACTERIAL TAXONOMY DOMINIUM
REGNUM
PHYLUM
CLASSE
ORDO
FAMILIA
GENUS
SPECIES
SUBSPECIES (STRAIN)
95%
99% 100%
Genes identity
MOLECULAR BACTERIOLOGY: most abundant PHYLA in the GUT (>70%)
Eckburg et al, Science 2005
BACTEROIDETES
FIRMICUTES
HUMAN GUT
ENTEROTYPE
of people as lean or obese can be made solely on the basis of their gut microbiota with 90% accuracy46,47, but they do not separate into distinct microbiota-based clusters on commonly used principal coordinates plots, which are used to identify statistical differences between groups. Thus, multiple statistical techniques are needed to show fully the differ-ences in the microbiota between different physiological states (Fig. 2).
Some differences in the microbiota can contribute directly to disease states. Gnotobiotic mice that were raised germ-free then colonized with the microbiota from an obese mouse gained fat more rapidly than those colonized with the microbiota of a lean mouse7,45. A phenotype can emerge from different compositional backgrounds, which may indicate that specific components of the microbiota can exert large effects or that many different changes can lead to the same functional result.
Differences in faecal microbial community diversity, composition and function have also been correlated with Crohn’s disease9, ulcerative coli-tis10, irritable bowel syndrome (IBS)48, Clostridium difficile- associated disease (CDAD)49 and acute diarrhoea50. Sometimes, the nature of the microbiota deviation from health is consistent across individuals with the same disease. For instance, a twin study of IBD found marked and reproducible deviations in patients with ileal Crohn’s disease relative to the controls, and more subtle, but characteristic, changes in patients with colonic Crohn’s disease51, and specific functional differences were also observed from metabolic profiling of the same samples24. Other diseases are associated with marked deviations from health that are inconsistent across individuals. For instance, individuals with recurrent CDAD had a phylum-level diversity that was very different from controls but not similar to each other49. Many disease studies are confounded by extensive use of treatments, such as antibiotics, that may obscure true disease-asso-ciated changes, highlighting the urgent need for prospective longitudinal studies that establish cause and effect.
Parallels between host physiological statesStudies of the microbiota often target one specific disease or state, but comparisons of the microbiota across many diseases can show common changes in the gut environment. Disturbed mucous lay-ers that line the intestinal cell wall and concomitant inflammation are seen in individuals with IBD, coeliac disease, HIV enteropathy, acute diarrhoea, diverticulosis, carcinoma and IBS52. Given these parallels, an increase or decrease in abundance of similar microbes across different disturbances might be expected53, but elucidation of these differences may require detailed biogeographical studies along the length of the gut — once safe and reliable means for such comprehensive sampling are developed.
Perturbed adult gut microbial communities are intriguingly simi-lar to infant gut microbial communities. Both systems may represent successional communities in which the same opportunistic or fast-growing species can predominate53. For instance, C. difficile is a normal gut resident that can cause disease when antibiotics com-promise the stable adult gut communities; it also colonizes 2–65% of infants, although most infants are asymptomatic54,55. Clostridium bolteae and Clostridium symbiosum are also associated with a dis-turbed gut and systemic infection, and are found in the infant gut53. The microbiota of individuals with ileal Crohn’s disease can also resemble that of infants: both have increased levels of Ruminococcus gnavus and Enterobacteriaceae in their stools, and an under-repre-sentation of the genera that are prevalent in healthy adults, including Faecalibacterium and Roseburia51. These examples show the impor-tance of understanding whether generally opportunistic members of the gut microbiota have a selective advantage during early succession or disruption caused by disease, and therefore whether they are the side effects of disease rather than causal agents.
Resilience of stable states Resilience is the amount of stress or perturbation that a system can tolerate before its trajectory changes towards a different equilib-rium state56. Macroecosystem studies of human interference such as
resource exploitation, pollution, land-use change and global warm-ing have shown how ecological systems can be transformed into less productive or less desirable states56. For example, in a tropical lake community, a regime in which submerged plants dominate is pre-ferred to one with extensive free-floating plant cover because dense mats of floating plants create anoxic conditions that reduce animal biomass and diversity. However, pollution can cause floating plants to predominate because they are better at competing for light and can exclude submerged plants when the nutrient load is high. By under-standing the environmental drivers of conversion between states, interventions can be used to induce a regime change. For example, harvesting the floating plants once can induce a permanent shift to the submerged-plant-dominant state, but only if the nutrient loading is not too high57.
Environmental studies can provide examples of the microbial response to perturbations, and perhaps an insight into how the gut microbiota might react. During the Deepwater Horizon oil spill in the Gulf of Mexico, the microbial community structure and func-tional gene repertoire in the deep-sea oil plume shifted, transiently, to an enrichment of microbes that were capable of hydrocarbon-degradation58,59. This is similar to the effect of an extreme dietary change on the gut microbiota of mice that were moved from a low-fat, plant-rich diet to a high-fat, high-sugar diet7. In both cases, the
Firmicutes
Phylum Function
ActinobacteriaBacteroidetesProteobacteriaFusobacteriaTenericutesSpirochaetesCyanobacteriaVerrucomicrobiaTM7
Central carbohydrate metabolismCofactor and vitamin biosynthesisOligosaccharide and polyol transport systemPurine metabolismATP synthesisPhosphate and amino acid-transport systemAminoacyl transfer RNAPyrimidine metabolismRibosomeAromatic amino-acid metabolism
Figure 4 | Functional redundancy. The functional redundancy in microbial ecosystems may mirror that in macroecosystems. As shown in the HMP data set14, oral communities (top panels) and faecal communities (bottom panels) analysed using 16S rRNA (coloured by microbial phyla, left panels) show tremendous abundance diversity. The same samples analysed by shotgun metagenomics (panels on right) have remarkably similar functional profiles. Reprinted with permission from ref. 14.
1 3 S E P T E M B E R 2 0 1 2 | V O L 4 8 9 | N A T U R E | 2 2 5
REVIEW INSIGHT
© 2012 Macmillan Publishers Limited. All rights reserved
Lozupone et al. Nature 2012
Phylum level diversity can have a marked variation even across healthy adults in the same population. Each individual has many unique phylotypes not found in the other.
INDIVIDUAL ENTEROTYPE
U.S. adults sampled >5 times up to 296 weeks apart revealed that they harbored 195±48 bacterial strains, representing 101 ± 27 species
..in stable conditions, microbiota is stable, with 60% of strains remaining over the course of 5 years: stable core of dominant species
David – Nature 2013
Ø The human gut microbiome can rapidly switch between herbivorous and carnivorous functional profiles Ø It may reflect past selective pressures during human evolution Ø Microbial communities that could quickly, and appropriately, shift their functional repertoire in response to diet change would have subsequently enhanced human dietary flexibility
Ø Examples of this flexibility may persist today in the form of the wide diversity of modern human diets
Microbiota composition is affected by life events Ottman et al. Functionality of the human microbiota
healthy
obese
65 to 80 years
>100 years
healthy
antibiotic treatment
malnutrition breast-fed
formula-fed
solid food
Baby Toddler Adult Elderly
16S
16S
16S
DNA 16S
DNA
16S
16S
DNA
16S
DNA
16S
16S
16S
Unborn
FirmicutesBacteroidetesActinobacteriaProteobacteriaothers
FIGURE 1 | Human microbiota: onset and shaping through life stages andperturbations. The graph provides a global overview of the relative abundanceof key phyla of the human microbiota composition in different stages of life.Measured by either 16S RNA or metagenomic approaches (DNA). Data
arriving from: Babies breast- and formula-fed (Schwartz et al., 2012), baby solidfood (Koenig et al., 2011), toddler antibiotic treatment (Koenig et al., 2011),toddler healthy or malnourished (Monira et al., 2011), adult, elderly, andcentenarian healthy (Biagi et al., 2010), and adult obese (Zhang et al., 2009).
foods. Diet-related diseases such as allergies and obesity are alsocharacterized by microbiota changes. Obesity is characterized bya typical Firmicutes to Bacteroides ratio. Energy harvest potentialand short chain fatty acids (SCFA) are determined by the micro-biota composition and have a direct effect on the host epithelialcell energy availability. A microbiota stimulated with probioticmicrobes can even decrease the incidence of infant diarrhea andatopic eczema due to host immune stimulation (Niers et al., 2009;Sjogren et al., 2009).
Numerous meta-omics approaches have vastly increased theknowledge available on the genome, activity and functionality ofthe complex ecosystem residing in the human gut. By far the mostcommonly applied technique is metagenomics, which is based ondirect isolation and, in most cases, sequencing of the completegenetic material obtained from an environmental sample, such asthe intestine. However, one of the biggest drawbacks of this tech-nique is its inability to display the actual metabolic activity due tothe fact that it detects both expressed and non-expressed genes.In addition, it may generate information from dead cells as it isknown that more than half of the cells in fecal samples are non-viable or heavily damaged (Ben-Amor et al., 2005). Instead offocusing on microbiota composition the purpose of this review isto combine the available knowledge on microbial genomics with
reports on the functional metagenomics, i.e., transcriptomics andproteomics approaches. This combination is expected to providea refined understanding of the role of the microbiota and itscapabilities in regulating human health.
ROLE OF THE MICROBIOTA IN EARLY AND LATE LIFEEARLY LIFEDuring natural birth, a newborn is exposed to the environmental,mainly maternal, microbiota which commences the acquisitionof what we assume is a normal microbiota. The mode of deliv-ery strongly affects the composition of the microbiota. In the caseof caesarean delivery (C-section), other environmental bacteriaform the basis for the microbiota instead of vaginal and faecalbacteria from the mother, reportedly resulting in a substantialreduction of bifidobacteria (Biasucci et al., 2008). In a compari-son of the microbiota of babies delivered either vaginally or viaC-section, it was shown that the newborns harbored undiffer-entiated bacterial communities across skin, oral, nasopharyngeal,and gut habitats regardless of delivery mode, and that the micro-biota of C-section babies was similar to the skin communities ofthe mothers whereas vaginally delivered infants acquired bacterialcommunities resembling the vaginal microbiota of their mothers(Dominguez-Bello et al., 2010). Other factors influencing the
Frontiers in Cellular and Infection Microbiology www.frontiersin.org August 2012 | Volume 2 | Article 104 | 2
Ottmann N et al. Front Cell Infect Microb 2012
…specific effects in each GI tract!
EFFECTS OF GUT MICROBIOTA ON HOST HEALTH
Ø Barrier effect Ø Immunocompetence/Tolerance Ø Synthesis
Ø Metabolism Ø Drug metabolism Ø Behavior conditioning
Lifelong immunostimulation by enteric commensal and pathogenic bacteria
Maynard CL et al. Nature 2012
‘‘NUTRIENT SENSOR PATHWAY’’
Tilg H, J Hepatology 2010
Hologenome theory of evolution The object of genomic natural selection is not a single organism, but the organism and its microbial communities
Rosenburg et al, 2007
• Vibrio shiloi-mediated bleaching of the coral Oculina Patagonica (Coral probiotic hypothesis)
Rosenburg and Zilber, 2008
• Commensal bacteria play a role in mate choice by the fruit fly Drosophila Melanogaster (microbial communities of the fly altered hydrocarbons, which function as insect pherormones)
Sharon et al; 2010 Ringo et al, 2011
Holobiont: any organism and all its associated symbiotic microbes (parasites, mutualists, synergists, amensalists)
Hologenome: genes of host and symbiotic microbes
Microbiota
GUT BARRIER
Acquired and
Innate immunity
Mucosal Barrier
Epithelial barrier
Vascular and lymphatic systems
Neuroenteric system Digestive enzymes
Endocrine system
• The inner mucus layer is dense and does not allow bacteria to penetrate, thus keeping the epithelial cell surface free from bacteria
• The inner mucus layer is converted into the outer layer, which is the habitat of the commensal flora
Epithelial cells
Loosely adherent mucus layer
Firmly adherent mucus layer
Good bacteria
IMPORTANCE OF THE MUCUS LAYERS
Johansson, M.E., Proc Natl Acad Sci U S A, 2010
GUT MICROBIOTA HAS TO BE UNDER CONTROL
Ø Mucosal barrier integrity Ø Secretion of :
Ø Gastric acid Ø Biliary salts Ø Antimicrobial substances
Ø Mucosal pH Ø Local mucosal and systemic immunity Ø Intestinal motility Ø Interactions among different bacteria species
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
• Burns and trauma • Major vascular surgery • Sepsis • MOF • Renal failure • Diabetes/Metabolic syndrome • Joint disease • Autoimmune diseases • Psychiatric pathologies • Heart and lung diseases
• Coeliac disease • IBD/IBS • Food allergy • Acute gastroenteritis • Radiation enteritis • Intestinal by-pass • Intestinal obstruction • Peritonitis • Wipple’s disease • Liver Diseases
Human diseases associated to LEAKY GUT/DYSBIOSIS
Digestive diseases associated to DYSBIOSIS/LEAKY GUT
• Gastrointestinal infections • Irritable Bowel Syndrome • Intestinal Bacterial Overgrowth • Diverticulosis • Inflammatory Bowel Diseases • Gastro-intestinal Cancers • Food Intolerance/Allergy • Celiac disease • Liver diseases • Pancreatic diseases • Obesity, Diabetes and Metabolic Syndrome
Diet and Nutritional Support Ø Caloric amount, minerals, vitamins, sweeteners..
Ø Diet composition (fibers/high glicemic index/saturated fatty acids…)
Removal of predisposing conditions Ø Treat diabetes, endocrine, other motility disorders.. Ø Surgery or prokinetics when indicated
Ø Stop PPI or other antiacid, NSAIDs, antibiotic, immunodepressant….
Intervention Ø Antibiotics Ø Biotherapy (prebiotics, probiotics, symbiotics, postbiotics) Ø Microbiota Transplantation
How (re)modulate gut microbiota?
Need to know: mechanisms of Probiotic/Host interaction
Immunological benefits: Ø Activate macrophages to increase antigen presentation to B cells and IgA production
Ø Modulate cytokine profiles
Ø Induce hyporesponsiveness to food antigens
Nonimmunological benefits: Ø Digest food and compete for nutrients with pathogens
Ø Alter local pH to create an unfavorable local environment for pathogens
Ø Produce bacteriocins to inhibit pathogens
Ø Scavenge superoxide radicals
Ø Stimulate epithelial mucin production
Ø Enhance intestinal barrier function
Ø Compete for adhesion with pathogens
Ø Modify pathogen-derived toxins WGO 2011
Needs for a Specie (Strain)-specific Microbial Therapy
Different action for each Probiotic: Knowledge of micro-organism functions and host genetic
modulation by different Species/Strain is crucial
From Gopubmed, 2013
Probiotics: fastly growing research area
Probiotics available on the market
FAO/WHO, 2001
Lactobacillus spp • casei spp (Rhamnosus, DN..) • reuteri • acidophilus • shirota • delbrueckii, sp. Bulgaricus • brevis • plantarum • …
Bifidobacterium spp • bifidum • infantum • longum • thermophilum • lactis • …
Cocci gram-positive • Streptococcus thermophilus • Enterococcus faecium • Streptococcus intermedieus • Streptococcus alfa-emoliticus • …
Bacillus gram-negative • Escherichia coli Nissle (1917)
Bacillus gram-positive • Bacillus clausii • Bacillus subtilis
Yeast • Saccharomyces boulardii • Kluyveromyces marxianus fragilis • …
ROLE OF CURRENT PROBIOTICS IN GASTROINTESTINAL DISORDERS
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori and C. difficile treatment
ð Treatment of Necrotizing enterocolitis
ð Treatment of Sugar Intolerance
ð Prevention and treatment of Pouchitis
ð Maintenance of remission of IBD
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
LACTOBACILLUS CASEI sp RHAMNOSUS
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori and C. difficile treatment
ð Treatment of Necrotizing enterocolitis
ð Treatment of Sugar Intolerance
ð Prevention and treatment of Pouchitis
ð Maintenance of remission in Ulcerative Colitis
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
SACCHAROMYCES CEREVISAE sp BOULARDII
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori and C. Difficile treatment
ð Treatment of Traveller’s diarrhea
ð Treatment of Necrotizing enterocolitis
ð Prevention and treatment of Pouchitis
ð Maintenance of remission in IBD
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
ESCHERICHIA COLI sp NISSLE 1917
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori treatment
ð Treatment of Necrotizing enterocolitis
ð Treatment of Sugar Intolerance
ð Prevention and treatment of Pouchitis
ð Maintenance of remission of Ulcerative Colitis
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
BACILLUS COAGULANS GBI-30, 6086
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori treatment
ð Treatment of Necrotizing enterocolitis
ð Treatment of Sugar Intolerance
ð Prevention and treatment of Pouchitis
ð Treatment and maintenance of remission of Ulcerative Colitis
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
BIFIDOBACTERIUM INFANTIS 35624
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori treatment
ð Treatment of Necrotizing enterocolitis
ð Treatment of Sugar Intolerance
ð Prevention and treatment of Pouchitis
ð Treatment and maintenance of remission of Ulcerative Colitis
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
Multistrains BACILLUS CLAUSII sp OC, NR, SIN, T
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori treatment
ð Prevention of Respiratory Tract Infection
ð Treatment of Sugar Intolerance
ð Prevention and treatment of Pouchitis
ð Treatment and maintenance of remission of Ulcerative Colitis
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
LACTOBACILLUS DELBRUECKII sp BULGARICUS + STREPTOCOCCUS TERMOPHILUS
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori and C. difficile treatment
ð Treatment of Necrotizing enterocolitis
ð Treatment of Sugar Intolerance
ð Prevention and treatment of Pouchitis
ð Maintenance of remission in IBD
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
Multispecies/strains combination VSL 3
ð Reduction of Antibiotic-associated Diarrhea
ð Prevention and treatment of Infectious Diarrhea
ð Adjuvant for H. pylori treatment
ð Treatment of Necrotizing enterocolitis
ð Treatment of Sugar Intolerance
ð Prevention and treatment of Pouchitis
ð Treatment and maintenance of remission of Ulcerative Colitis
ð Treatment of IBS
Practice guidelines on Probiotics usage World Gastroenterology Organization (2011)
Quali sono le evidenze di efficacia dei probioIci nella diarrea acuta?
Probiotics plus rehydration therapy reduce the duration and severity of diarrhoea
- 24.76 hours (IC95% 15.9-33.6)
Total patients n=8014 • 6489 children • 352 adults • 1173 age ndd
Patients: adults and children
… good results but more research is needed to identify a specific probiotic for each subjects
Duration of diarrhoea
Conclusions
Allen SJ, Cochrane Database Systematic Review 2010
Infectious acute diarrhea
Prevention of traveler’s diarrhea
ProbioIcs significantly prevent TD (RR 0.85, 95% CI 0.79,0.91, p<0.001)
Enterococcus faecium
LAB SF68
L. rhamnosus GG o Lactobacillus
paracasei B 21060
Saccharomices boulardii, ceppo di
S.cerevisiae
108 cfu, tid
109 cfu bid
109 cfu per capsule or
250 mg, 2-6 capsule/die
1b
2b
WGO: Organizzazione Mondiale di Gastroenterologia Cfu: unità formanti colonie WGO, 2011
1b
Evidence-based adult indications for probiotics
Disorder Probiotic strain Recommended dose Evidence level
Treatment of acute infectious diarrhea
Evidence-based pediatric indications for probiotics
Disorder Recommended dose Probiotic strain Evidence level
Treatment of acute infectious diarrhea
L. rhamnosus GG
Saccharomyces boulardii,
ceppo di S. cerevisiae
Indian Dahi: Lactococcus
lactis, L. lactis cremoris,
Leuconostoc mesenteroides
cremoris
1010 – 1011 cfu, bid
200 mg, tid
1010 cfu each
bid o tid
1a 1a
2b
WGO: World Gastroenterology Organisation WGO, 2011
Meta-‐analysis of RCTs; ESPGHAN/ESPID recommendaIon
Quali sono le evidenze di efficacia dei probioIci nella prevenzione della diarrea da anIbioIci?
Cremonini and Gasbarrini, Alim Pharm Ther 2002
[ A meta-analysis of 7 RCT shows a strong benefit of probiotic administration on AAD
[ The combined RR was 0.39 (95% CI: 0.27-0.57) of probiotics efficacy when compared to placebo
Probiotics and Antibiotic-associated Diarrhea
WGO: Organizzazione Mondiale di Gastroenterologia Cfu: unità formanti colonie WGO, 2011
Prevention of antibiotic associated diarrhea in adults
E. faecium LAB SF68
S. boulardii, ceppo
di S. cerevisiae
L. rhamnosus GG
L. casei DN-114 001 in latte fermentato
Bacillus clausii
L. acidophilus
CL1285 + L. casei LBC80R
1 g o 4 x109 cfu die
1010- 1011 cfu bid
1010 cfu bid
2x109 spore tid
5x1010 cfu uid o bid
1b
1b
1b
1b
1b
1b
108 cfu, bid
Evidence-based adult indications for probiotics
Disorder Probiotic strain Recommended dose Evidence level
WGO, 2011
S. boulardii, ceppo di S. cerevisiae
L. rhamnosus GG
Bifidobacterium
lactis Bb12 + Streptococcus thermophilus
L. rhamnosus GG
(ceppo E/N, Oxy e Pen)
250 mg, bid
1010 cfu, uid o bid
107 + 106 cfu/g di formula
2 x 1010 cfu, bid
1a
1b
1b
1b
Evidence-based pediatric indications for probiotics
Disorder Probiotic strain Recommended dose Evidence level
Meta-‐analysis of RCTs
Prevention of antibiotic-associated diarrhea
C. difficile-associated diarrhea
v ProbioIcs have a significant protecIve effect for CDAD (RR 0.59 95% CI 0.41-‐0.85, z = 2.8, p = 0.005.)
v Only S. boulardii showed significant reducIons in recurrences of CDAD
Prevention of C. difficile diarrhea
1010 cfu, bid
2 x 1010 cfu each strain, uid
109 cfu each strain, uid
5x109 cfu uid o bid
2-3x109 for 28 days, Followed for another
4 weeks
1b
1b
2b
1b
1b
WGO: Organizzazione Mondiale di Gastroenterologia Cfu: unità formanti colonie WGO, 2011
L. casei DN-114 001 in latte fermentato
L. acidophilus + B.
bifidum
L. rhamnosus HN001 + L. acidophilus NCFM
L. acidophilus CL1285
+ L. casei LBC80R
S. boulardii,
ceppo di S. cerevisiae
Evidence-based adult indications for probiotics
Disorder Probiotic strain Recommended dose Evidence level
Quali sono le evidenze di efficacia dei probioIci nell’IBS?
• Pooling of 8 trials for the outcome of clinical improvement yielded a significant RR of 1.22 (95% CI 1.07–1.4; P=0.0042)
• Homogenous studies (Cochran Q test: P=0.4482
Probiotics may improve symptoms of IBS and can be used as supplement to standard therapy
Irritable Bowel Syndrome
Nikfar - Dis Colon Rectum 2008
• Pooling of 8 trials for the outcome of clinical improvement yielded a significant RR of 1.22 (95% CI 1.07–1.4; P=0.0042)
• Homogenous studies (Cochran Q test: P=0.4482
Probiotics may improve symptoms of IBS and can be used as supplement to standard therapy
Irritable Bowel Syndrome
Nikfar - Dis Colon Rectum 2008
“…There are some limitations of this meta-analysis, such as characteristics
of patients (age, sex, lifestyle, compliance), species, dosages,
treatment durations, and different endpoints of studies…”
• Modest improvement in overall symptoms after several weeks of treatment:
Ø For dichotomous data from 7 trials: OR 1.6 (95% CI, 1.2 to 2.2)
Ø For continuous data from 6 trials: standardised mean difference (SMD) 0.23 (95% CI, 0.07 to 0.38)
• Metanalysis of 7 RCT (398 pts): significant improvement in ABDOMINAL PAIN: OR 2.88 (95% CI, 1.84 to 4.50)
• 2 studies (101 participants) reported on improvement in abdominal pain. However, high heterogeneity (I2 = 63%) suggested pooling was inappropriate.
Irritable Bowel Syndrome
Hoveyda, BMC Gastroenterology 2009
• Modest improvement in overall symptoms after several weeks of treatment:
Ø For dichotomous data from 7 trials: OR 1.6 (95% CI, 1.2 to 2.2)
Ø For continuous data from 6 trials: standardised mean difference (SMD) 0.23 (95% CI, 0.07 to 0.38)
• Metanalysis of 7 RCT (398 pts): significant improvement in ABDOMINAL PAIN: OR 2.88 (95% CI, 1.84 to 4.50)
• 2 studies (101 participants) reported on improvement in abdominal pain. However, high heterogeneity (I2 = 63%) suggested pooling was inappropriate.
Irritable Bowel Syndrome
Hoveyda, BMC Gastroenterology 2009
“…Trials varied in relation to the length of treatment (4-26 weeks), dose, organisms and strengths of
probiotics used…”
• 19 RCTs/18 papers in 1650 patients
PROBIOTICS: EFFECT ON OVERALL SYMPTOMS
Symptoms described as a dichotomous outcome
Irritable Bowel Syndrome
Moayyedi – Gut 2010
Ø Signif icant effect in reducing symptoms (RR of persistance: 0.71; 95%CI 0.57-0.9)
Ø No difference between different strains
• 19 RCTs/18 papers in 1650 patients
PROBIOTICS: EFFECT ON OVERALL SYMPTOMS
Symptoms described as a continuous outcome
Irritable Bowel Syndrome
Moayyedi – Gut 2010
Ø Probiotics had a statistically significant effect in improving symptoms compared with placebo (SMD . 0.34; 95% CI 0.60 to 0.07)
Ø Lactobacillus: no effect on IBS symptoms Ø Bifidobacterium: non significant trend Ø Combination: significant effect
PROBIOTICS: EFFECT ON INDIVIDUAL SYMPTOMS
Irritable Bowel Syndrome
Moayyedi – Gut 2010
ABDOMINAL PAIN (10 RCTs, 834 patients) Ø Significant efficacy - SMD= -0.51; 95% CI -0.91 to -0.09, p=0.016 Ø Significant heterogeneity
BLOATING (8 RCTs, 682 patients) Ø Trend – SMD= -0.54; 95% CI -1.10 to -0.02, p=0.058) Ø Significant heterogeneity FLATULENCE (6 RCTs, 566 patients) Ø Significant efficacy – SMD=-0.22; 95%CI -0.42 to -0.01, p=0.04) Ø No significant heterogeneity)
URGENCY (3 RCTs, 394 patients) Ø No significant efficacy –SMD=-0.08; 95%CI -0.3 to -0.14, p=0.49)
Irritable Bowel Syndrome
Ford – AJG 2014
• 35 RCTs, 3,452 patients
• Low risk of bias in 14 RCTs
• The RR of IBS symptoms persisting with probiotics vs. placebo was 0.79 (95 % CI 0.70 – 0.89)
• Probiotics had beneficial effects on global IBS, abdominal pain, bloating, and flatulence scores.
Irritable Bowel Syndrome
Ford – AJG 2014
• 35 RCTs, 3,452 patients
• Low risk of bias in 14 RCTs
• The RR o f IBS symptoms persisting with probiotics vs. placebo was 0.79 (95 % CI 0.70 – 0.89)
• Probiotics had beneficial effects on global IBS, abdominal pain, bloating, and fl atulence scores.
Alleviates some symptoms of irritable bowel syndrome
108 cfu, uid
1010 cfu, bid
1010 cfu, die
1010 cfu, die
1010 cfu, die
2x109 cfu, die
1b
1b
2b
1b
1b
2b WGO: Organizzazione Mondiale di Gastroenterologia
Cfu: unità formanti colonie WGO, 2011
Bifidobacterium infantis 35624
B. animalis DN- 173 010 in
fermented milk
L. acidophilus SDC 2012, 2013
L. rhamnosus GG,
L. rhamnosus LC705, B. breve Bb99 e
Propionibacterium freudenreichii ssp.
shermanii
B. longum 101 (29%), L. acidophilus 102 (29%),
Lactococcus lactis 103 (29%) e S. thermophilus 104 (13%)
Bacillus coagulans GBI-30, 6086
Evidence-based adult indications for probiotics
Disorder Probiotic strain Recommended dose Evidence level
Quali sono le evidenze di efficacia dei probioIci nella malaYa diverIcolare?
Unlu – Int J Colorectal Dis 2012
v Systematic review of RCTs
v 2 trials investigating probiotics
E. Coli (strains 01, 02, 055 and 0111) + Proteus vulgaris
VSL#3
v The use of probiotics decreases symptoms but does not reduce recurrence
v 210 pts - symptomatic uncomplicated diverticular disease (SUDD)
v 4 groups of treatment (10 days/month for 12 months) Ø Mesalazine + placebo Ø Lactobacillus casei subsp. DG + placebo Ø Lactobacillus casei subsp. DG + mesalazine Ø Placebo + placebo
• Both cyclic mesalazine and Lactobacillus casei subsp. DG alone or in combination are significantly better than placebo for maintaining remission in SUDD
Quali sono le evidenze di efficacia dei probioIci nell’eradicazione dell’infezione da H. pylori?
Ø 14 RCTs included
Ø First or second-line triple eradication therapy
Ø Data for occurrence of total side effects can obtain from 7 RCTs
Ø 8 trials administrated single probiotic strain (4 different probiotic strains) and 6 trials administrated combining probiotic preparations
Tong – APT 2007
Side effects in probiotic supplementation were lesser than to without probiotics (24.7% vs 38.5%, OR 0.44)
Tong – APT 2007
Most of the trials reported lower incidences of diarrhoea (6.0% vs 16.1%, OR 0.34) in probiotic group
Tong – APT 2007
I probioIci sono sempre sicuri?
Gut Barrier disfunction
Intestinal permeability (Leaky gut)
Villus/crypt tight junctions in health
Intes;nal lumen
Villus cells
Tight junc;ons
Crypt cells Tight junc;ons
Hollander D et al. Scand J Gastroenterol, 1992
Villus/crypt tight junctions in health
Villus/crypt tight junctions in disease
Intes;nal lumen
Villus cells
Leaky ;ght junc;ons
Crypt cells Tight junc;ons
Hollander D et al. Scand J Gastroenterol, 1992
Villus/crypt tight junctions in disease
§ Burns and trauma § Major vascular surgery § Sepsis § MOF § Renal failure § Diabetes mellitus § Joint disease § Autoimmune diseases § Psychiatric pathologies § Heart and lung diseases
§ Coeliac disease § IBD/IBS § Food allergy § Acute gastroenteritis § Radiation enteritis § Intestinal by-pass § Intestinal obstruction § Peritonitis § Wipple’s disease § Liver Diseases
DISEASES RELATED TO BARRIER DEFECTS
GASTROINTESTINAL EXTRAINTESTINAL
§ 298 patients with predicted severe acute pancreatitis
§ Multispecies probiotic preparation (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactococcus lactis, Bifidobacterium bifidum, and Bifidobacterium lactis) bid/28 days
§ Infectious complications: 30% probiotics VS 28% placebo (RR 1.06, 95% CI 0.75–1.51)
§ Deaths: 16% probiotics VS 6% placebo (RR 2.53, 95% CI 1.22–5.25)
§ Bowel ischaemia (pts): 9 probiotics VS 0 placebo (p=0.004)
Besselink – Lancet 209
§ 298 patients with predicted severe acute pancreatitis
§ Multispecies probiotic preparation (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactococcus lactis, Bifidobacterium bifidum, and Bifidobacterium lactis) bid/28 days
§ Infectious complications: 30% probiotics VS 28% placebo (RR 1.06, 95% CI 0.75–1.51)
§ Deaths: 16% probiotics VS 6% placebo (RR 2.53, 95% CI 1.22–5.25)
§ Bowel ischaemia (pts): 9 probiotics VS 0 placebo (p=0.004)
Besselink – Lancet 209
Enache-Angoulvant et al. – Clin Infect Dis 2005
§ 92 cases of Saccharomyces invasive infection
§ S. boulardii accounted for 51.3% of fungemias
§ Saccharomyces invasive infection is clinically indistinguishable from an invasive candidiasis
§ S. cerevisiae clinical isolates exhibited low susceptibility to amphotericin B and azole derivatives
Bittersweet Saccharomyces boulardii
Esistono altri Ipi di modulazione di gut microbiota?
Diet and Nutritional Support Ø Caloric amount, minerals, vitamins, sweeteners..
Ø Diet composition (fibers/high glicemic index/saturated fatty acids…)
Removal of predisposing conditions Ø Treat diabetes, endocrine, other motility disorders.. Ø Surgery or prokinetics when indicated
Ø Stop PPI or other antiacid, NSAIDs, antibiotic, immunodepressant….
Intervention Ø Antibiotics Ø Biotherapy (prebiotics, probiotics, symbiotics, postbiotics) Ø Microbiota Transplantation
How to (re)modulate gut microbiota?
Prebiotic Dietary indigestible ingredient which selectively stimulates growth and activity of one or multiple microbial species Probiotic Non pathogenetic microorganisms that, when ingested, excert a positive influence on host by altering his microbial balance Symbiotic Mix of probiotics and prebiotics that increase survival of the probiotic, making immediately available its substrate for fermentation
BIOTHERAPY (I)
Postbiotic A metabolic byproduct generated by a probiotic micro-organism that influences the host’s biology Functional food Any modified food or food ingredient that provides a health benefit beyond that ascribed to any specific nutrient/nutrients it contains. It must remains a food and demonstrates its effect in amounts normally expected to be consumed in the diet. Any food that contains probiotics or prebiotics is a functional food. Example of a functional food: live-culture yogurt that contains probiotic bacteria, prebiotics and other dietary nutrients
BIOTHERAPY (I)
Ramirez-Farias C Br J Nutr 2009
Inulin and F. prausnitzi ü Analysis of faecal microbiota composition of human
volunteers after ingestion of inulin (10 g/d) for a 16-d VS control period
ü Significant increase of Faecalibacterium prausnitzii (from 10 to 15% during inulin intake, P=0.019)
..how to feed NEXT GENERATION PROBIOTICS?
1. Faecalibacterium Prausnitzii 2. Akkermansia Muciniphila 3. Eubacterium halii 4. …
DOMINIUM PROKARIOTA
REGNUM BACTERIA
PHYLUM FIRMICUTES
CLASSE CLOSTRIDIA
ORDO CLOSTRIDIALES
FAMILIA CLOSTRIDIACEAE
SPECIES FAECALIBACTERIUM
SUBSPECIES FAECALIBACTERIUM PRAUSNITZII
Faecalibacterium prausnitzii
Ø Gram positive, anaerobic bacterium. Among the most abundant anaerobic bacteria in the human gut microbiota, with a proportion of around 5% of total bacteria in faeces Ø It produces SCFA, particularly Butyrate, primary energy source for intestinal epithelial cells and crucial for maintenance of barrier integrity Ø It has a strong anti-inflammatory effect both in vitro and in vivo
Cao Y, Gastroenterol Res Pract. 2014 Duncan, Appl Environ Microbiol, 2002
Faecalibacterium prausnitzii
ü 11 studies included ü Significantly lower F. prausnitzii
counts in IBD patients versus controls
Reduction of F. prausnitzii links to dysbiosis of gut microbiota in IBD
patients, especially CD patients with ileal involvement
Cao, Gastroenterol Res. Pract 2014
DOMINIUM PROKARIOTA
REGNUM BACTERIA
PHYLUM VERRUCOMICROBIA
CLASSE VERRUCOMICROBIAE
ORDO VERRUCOMICROBIALES
FAMILIA VERRUCOMICROBIACEAE
SPECIES AKKERMANSIA
SUBSPECIES AKKERMANSIA MUCINIPHILA
Akkermansia muciniphila
Akkermansia muciniphila
ü Gram-negative bacterium, mucin-degrading bacteria that resides in the mucus layer ü Its genome contains numerous candidate mucinase-encoding genes ü A. muciniphila produces several proteins involved in the different steps of mucin degradation
Tilg, GUT 2014 Everard, PNAS 2013 Van Passel MW, Plos One 2011 Derrien M, Appl Environ Microbiol. 2008 Collado MC, Appl Environ Microbiol. 2011
ü Akkermansia muciniphila decreased in obese and was inversely correlated with body weight in rodents and humans
ü Akkermansia muciniphila treatment improved metabolic parameters in obese mice models
ü A. muciniphila treatment reversed fat gain, serum LPS levels, gut barrier function and insulin resistance by increasing endocannabinoids and gut peptides ü Metformin and RYGB surgery increased the abundance of Akkermansia muciniphila
Diet and Nutritional Support Ø Caloric amount, minerals, vitamins, sweeteners..
Ø Diet composition (fibers/high glicemic index/saturated fatty acids…)
Removal of predisposing conditions Ø Treat diabetes, endocrine, other motility disorders.. Ø Surgery or prokinetics when indicated
Ø Stop PPI or other antiacid, NSAIDs, antibiotic, immunodepressant….
Intervention Ø Antibiotics Ø Biotherapy (prebiotics, probiotics, symbiotics, postbiotics) Ø Microbiota Transplantation
How (re)modulate gut microbiota?
FECAL MICROBIOTA TRANSPLANTATION
v Oral probiotic doses are typically 3–4 orders of magnitude lower than the 100 trillion native micro-organisms contained within the colon
v Fecal microbiota transplantation is the transfer of gut microbiota from a healthy donor to introduce or re-establish a stable microbial community in the gut
v Previously known as “fecal bacteriotherapy”
v First FMT reported in fourth century China (Ge Hong)
Borody – Nat Rev Gastro 2011 Aroniadis – Curr Opin Gastro 2013
FECAL MICROBIOTA TRANSPLANTATION and C. DIFFICILE INFECTION
Borody – Nat Rev Gastro 2011 Aroniadis – Curr Opin Gastro 2013
v Premise of FMT is to repair or replace the disrupted native microbiota
v 1958 First report of FMT for CDI
v 2013 First guidelines of FMT for CDI
v Good standardization of technique over the last years
FECAL MICROBIOTA TRANSPLANTATION and C. DIFFICILE INFECTION
v Until 1989 enema was the most common technique
v Alternative methods carried out over the years: Ø Nasogastric/Nasoduodenal tube Ø Gastroscopy Ø Colonoscopy Ø Self-administered enemas
v Colonoscopic approach is favored over fecal enema (reaching of entire colon)
Borody – Nat Rev Gastro 2011 Aroniadis – Curr Opin Gastro 2013
Route of administration
FMT and CDI: the evidences
Kassam – AJG 2013
v 11 studies, 273 CDI patients treated with FMT, no RCTs
v No statistically significant heterogeneity between studies (Cochran Q test P = 0.13, I 2 = 33.7 % )
v No difference in clinical outcomes between anonymous vs. patient selected donors
v No reported adverse events
v Follow-up: weeks to years
FMT and CDI: the evidences
Kassam – AJG 2013
v Clinical resolution in 245 pts (UPR 89.7 % ; WPR 89.1 % (95 % CI 84 to 93 % ))
v Lower GI FMT delivery led to a trend towards higher clinical resolution rates than the upper GI route
UPR: unweighted pooled resolution rates WPR: weighted pooled resolution rates
Microbial transplantation and Clostridium Difficile
Van Nood – NEJM 2013
v After enrollment of 43 pts the study was stopped (interim analysis)
v CDI Resolution (pts) Ø Infusion group: 13/16 (81%) after the first infusion (2/3 after a second infusion
Ø Vancomycin group: 4/13 (31%)
Ø Vanco + bowel lavage: 3/13 (23%)
P<0.001 (both comparisons with infusion group)
RESULTS
FMT and CDI: the evidences
Cammarota et al – JCG 2014
v 20 full-text case series, 15 case reports, and 1 RCT
v A total of 536 patients were treated
v 467 (87%) patients experienced resolution of diarrhea
FMT and CDI: the evidences
Cammarota et al – JCG 2014
v Diarrhea resolution rates varied
according to the site of infusion
Ø 81% in the stomach Ø 86% in the duodenum/jejunum Ø 93% in the cecum/ascending colon Ø 84% in the distal colon
v No severe adverse events were
reported
Borody – Nat Rev Gastro 2011
FECAL MICROBIOTA TRANSPLANTATION
Emerging application and future dreams
v OBESITY and METABOLIC SYNDROME
v IBD v IBS
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