BACILLUS CLAUSII Taxonomic Rank

Agadir July the 7th 2019

Probiotics: State of the Art

Francesco Franceschi Internal Medicine Institute

Gemelli University Hospital, Catholic University of Rome, Italy Phylogenetic Diversity of Intestinal Microbiota

2 major phyla: Firmicutes and Bacteroidetes (80 to 90 % of the microbiota)

Faecalibacterium prausnitzii Clostridium leptum (cluster IV) Firmicutes Enterococcus

Lactobacillales 60 to 80 % Streptococcus thermophilus Clostridium Lactobacillus coccoides (cluster XIVa)

Bacteroides thetaiotaomicron Actinobacteri Bifidobacterium

Bacteroidetes Proteobacteria 20 to 40 %

Dethlefsen et al, Nature, 2007 Escherichia coli Ley et al, Science, 2008 Tap et al, Environ Microbiol, 2009 A shared phylogenetic core 57 species present in 90% 18 species present in 100% of subjects of subjects Faecalibacterium prausnitzii SL3 3 Roseburia intestinalis M50 1 Bacteroides vulgatus ATCC 8482 Bacteroides sp. 9_1_42FAA Ruminococcus sp SR1 5 Coprococcus comes SL7 1 Bacteroides sp. 2_1_7 Bacteriodes xylanisolvens XB1A Ruminococcus torques L2-14 Bacteroides sp. 2_2_4 Bacteroides sp. D4 Bacteroides dorei Ruminococcus obeum A2-162 Ruminococcus lactaris Bacteroides capillosus Bacteroides finegoldii Clostridium sp M62 1 Clostridium nexile Qin et al. Nature 2010 EUBIOSIS

EU= good BIOS= life

• Composition: Diversity Richness Relative Abundance

Our gut is a sophisticated ecosystem that is regulated by the logic of RELATIONAL ARMONY

Microbiota and Host live in a COOPERATIVE SYSTEMIC AGGREGATION MODEL

EUBIOSIS

Failure of MICROBIOTA-HOST balance

Quali-quantitative alterations of oral, esophageal, gastric, small bowel and/or colonic microbiota

DYSBIOSIS

Digestive and extradigestive diseases How can we modulate GUT microbiota? Diet and Nutritional Support Caloric amount, minerals, vitamins.. Diet composition (High fibers/low carb/low saturated fatty acids…) Removal of predisposing conditions Treat diabetes, endocrine, other motility disorders.. Surgery or prokinetics when indicated Stop PPI or other antiacid, NSAIDs, antibiotic, immunosoppressant, antidepressant…. Intervention Antibiotics Biotherapy (prebiotics, probiotics, symbiotics) Fecal Microbiota Transplantation BACILLUS CLAUSII Taxonomic rank

DOMINIUM PROKARIOTA REGNUM BACTERIA PHYLUM FIRMICUTES CLASSE BACILLI ORDO BACILLALES FAMILIA BACILLACEAE GENUS BACILLUS SPECIE BACILLUS CLAUSII SUBSPECIE BACILLUS CLAUSII OC, NR, SIN, T BACILLUS CLAUSII •On the market since 1958 •Long time on the field demonstration of efficacy and safety •Well known basic aspects:

- Primary characteristics • Taxonomy

– Functional properties • Gastric resistance • Resistance to biliary salts • Adhesivity to intestinal epithelial cells

– Antibiotic resistance (chromosomal)

– Mechanisms of action

– Safety and stability BACILLUS CLAUSII Primary characteristics

 Bacillus clausii: Taxonomic identification ratified by the Pasteur Institute (Paris)

The genome of one reference strain of B. clausii has been fully sequenced (published March 2005: http://wishart.biology.ualberta.ca/BacMap/ )

 Alkalophilic, Gram-positive micro-organism

 Non-pathogenic, ubiquitous bacteria

 As a member of the genus Bacillus, it is able to form spores, which are highly resistant to both physical and chemical factors Probiotics “Live Microorganisms that, when administered in adequate amounts, confer health benefit to the host, expecially by improving intestinal microbial balance” Bacillus clausii Lactobacillus spp Bifidobacterium spp Other (E.coli; S. cerevisiae; S. thermophilus…)

Single strain Multiple strains Guarner F et al, Nutr Hosp. 2010;25:700-704 Guarner F et al, Nutr Hosp. 2010;25:700-704 Guarner F et al, Nutr Hosp. 2010;25:700-704 Optimal Characteristics for a Probiotic

Resistance to gastric acidity and bile Synthesis of antimicrobial substances Competitive interaction with pathogens for “space and food” (nutrients and microbial adhesion sites) Immunomodulation: Adjuvant-like effects on intestinal and systemic immunity Balance the generation of pro- and anti-inflammatory cytokines Modification of toxins or toxin receptors Safety and stability

B. Clausii exerts all those characteristics Optimal Characteristics for aProbiotic

B. Clausii exerts all those characteristics BACILLUS CLAUSII Endospore-forming bacteria

• Sporulation resistance to acidity, heat..

Nakano Ann Rev Microbiol 1998, Jadamus Anim Physiol Nutr 2002, Casula Appl Env Micro 2002 BACILLUS CLAUSII Endospore-forming bacteria

• Sporulation resistance to acidity, heat..

• Germination in jejunum/ileum

Nakano Ann Rev Microbiol 1998, Jadamus Anim Physiol Nutr 2002, Casula Appl Env Micro 2002 BACILLUS CLAUSII Functional Properties

B. Clausii spores are able to germinate after acid challenge and grow as vegetative cells both in presence of bile and under limited oxygen availability

Cenci et al, J Applied Microbiology 2006 Vegetative forms: treatment comparison in daily

measurement (log10-transformed values) Optimal Characteristics for a Probiotic

B. Clausii exerts all those characteristics Bacillus clausii displays antimicrobial properties The antimicrobial compounds present in the cell-free supernatant were relatively thermostable, because activity remained after 30 minutes of incubation at 85°C The antimicrobial is a bacteriocin-like substance, because its antimicrobial activity was sensitive to pronase treatment

Maria C. Urdaci et al, J Clin Gastroenterol 2004;38:S86–S90) Inhibition of S. aureus (halo of inhibition)

O/C - 37°C O/C - 30°C 3 days 3 days

S. aureus Spot of B. clausii 22 Bacillus Clausii

• B. Clausii produces the type A lantibiotic «Clausin» that interacts with lipid intermediate in biosynthesis of bacterial cells wall polymers

• Lantibiotics are a class of bacteriocins that target peptidoglican lipid II intermediate, its form pores in cytoplasmic membranes leading to bacterial lysis

Bouhss et al, Biophys J 2009

ANALYSIS – RMN and confirmation of the structure

A new Lantibiotic = CLAUSIN

24 MIC μg/ml 4 4 8 2 CLAUSIN 1 >256

> 256 > 256 > 256 > 256 >256 nd

MIC μg/ml C. perfringens ATCC 13124 0.5 C. difficile VPI 1 CLAUSIN inhibits C. difficile ATCC 9680 1 C. difficile growth C. difficile ATCC 43539 2 C. difficile S384 1

Urdaci et al Optimal Characteristics for a Probiotic

B. Clausii exerts all those characteristics

Effect of C. difficile VPI 10463 supernatant (Cd-SN) on Vero cell viability in the presence or absence of B. clausii O/C supernatant (OC-SN).

B. Clausii supernatant significantly inhibits the cytotoxic effect induced by C. difficile and b. cereus toxin Probiotics and Antimicrobial Proteins https://doi.org/10.1007/s12602-018-9436-5

Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1- and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced Diarrhea Biswaranjan Pradhan1,2 & Dipanjan Guha1 & Aman Kumar Naik1 & Arka Banerjee1,3 & Subodh Tambat4 & Saurabh Chawla1 & Shantibhusan Senapati5 & Palok Aich1

Histolgy and histological score in animals treated with B. clausii Optimal Characteristics for a Probiotic

B. Clausii exerts all those characteristics Swiss and C57 Bl/6j murine cells +

B. clausii

NOS II synthetase activity CD4+ T-cell proliferation

IFN-γ production Bacillus Clausii

• B. clausii induces TNF-a production from human monocytes through depolymerization of b-glucan oligomer with its b-glucanase

• It induces activation of Th1 immune responses and of a miscellaneous of inflammatory response and cellular defense genes Miyanashi, J Biosci Bioeng 2003 Di Caro and Gasbarrini, Eur J Gastro Hep 2005 10 subjects (22.3 years) with allergic rhinitis

B. clausii 2 billions 3 vials/day for 4 weeks.

IL-4 IFN-g TGFbeta IL10 (p=0.004) (p=0.038) P=0.039 (p=0.009) 6 males (38±5 years) with mild oesophagitis

1 month Esomeprazole 1 month Esomeprazole + B. clausii

Duodenal biopsies for Gene Microarray Genes Expression profile using B.clausii

Comparison the expression profiles of the samples before and after treatment

158 Up-regulated genes

265 Down-regulated genes

Di Caro and Gasbarrini, Eur J Gastro Hep 2005 BACILLUS CLAUSII … and gut barrier

4.3 fold change b-Defensin 126 vs controls

Defensins: “natural occuring antibiotics”. They are cysteine-rich cationic polypeptides important in the immunologic response to invading microorganisms.

Optimal Characteristics for a Probiotic

B. Clausii exerts all those characteristics EFFECT OF THE ADMINISTRATION OF BACILLUS CLAUSII ON MICE TREATED WITH THE COLORECTAL CARCINOGEN MNU

60 mice C57/BL6 6 week old

B. Clausii I.R. MNU for 3 weeks

Sacrificed 6 weeks after Placebo

Franceschi et al, preliminary data EFFECT OF THE ADMINISTRATION OF BACILLUS CLAUSII ON MICE TREATED WITH THE COLORECTAL CARCINOGEN MNU

Results - 1

0% animals showed dysplasia B. Clausii

40% of animals showed dysplasia Placebo

Franceschi et al, preliminary data EFFECT OF THE ADMINISTRATION OF BACILLUS CLAUSII ON MICE TREATED WITH THE COLORECTAL CARCINOGEN MNU

Results - 2

RPL11 Slc25a20 ENPP5 Kat2b MEKK3

B. Clausii DHH Serpinb3a NME2 NUP210 BCL-3

Franceschi et al, preliminary data EFFECT OF THE ADMINISTRATION OF BACILLUS CLAUSII ON MICE TREATED WITH THE COLORECTAL CARCINOGEN MNU

Results - 3

RPL11: Oncosuppressor gene Slc25a20: Transport and oncosuppressor gene ENPP5: Antiproliferative and pro-apoptotic function Kat2b: Co-activator of the oncosuppressor p53 MEKK3: Pro-apoptosis

DHH: Increased in several tumors, including CRC Serpinb3a: Neoplastic marker NME2: Increased in CRC NUP210: Increased in different kind of tumors BCL-3: Marker of proliferation and prognosis

Franceschi et al, preliminary data Optimal Characteristics for a Probiotic

B. Clausii exerts all those characteristics Safety of Probiotics (Functional foods-Dietetics-Farmaceutical)

Lack of Pathogenicity and Infectivity Essential conditions for Probiotic definition

Acute/Chronic toxicity test

Not transmissible Antibiotic-resistance BACILLUS CLAUSII Safety profile Bacillus clausii spores granted its first Marketing Authorization in Italy on 03-Nov-1958 and is currently marketed in >60 countries: Argentina, Aruba, Belarus, Brazil, Chile, Colombia, Costa Rica, Croatia, Cuba, Dominican Republic, Ecuador, El Salvador, Georgia, Guatemala, Gulf Counries, Honduras, Hungary, India, Italy, Jamaica, Jordania, Lebanon, Kazakhstan, Mexico, Morocco, Netherlands Antilles, Panama, Pakistan, Peru, Philippines, Trinidad & Tobago, UAE, Ukraine, Uruguay, Venezuela, Vietnam, Turkey…

During a reference period of ~18 years from 1 January 1995 to 30 November 2013 a total of approximately 4.500 millions of vials were sold in pharmacies

Very Good Safety Profile Genetic of antibiotic resistance in B. Clausii

Antibiotic resistance is chromosomal

Courvalin P, DLD 2006 No risk of antibiotic-resistance transfer For what conditions we may use B. clausii in the clinical practice? ROLE OF CURRENT PROBIOTICS IN GASTROINTESTINAL DISORDERS

Reduction of Antibiotic-associated Diarrhea Prevention and treatment of Infectious Diarrhea Adjuvant for H. pylori and C. difficile treatment Treatment of Necrotizing enterocolitis Treatment of Sugar Intolerance Prevention and treatment of Pouchitis Maintenance of remission of IBD Treatment of IBS Practice guidelines on Probiotics usage World Gastroenterology Organization (2017) Multistrains BACILLUS CLAUSII sp OC, NR, SIN, T

Reduction of Antibiotic-associated Diarrhea Treatment of Infectious Diarrhea Adjuvant for H. pylori treatment Prevention of Respiratory Tract Infection Treatment of IBS with intestinal bacterial overgrowth Treatment of Sugar Intolerance Prevention and treatment of Pouchitis Treatment and maintenance of remission of Ulcerative Colitis Antibiotic effect on GUT microbiota

1-week Clyndamicin

healthy young adults (HY) HY healthy elderly people (HE) HE elderly people receiving clyndamicin (CE) CE Woodmansey E, Appl Environ Microb 2004 Antibiotics associated side effects

1. Diarrhea, bloating, nausea, constipation, dysgeusia, … 2. Overgrowth of pathogens (C. diff) 3. Antibiotic-resistance Antibiotic-related diarrhoea

Antibiotic-related diarrhoea

Direct effect of Alteration of the gut antibiotics on the gut flora system

 Bacterial population Overgrowth of and SCFA production pathogens

Functional Intestinal Infection diarrhea (ie C. Diff)

Gorkiewicz G, Int J Antimicrob Agents. 2009 Beaugerie, BP&RCG 2004 Incidence of antibiotic associated diarrhea (AAD) in trials

Mc Farland VL, Future Microbiology. 3.5 (Oct. 2008) Incidence of Clostridium Difficile

Mc Farland VL, Future Microbiology. 3.5 (Oct. 2008) Probiotics and Antibiotic-associated Diarrhea

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

Cremonini and Gasbarrini, Alim Pharm Ther 2002 Puddu et al. CLINICAL EXPERIENCE WITH B. SUBTILIS International Pediatrics; 6: 1980

93 children (47 males, 3-14 years old) with bacterial infections of the tonsils or the middle ear, nasal and paranasal cavities requiring antibiotic treatment.

• 39 pts: Ampicillin (100 mg/kg/day) • 19 pts: Erythromycin (30 mg/kg/day) • 24 pts: Tetracycline (20 mg/kg/day) • 11 pts: Thiamphenicol (50 mg/kg/day) for 10 consecutive days

During antibiotic treatment subjects were randomly assigned to receive either 4 vials/p.o./day of a preparation containing 1 billion spores of Bacillus subtilis Puddu et al. CLINICAL EXPERIENCE WITH B. SUBTILIS International Pediatrics; 6: 1980

TABLE 1 - Incidence of gastrointestinal problems in children treated with antibiotics and Bacillus subtilis (group A) and with antibiotics alone (group B)

No. Nausea Vomiting Abdom. pain Diarrhea Aphthae Total

Group A 45 2 - 2 - 5 Group B 48 2 1 9 5 1 18 p<0.01 120 H. pylori-positive patients were randomly screened to receive:

• standard 7 days triple therapy with rabeprazole 20 mg b.d., clarithromycin 500 mg b.d. and amoxicillin 1 g b.d. and B. clausii t.d.s. (each preparation containing 2 x 10(9) spores) for 14 days starting from the first day of treatment

• standard 7 days triple therapy with rabeprazole 20 mg b.d., clarithromycin 500 mg b.d. and amoxicillin 1 g b.d. and PLACEBO for 14 days starting from the first day of treatment

B. clausii and Antibiotic-associated Diarrhoea

Bacillus clausii supplementation (6x109/d), compared to placebo, determines a significant reduction of frequency and intensity of

 Diarrhea  Nausea  Epigastric pain in asymptomatic patients who underwent to H. pylori eradication (Amoxicillin and Metronidazole)

Nista and Gasbarrini, Aliment Pharmacol Ther 2004 B. Clausii reduce incidence of Diarrhea during a 1-week course of antibiotics

Group A (Bacillus c.) Group B (placebo)

Nista, Franceschi and Gasbarrini, Aliment Pharmacol Ther 2004 Multistrains BACILLUS CLAUSII sp OC, NR, SIN, T

• The use of specific Probiotics is mandatory • Spore form, Bifidobacteria and lactic producing are the three most commonly used strains. Effects of Bacillus clausii on Infectious Diarrhea

7 days of B.Clausii 4x109/d vs no treatment in children (mean age 3 years) with acute Adenovirus/Rotavirus diarrhea

Faster eradication of adenovirus/rotavirus Persistent gut colonization Faster restoring of normal enteric ecoflora More consistant body weight recovery

Arzese et al, Mom Med Chir 2002

Review Bacillus clausii for the Treatment of Acute Diarrhea in Children: A Systematic Review and Meta- Analysis of Randomized Controlled Trials

Gianluca Ianiro 1,*, Gianenrico Rizzatti 1, Manuel Plomer 2, Loris Lopetuso 1 ID ,

Franco Scaldaferri 1, Francesco Franceschi 1, Giovanni Cammarota 1 and Antonio

Gasbarrini 1

Effect of Bacillus clausii on mean duration of diarrhea Effect of Bacillus clausii on mean stool frequency Effect of Bacillus clausii on mean duration of hospitalization 131 children with acute diarrhea (aged 6 months-12 years)

Group 1 Group 2 • 69 pts • 62 pts • ORS+ Zinc • ORS+ Zinc • B. clausii 2 billions 2/day for 5 days

Multistrains BACILLUS CLAUSII sp OC, NR, SIN, T

Fermentation and gas production Lin, JAMA 2004 B. Clausii in Small Intestine Bacterial Overgrowth

Treatment Scheme ITT PP

Levofloxacin 60% 64%

Nitroimidazolic Compound 40% 42%

Levofloxacin+NC 70% 81%

BacillusBacillus Clausiiclausii 47%47% 47%47%

Lauritano, Gasbarrini et al, Am J Gastro 2009 CLINICAL CASE - I How to use B. clausii in the clinical practice? Clinical Case 1

13 yrs old male Clinical hystory of epigastric pain and dyspepsia; worsening of symptoms in the last month Submitted to upper GI endoscopy with diagnosis of duodenal ulcer Rapid urease test for H. pylori positive (confirmed by hystology) 7-day therapy with amoxicillin 1 g bid, tinidazole (500 g bid), omeprazole (20 mg bid) Discontinuation of therapy after 4 days for severe diarrhea and bloating Antibiotic-related Diarrhoea

Direct effect of Alteration of the gut antibiotics on the gut flora system

Bacterial population Overgrowth of with specific functions pathogens

Functional Intestinal diarrhoea Infection

Beaugerie, BP&RCG 2004 Clinical Case 1

After 4 weeks pts was submitted to 13C-urea breath test that shows persistent positivity for H. pylori

Rescue 10 day bismuth quadruple therapy and omeprazole + Bacillus clausii 6x109/day x 14-day (starting together with antibiotics) Patient completed antibiotic therapy complaining only mild diarrhea at days 6 and 7 After 6 weeks from end of therapy pts was submitted to 13C-urea breath test that shows H. pylori eradication B. Clausii reduce incidence of Diarrhea during a 1-week course of antibiotics

Group A (Bacillus c.) Group B (placebo)

Nista, Franceschi and Gasbarrini, Aliment Pharmacol Ther 2004 TAKE HOME MESSAGES There is no chronological threshold or age at which the composition of the microbiota suddenly alters; rather, changes occur gradually with time…

Nikoletopoulou V et al. Trend in Endocrinology and Metabolism 2014 Dysbiosis • Gastrointestinal infections • IBS and IBD • SIBO and CBO • Diverticulosis • Gastro-intestinal Cancers • Food Intolerance/Allergy • Celiac diseaseDysbiosis • Liver and Pancreatic diseases • Obesity, Diabetes and Metabolic Syndrome • Gynecological, Rheumatological, Cardiovascular, Neuropsichiatric disorders… Dysbiosis B. clausii Dysbiosis Thanks!