Mutaflor. E. coli strain Nissle 1917

Transcription

1 Mutaflor E. coli strain Nissle 1917

2 A Bacterium Becomes a Medicinal Product The discovery of Mutaflor The discovery and development of Mutaflor goes back to Professor Alfred Nissle ( ) from Freiburg. During World War I, when searching for E. coli-bacteria with an antagonistic activity (growth inhibition) against intestinal pathogens, he isolated a special E. coli strain from the stool of a war participant. As opposed to most of his comrades, the latter had remained completely gut-healthy during the military campaign in the Balkans, contracting neither dysentery, typhoid fever, paratyphoid fever nor any other enteritides. In honor of Professor Alfred Nissle, the strain bears the designation Escherichia coli stain Nissle 1917, abbreviated EcN. Nowadays we apply the most modern processing methods to culture these bacteria. They are grown in fermenters and then carefully lyophilized. This keeps the microbes alive for several months when stored in a cold environment. Mutaflor the broad application range of E. coli strain Nissle 1917 Current registrations, clinical studies and casuistics demonstrate the broad range of Mutaflor applications. This reaches from chronic bowel diseases to the colonization prophylaxis of premature infants and full-term neonates all the way to the treatment of extraintestinal diseases. The limitations of antibiotic therapy, which are becoming more and more visible by the development of multiresistant pathogenic microorganisms, heighten the interest in the probiotic medicinal product Mutaflor, containing E. coli strain Nissle 1917 as the active substance. New knowledge about the medicinal relevance of the microbiome to human health have been added. In this brochure, we present an overview on the latest non-medical (pre-clinical) findings concerning the unique properties of E. coli strain Nissle 1917, as well as on the clinical experiences that have been made with Mutaflor treatment of patients. Electron microscopic image of E. coli strain Nissle Magnification: 1: 10,000. 2

3 Table of Contents: I The Development and Significance of Gut Microbiota... 4 The natural development of the intestinal microbiota The normal Escherichia coli bacteria of the gut... 4 Functions of the gut microbiota Pathophysiological role of the gut bacteria... 5 II Strain-Specific Properties of E. coli Strain Nissle Relevance to clinical practice... 6 III Mechanisms of Action of E. coli Strain Nissle Communication strategies of E. coli strain Nissle Positive influence of E. coli strain Nissle 1917 on the intestinal epithelium Antagonism action of E. coli strain Nissle E. coli strain Nissle 1917 strenghtens the intestinal barrier... 9 E. coli strain Nissle 1917 forms short-chain fatty acids from fiber substances Defensins strengthen the body's innate immune defenses E. coli strain Nissle 1917 protects against infections The healthy gut microbiota prevents against atopic diseases IV Prophylaxis of Unwanted Microbial Gut Colonization with Mutaflor Suspension Increased risk of infection in newborns Mutaflor the natural protection against infection V Therapy of Inflammatory and Functional Intestinal Diseases with Mutaflor Diarrhea Chronic constipation Ulcerative colitis Crohn's disease Irritable bowel syndrome Mutaflor in other diseases VI Application and Dosage Recommendations VII Medicinal Product Safety VIII References Status of the information: January

4 I The Development and Significance of Gut Microbiota The natural development of the intestinal microbiota According to general conceptions, the intestines are sterile until the moment of birth. The initial bacterial colonization of the gastrointestinal tract of newborns sets in immediately after birth. The primary intestinal colonization proceeds randomly. Recent molecular biological analyses have started a discussion as to whether microbial colonization might already occur during embryogenesis. E. coli is the typical initial colonizer of the 1; 2; 3 human gut The colon's oxygen content, still high directly after birth, begins to decrease by colonization with E. coli. Oxygen consumption is caused by the metabolic activities of E. coli leading to anaerobic conditions in the large bowel. Only then does the further colonization of the intestinal tract by strictly anaerobic bacteria begin (e. g. bacteroides, bifidobacteria). Whether physiological or pathogenic bacteria initially colonize the intestine depends on the environmental conditions that prevail at the time of birth (for example, hospital milieu). 4 The normal Escherichia coli bacteria of the gut As E. coli is capable of growing under both aerobic and anaerobic conditions, the bacterium has a pioneering role in the gradually progressing colonization of the gut in newborns [Fig. 1]. Normal E. coli-bacteria preferentially colonize the mucous layer (mucin layer) lying on top of the mucosa of the colon. This is where they form a microbial barrier against invading foreign bacteria and owing to their pronounced immunogenic potency, exercise the gut-associated immune system. Read more about risks of infection and colonization prophylaxis in premature and full-term infants from page 12 onwards. Fig. 1: Sequential microbial colonization of the intestines of newborns by aerobic and anaerobic bacteria (after Hoogkamp-Korstanje et al. 1979) Aerobes: E. coli Lactobacillus Enterococcus Bacterial cell count (log/g feces) 8 6 Anaerobes: Bacteroides Bifidobacterium Hours Days 4

5 Functions of the gut microbiota Consisting of about bacteria in adults, the gut microbiota represents a complex ecological entity that possesses a high metabolic activity. It thus numerically surpasses the body's own approx cells by a factor of 10. By its weight and metabolic capacity, it is comparable with the liver. The intestinal microflora supports the human organism with metabolic, trophic and protective functions. 6; 7 It controls the cell proliferation and differentiation of the intestinal epithelium and promotes the development and homoeostasis of the immune system. The gut microbiota protects against infections caused by pathogenic microbes and also plays an important role in sustaining immunological tolerance as long as we live. The short-chain carboxylic acids synthetized by the gut microbiota (primarily acetic, propionic and butyric acid) serve in nourishing epithelial cells of the colon [Fig. 2]. Pathophysiological role of the gut bacteria The significance of the normal gut microbiota will become particularly obvious once the microecological balance is disturbed. Apart from the obligatory gastro intestinal pathogens, some regular intestinal bacteria might also assume pathogenetic relevance: in the development of a post-infectious irritable bowel syndrome by inducing and sustaining chronic inflammatory bowel diseases and associated extraintestinal manifestations to the devolpment of the metabolic syndrome in the development of the antibiotic-associated diarrhea and pseudomembranous colitis in the bacterial overgrowth syndrome of the small bowell in case of a disturbance of the intestinal barrier function ( leaky gut ) Associations between gut microbiota and extraintestinal diseases such as urinary tract infections, allergies, food intolerances and joint disease are also being discussed. 8; 6 from the ileum oligosaccharides, polypeptides, glycoproteins (mucin) Fig. 2: Nutrient-supply of the colonocytes with short-chain carboxylic acids COLON LUMEN WITH MICROFLORA short-chain carboxylic acids (acetic, propionic and butyric acid) microbial cleavage and fermentation COLONOCYTES CO 2 Energy metabolism ATP Cellular respiration Na + resorption Mucin synthesis Protein synthesis Cell turnover SEROSA, BLOOD VESSELS Acetic, propionic and butyric acid Colon motility Blood circulation to the liver 5

6 II Strain-Specific Properties of E. coli Strain Nissle 1917 The E. coli strain Nissle 1917 (EcN) contained in Mutaflor disposes of a number of properties which are important to its survival, colonization ability, and persistence in the intestinal ecosystem, and to its therapeutic efficacy. 9; 10; 11 These properties are listed in Table 1. Fimbriae and flagellae are of significance for the attachment to the mucin layer of the human intestinal flagellum were capable of stimulating the production of HBD-2 in the epithelium cells of the colon. The structural flagellum protein flagellin, which forms the actual flagellum, i. e. the so-called flagellum filament, was identified to be the effective component [Fig 4]. The EcN strain expresses two microcins 14 and exerts antagonistic actions against foreign 15; 16; 17; 18; 19; 20 bacteria. mucosa. 12 Studies which were designed to elucidate the molecular principles of EcN s action on the gut mucosa, revealed that EcN is able to induce in the colonocytes the synthesis of the antimicrobial peptide human beta-defensin-2 (HBD-2), and that the HBD-2-stimulating activity relies on the presence of a structurally bound component of the EcN strain, namely the bacterium's own flagella apparatus. 13 Experiments with various defect mutants revealed that only EcN bacteria able to synthetize an intact Relevance to clinical practice As a consequence for clinical practice, the induction of the body's own defense agents through the action of E. coli strain Nissle 1917 is equivalent to an increase of the general antimicrobial defense capacity of the intestinal mucosa. This is of special significance to certain gastrointestinal diseases, such as the chronic inflammatory bowel diseases in which the mucosal synthesis of antimicrobial peptides is affected by disease. No pathogenicity factors Previous molecular genetically discovered gene clusters of the chromosome which encode the phenotypic traits 2 antagonistically active microcins 3 different types of fimbriae enable adherence and biofilm formation 7 different ironincorporation systems as important fitness factors Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ 3 kb 5 kb ybt mch mcm fim cbt ent Chromosome chu aer cit rfb kps foc fla csg 2 strain-specific plasmids, no transmissible, without antibiotic resistance genes Capsule (K5) Special immunomodulatory LPS variant of serotype 06, previously not identified in any other strain Serotype 06:K5:H1 Flagella (H1) account for active motility, adherence to mucin, and induction of the HBD-2 system Fig. 3: Phenotypical characteristics of E. coli strain Nissle 1917 and its gene loci in the bacterial chromosome. The important structural components of E. coli strain Nissle 1917 possessing signal-mediating properties are highlighted in red. 06 antigen, adhesive fimbriae (F1A, F1C and Curli fimbriae) and flagellae (H1 antigen). 6

7 Junction proteins Filament cap Filament made of flagellae Fig. 4: Structure of a flagella apparatus of E. coli. The structural protein flagellin which builds up the flagellum filament was identified as the inductor of the HBD-2 synthesis by E. coli strain Nissle 1917 in epithelium cells of the colon. Outer membrane Hook Peptidoglycane Inner membrane Mot A-B complex Basal body C ring Export apparatus Properties of E. coli strain Nissle 1917 Mobility due to type H1-flagella Ability to colonize due to specific interaction of its flagellae with the mucin layer Ability to form biofilms due to F1A-, F1C- and Curli-fimbriae Antagonistic activity directed against foreign microbes due to antimicrobial microcins Vitality and resilience ( biological fitness ) due to multiple iron uptake systems Immunomodulatory effects due to strain-specific surface structures (special LPS = lipopolysaccharide) and synthesis of immunomodulatory signal substances Anti-inflammatory effects Stabilizing effect on the barrier function of the intestinal mucosa Table 1 Read more about gastroenterological diseases and the clinically corroborated effects of E. coli strain Nissle 1917 from page 17 onwards. 7

8 III Mechanisms of Action of E. coli Strain Nissle 1917 Communication strategies of E. coli strain Nissle 1917 E. coli strain Nissle 1917 communicates with the intestinal epithelium and the gut microbiota ( cross talk and quorum sensing ) [Fig. 6]. Studies showed that this communication involves the regulation of approximately 300 different genes in the enterocytes. 22 It was also shown that EcN inhibits the formation and release of pro-inflammatory cytokines within the intestinal epithelium 23 and stimulates the body's innate defense by means of an upregulation of defensins Strengthening the barrier function of the intestinal epithelium, adherence, biofilm formation, suppression of pathogens Cross Talk with eukaryotic cells Antagonism / competition Relative defensin expression (HBD-2) E. coli K-12 EPEC E. coli strain Nissle 1917 Fig. 5: In Co-cultures of CaCo2 cells and E.coli strains, E. coli strain Nissle 1917 induces the expression of HBD-2 most effectively (after Wehkamp J. et al., 2004). 24 Intestinal epithelium Enteric lumen Cross talk and quorum sensing, interaction between commensals and pathogens. Establishment of normal microbiota Enteric microbes Pathogens Commensals E. coli strain Nissle 1917 Signal molecules Basal membrane Intestinal epithelium cells Positive influence of E. coli strain Nissle 1917 on the intestinal epithelium An increased permeability of the intestinal mucosa ( leaky gut ) for large-molecular antigens and even whole microorganisms from the gastrointestinal tract is suspected to be of pathophysiological importance in various types of diseases. E. coli strain Nissle 1917 has a positive influence on a damaged enteric mucosa displaying an increased permeability. This property is capable of improving the clinical symptoms in patients with liver cirrhosis. 25 EcN is capable of strengthening certain structural elements which account for the cohesion of enterocytes in the gut Mucin layer Desmosomes and tight junctions Fig. 6: Communication strategies of E. coli strain Nissle 1917 (modified after Hacker et al., 2000). 21 mucosa, and stabilzes the intestinal barrier function. For example, EcN elicits both a curative and a prophylactic effect in cases when the intestinal barrier is disturbed by enteropathogenic E. coli-bacteria. 26 An increased mucosal permeability for the dye Evans Blue effected by dextran sodium-sulfate in the colitis mouse model can be considerably decreased by a pretreatment with EcN. 27 8

9 E. coli strain Nissle 1917 Cell to cell communication Direct antagonistic activity Growth inhibition / killing of pathogenic bacteria and fungi (e. g. EHEC, Candida) Synthesis of mucin, defensin, cathelicidin, calprotectin Inhibition of invasion (Salmonella, EIEC, AIEC, Shigella, Yersinia, Listeria, Candida) Immunomodulation, e. g. IL-10-induction Anti-inflammatory effects (inhibition of IL-5, IL-6, IFN-γ, TNF-α-action on IL-8) Strengthening effect on a disturbed epithelium permeability barrier Prevention of leaky-gut symptoms in case of a disturbed barrier function Improvement of the ion transport capacity of the intestinal cells INTESTINAL EPITHELIUM Fig. 7 Action mechanisms and interactions of E. coli strain Nissle 1917 in the gut 6; 10; 11 (bacterial antagonism) and the enteric epithelium ( host-cells signaling, cross talk ). Antagonism action of E. coli strain Nissle 1917 E. coli strain Nissle 1917 suppresses the multiplication of pathogenic microorganisms and protects against the invasion of enteric pathogenic microorganisms, e. g. EHEC 18; 19; 28 as well as the potential adverse effects of a preceding intestinal infection (post-infectious irritable bowel syndrome). 29 E. coli strain Nissle 1917 strenghtens the intestinal barrier Tight junctions and certain desmosomes (adherence junctions) enable the cohesion of the intestinal epithelial cells. This phenomenon prevents the uncontrolled passage of substances from the intestines into the body (leaky gut-syndrome). Furthermore: E. coli strain Nissle 1917 has a direct stimulating effect on the smooth musculature of the intestines. 30 E. coli strain Nissle 1917 reduces the mutagenic activity of various potentially carcinogenic compounds. 31 An increased COX-2-activity, often brought in connection with inflammatory bowel diseases or the development of colonic carcinoma, and thus increased prostaglandin E 2 -levels, are decreased in vitro in the presence of E. coli strain Nissle Tight junction Zonula adhaerens Macula adhaerens Fig. 8: Electron microscopic image: Intestinal epithelial cells with tight juncton sealling. 9

10 Action Principles of E. coli Strain Nissle 1917 Controls without bacteria EPEC 120 min EcN 120 min Separate incubation EPEC + medium 60 min EPEC + EcN 60 min EPEC + EcN 120 min Concomitant incubation Fig. 9: Representation of the induction of ZO-2-gene expression in T84 epithelial cells and the incorporation of the ZO-2-protein into the tight junction. Staining with the ZO-2-specific fluorescent antibody shows (control without bacteria) that the ZO-2 protein is predominately localized in the membranes of the epithelium cells. This becomes evident by the strong light-red color of the cell contours. An incubation with E. coli strain Nissle 1917 (EcN) for 120-minutes did not alter the distribution of the ZO-2 protein. In contrast, a 120-minute incubation with an enteropathogenic E. coli strain (EPEC) induced a redistribution of ZO-2 and a reduction of the tightness of the tight junctions. In case of a concomitant incubation of EPEC and EcN the EPEC bacteria could not exhibit their noxious effects (after Zyrek et al., 2007). 26 EcN enhances the expression of the tight-junction proteins zona occludens 1 and 2 and the desmosome protein named pinin, and thus stabilizes the 22; 26; 27 intestinal barrier [Fig. 8, previous page]. Interestingly, EcN is capable of reconstituting the tight junctions previously loosened by the attack of the enteropathogenic bacteria and thus reestablishing the integrity of the intestinal epithelium. As M. Alexander Schmidt and his colleagues working at the Institute of Infectiology of the University of Münster, Germany, were able to show by applying socalled DNA microarray technology, 22; 26 a co-incubation of intestinal epithelium cells (T84-cells) with EcN caused a time-dependent induction of gene expression for proteins which are involved in the structural make-up and integrity of the tight junctions and/or desmosomes. In summary, the contact between epithelial cells and EcN produced an alteration in the expression of more than 300 genes, whereby both an upregulation and downregulation of the gene activity of various epithelial hereditary factors has been determined. An increased gene expression of the tight junction proteins and/or desmosomal proteins ZO-2 and pinin was demonstrated in the T84-cells used, reaching a maximum after an incubation time of 120 minutes. 22 Further experiments conducted by the same working group 26 concerned with the molecular regulation of ZO-2-expression in epithelial cells were able to prove by applying high-resolution microscopic technology and specific fluorescence-labeled antibodies [Fig. 9] and protein-chemical analyses that EcN is capable of restoring an epithelial barrier previously destroyed by the infection with an enteropathogenic strain of E. coli (EPEC). In the course of this reaction the ZO-2- protein is redistributed from the cytoplasm to the cell membrane of the epithelial cell. This stabilizing effect 10

11 of EcN was reproduced in various cell culture experiments for both a prophylactic and a therapeutic applications. Significance for the clinical practice As a consequence for the clinical practice, it follows from these experimental results that the E. coli strain Nissle 1917 is capable of strengthening and regenerating the intestinal barrier function. This may be of central significance to the efficacy of EcN in various gastroenterological, hepatological and allergological-immunological diseases, which are associated with the leaky gut-phenomena and are caused by the increased permeability of the intestinal epithelium. E. coli strain Nissle 1917 forms short-chain fatty acids from fiber substances EcN is to a greater extent capable of forming shortchain fatty acids (formic, acetic, propionic, and butyric acid) from fiber substances. Acetic acid is predominant among these acids. It stimulates the ring musculature of the colon and promotes the blood circulation of the mucosa. Acetic acid consequently has a stimulating effect on intestinal motility. 30 The efficacy of EcN, for example, in cases of chronic obsti pation seems to be based on this action. In addition, the bacterial fermentation products, particularly butyric acid, are very energy-rich and indispensable to metabolism in the epithelium of the colon. E. coli strain Nissle 1917 protects against infections EcN enhances the in-vitro cytotoxicity of isolated murine macrophages against Leishmania donovani. This speaks in favor of a reinforcement of the defense against intracellular parasites. 33 In animal studies, the oral administration of EcN was shown to protect against systemic infections with Candida albicans and Listeria monocytogenes. In animals infected with Candida albicans, a drastic reduction of the microbial contamination of the main target organ, i. e. the kidney, was achieved. A comparable effect was observed in the livers and spleens of animals infected with L. monocytogenes. 34 The healthy gut microbiota prevents against atopic diseases The microbiota of the gut has an influence on the immune reactions regulated by TH1-lymphocytes and TH2-lymphocytes. A disturbed balance among the various helper-t-cells subsets appears to be responsible for the increased occurrence of atopic diseases. 8 Immunological studies dealing with the colonization of EcN in newborns have shown that the development of the infant's immune system is stimulated. 35 This may also be coupled with a reduction of the risk of developing an allergy. Defensins strengthen the body's innate immune defenses Defensins are the body's own antimicrobial peptides which are produced, for example, in the intestinal epithelium. They are part of the non-specific innate immune system and protect the body against a large number of pathogenic microorganisms. EcN induces the formation of human β-defensin-2 (HBD-2) in intestinal epithelium cells and thus strengthens the 13; 24 body's defense capacity. 11

12 IV Prophylaxis of Unwanted Microbial Gut Colonization with Mutaflor Suspension Increased risk of infection in newborns Despite the growing progress made in medicine, infections in neonatology still are of special significance. Newborns are exposed to a particularly high risk of infection because of the immaturity of their immune system and some of their organs, such as the skin, the lungs and the gastrointestinal tract. Infections belong to the essential causes of morbidity and mortality in the newborn In the period from January 2005 to December 2009, the National Reference Center for the Surveillance of Nosocomial Infections (NRZ) with the Hospital Infection Surveillance System (KISS) registered for the risk sector Neonatological Intensive Medical Care a total of 24,301 newborns (birth weight: < 1,500 g) with 901,037 days on ICU, 4,486 sepsis cases and 596 pneumonias. The further evaluation revealed that antibiotics were administered on ICU on a total of 270,671 days (30 %). The risk of infection increased with sinking gestation age and birth weight. Catheter-associated infections constituted an additional risk in intensive-medical care. Nosocomial infections prolong hospitalization periods and increase treatment costs in the hospital. The prophylaxis of infections and the introduction of new prevention measures are therefore absolutely necessary and for economic reasons as well. Infectious germs originate from the mother and the immediate environment of the child The first intestinal colonization of the infant's intestines with commensal or pathogenic bacteria depends, among other factors, on the vaginal and gut microbiota of the infant's mother as well as the microbiota on the surface of her skin. Bacteria in the environment during and after delivery also have an impact on the first intestinal colonization. Frequently perinatal-acquired infective agents Pathogenic E. coli Streptococci (Group B) Enterococci Staphylococci Haemophilus influenzae Listeria ssp. Herpes simplex virus Table 2 A study compared the microbial intestinal colonization of sixty newborns in three hospitals. The evaluation revealed a typical frequency distribution of the respectively prevailing bacterial species for each hospital. Opportunistic or obligate pathogens (e. g. Klebsiella pneumoniae ssp. ozaenae, Shigella ssp., Yersinia enterocolitica, etc.) were detected in the stool samples of several children. A certain extent of these species still prevailed until the children had been discharged from hospital. Common pathogens of postnatal infections Gram-positive Staphylococcus haemolyticus bacteria Staphylococcus epidermis Pneumococci Gram-negative Pathogenic E. coli bacteria Klebsiella ssp. Pseudomonas aeruginosa Enterobacter Shigella ssp. Salmonella ssp. Serratia ssp. Proteus spp. Yeasts Candida albicans Table 3 12

13 30 % of the E. coli strains isolated from the stool samples during the first week of life exhibited pathogenic features, e. g. ability to induce hemolysis. Candida albicans was determined in seven newborns in one hospital. Hospital-specific differences were also revealed when the meconium (first stool of a newborn) was analyzed. In two hospitals, 50 % of the children's melena stools have already been colonized with microbes. In the third hospital, even 95 % of all melena stools were contaminated. 3 Type and diversity of the microbial composition are decisively influenced by the environment. Particularly in premature infants, developmentally disordered or diseased newborns, the risk of colonization with potentially pathogenic bacteria and antibiotic-resistant hospital-acquired microorganisms increases strongly in the course of hospitalization. The increasing number of antibiotic resistances among pathogenic bacteria is alarming (e. g. VRE, EBL, MSRA). Avoiding colonization with unwanted bacteria Various measures are applicable in order to restrict the colonization of the newborn's intestines with pathogenic or potentially pathogenic bacteria: Hygiene measures minimize the transmission of pathogens (e. g. antiseptic measures, hand disinfection, disinfection of medical devices). Sparing, targeted application of antibiotics avoids the further spread of antibiotic-resistant microorganisms. The postnatal colonization with E. coli strain Nissle 1917 ( Mutaflor Suspension) acts antagonistically against pathogenic bacteria and thus protects against dysbiosis of the intestinal microbiota right from the beginning. Furthermore, EcN enhances the immune competence by an early stimulation of the development of the gut-associated immune system in the newborn. Mutaflor the natural protection against infection E. coli strain Nissle 1917 colonizes the gut of newborn infants The ability of EcN to colonize the intestine of full-term and premature neonates has been investigated in various studies. To this end, one ml Mutaflor suspension containing 10 8 EcN bacteria were daily administered to neonates orally on the first to the fifth day of life. It was shown that EcN colonizes in the intestines on a long-term basis. Colonization in full-term neonates In breast-fed full-term neonates, EcN bacteria were recovered in the stools of 75 % of the treated children from the third day onward. On the fourteenth day, EcN could be determined in all children. The Mutaflor strain was detectable in stools even sixteen days after the last application. 35 These results were corroborated by other studies which included comparison groups. The Mutaflor strain could still be detected in the feces even 6 12 months after the observation period and the application of the Mutaflor Suspension had ceased. 4; 36 Consequently, EcN colonizes the intestines of the newborn. Colonization in premature neonates In premature neonates (birth weight < 2,500 g) EcN was detectable in the stools of all children as early as after three days of treatment, and this remained unaltered until the end of the observation period on Day Results on the colonization of E. coli strain Nissle 1917 in the intestines of infants EcN is detectable in stools as early as on the second or third day EcN colonizes the intestines of infants on a long-term basis and thus protects against intestinal infection right after birth 13

14 Prophylaxis of Colonization with Mutaflor Suspension E. coli strain Nissle 1917 protects against the colonization with hazardous hospital-associated pathogens causing nosocomial infections This is the result of a study which examined the prophylactic effect of Mutaflor on infection and colonization under hospital conditions. Full-term neonates received 1 ml Mutaflor Suspension on five consecutive days. The results were compared with the data of a control group not treated with Mutaflor [Table 4]. Proven antagonism of E. coli strain Nissle 1917 against: Adherent invasive E. coli strains Enteroinvasive E. coli 0143:H- Enteropathogenic E. coli 0112 ab Uropathogenic E. coli 06:K15:H31 Candida albicans Legionella pneumophila strain Corby Bacterial species Citrobacter amalonaticus freundii Controls (N = 20) [%] 5 15 Mutaflor - Group (N = 16) [%] Listeria monocytogenes EGD Proteus vulgaris Salmonella enterica (two strains tested: Serovar Typhimurium C17 and SL 1344) Salmonella enteriditis Enterobacter agglomerans cloacae 10 5 Shigella flexneri strain M9OT Shigella dysenteriae Vibrio cholerae Escherichia coli 85* 100 Klebsiella pneumoniae ssp. ozaenae pneumoniae ssp. pneumoniae Kluyvera ascorbata 5 Yersinia enterocolitica 15 Table 4: EcN protects against hospital-associated pathogens. * A total of 22 different E. coli strains, 30 % of which are hemolysin producers. A variety of undesired pathogenic Enterobacteriaceae, including hemolytic E. coli-bacteria, could only be detected in non-treated neonates. The protective Mutaflor strain was the dominant E. coli strain in all colonized neonates. 4 EcN protects against enteroinvasive pathogenic bacteria and yeasts 16; 17 [Table 5] Yersinia enterocolitica (two strains tested: WA314 and WA-C) Table 5 Colonization prophylaxis in newborns The deliberate colonization with E. coli strain Nissle 1917 (EcN) and its prophylactic effects on infection and contamination with unwanted bacteria were examined in a double-blind placebo-controlled clinical study including 54 newborns ( Mutaflor group: N = 27, placebo group: N = 27). 36 All children were breast-fed in their first week of life. Stool samples were recovered on days 1, 2, 3, 5 and on day 21 as well as six months after birth. Mutaflor was detected in the infant s stools after the second day and remained to be so during the entire study period of six months, involving more than 90 % of the colonized children. Pathogenic and/or potentially pathogenic bacteria were found on the day of birth in both the Mutaflor group and in the placebo group in 19 % of the new- 14

15 Mutaflor Placebo 100 % of children with pathogenic bacteria Day 1 Day 2 Day 3 Day 5 Day 21 6 months Fig. 10: Colonization of the intestines of newborns with pathogenic and potentially pathogenic bacteria in the Mutaflor group and in the placebo group, respectively. 36 borns. As early as on the third day of life, however, the Mutaflor group displayed a significantly lower colonization rate of pathogenic microorganisms (p < 0.003). This advantage of Mutaflor administration increased until the last day of treatment (Day 5; p < 0.001) and remained to be so until 6 month after birth (p < 0.002) [Fig. 10]. In addition, the application of Mutaflor significantly reduced both the spectrum of microbial species and the cell counts of pathogenic and potentially pathogenic microbes (p = 0.008). Mutaflor tolerance was mostly rated as very good or good. Mutaflor improves cellular and humeral immunity of newborns A clinical study with 31 newborns revealed a significant increase of IgA- and IgM-levels in stool filtrates and in the blood serum subsequent to a colonization with E. coli strain Nissle There was no increase of IgG-levels in the blood serum. The response in serum occurred with a delay relative to the local response in the gut. 35 Premature infants treated with EcN (N = 34) displayed an enhanced activity of blood leukocytes in whole blood also in comparison with other types of E. coli. Untreated premature infants (N = 27) did not display this activity enhancement. 37 The immune system of EcN-treated newborns is thus better prepared for warding off infections. It responds quicker to invading bacteria foreign to the body. Mutaflor lowers the risk of infection in preterm infants after antibiotic therapy In certain circumstances, the intensive-medical care of preterm infants requires the rapid application of antibiotic substances. However, there is the risk of spreading antibiotic resistances and damaging the gut microbiota which is in the stage of development. Enterobacteriaceae (Klebsiella and Proteus), hazardous to health, emerged in the colon of 16 premature infants. 87 % of the premature infants had been previously treated with antibiotics. These bacteria disappeared after the oral administration of Mutaflor Suspension in 75 % of the infants treated

16 Colonization prophylaxis with Mutaflor Suspension Overview of health-related and economic benefit of Mutaflor Suspension in newborns: Targeted EcN colonization lowers the risk of infection A targeted intestinal colonization with the E. coli strain Nissle 1917 ( Mutaflor Suspension) prevents the colonization with harmful bacteria causing nosocomial infections as well as potentially pathogenic bacteria from the mother and thus protects against infections The oral application of Mutaflor Suspension prevents and/or reduces the colonization of the infant's intestines with problematic bacteria (colonization prophylaxis). This prophylactic defense against pathogenic bacteria was evidenced even six months after the administration of medication had been terminated (infection protection). stimulates immune defense reestablishes a healthy gut microbiota Improvement of secondary clinical symptoms Children treated with probiotics exhibit less problems during weaning (less flatulence, three-month colics and diarrheas) Irregular stools turn back to normal Advantages for mother and child Disorders affecting the gut microbiota and imbalances of the immune system are trigger factors for the development of atopic diseases. The E. coli strain Nissle 1917 in Mutaflor rebalances the gut microbiota and the immune system Mother and child can often return sooner to their accustomed social surrounding The survival and physical capacities of the newborns is strengthened Very good tolerance Mutaflor Suspension is tolerated very well by newborns Economic efficiency The risk of prolonged hospitalization due to perinatal and postnatal infections can be lowered. This will lessen the financial burden on the health insurances companies. 16

17 Therapy of Inflammatory and Functional Bowel Diseases with Mutaflor V The current marketing authorizations as well as the clinical studies and casuistics all reveal the broad application spectrum of Mutaflor. For you, we have compiled the most important fields of application and study results below. Please refer to the overview on page 22 for more information. Diarrhea The efficacy of Mutaflor Suspension in cases of acute and chronic diarrhea at the age of infants and toddlers was demonstrated in two placebo-controlled multicentric studies (N = 264). The duration of diarrhea was significantly reduced by 2.3 days in cases of acute diarrhea [Fig. 11] and by 3.3 days in cases 39; 40 of protracted diarrhea [Fig. 12]. Chronic constipation Two controlled clinical Mutaflor studies are available for the indication of chronic constipation. As a result of an 8-week study, patients having received Mutaflor had a significantly higher weekly stool frequency than patients who had received the placebo [Fig. 13]. 41 A second study proved that the efficacy of Mutaflor and lactulose was comparable after a treatment period of 12 weeks [Fig. 14] Mutaflor Placebo 8 Acute diarrhea Mutaflor Suspension Time gained: 2.3 days Placebo p = Stool frequency/weeks p < p < Days of treatment 2.5 days 4.8 days Weeks Fig. 13: Chronic constipation, Mutaflor vs. placebo, 8 weeks, N = Fig. 11: Mutaflor Suspension reduces the duration of acute diarrhea by 2.3 days (after Henker, J. et al., 2007). 39 Mutaflor Lactulose Mutaflor Suspension Placebo Mean duration of disease before therapy 5.8 days Time gained: 3.3 days p = Number of patients (%) n/weeks Days of treatment 2.4 days 5.7 days 0 Regular consistency Ease of defecation Side effects Stool frequency 0 Fig. 12: Mutaflor Suspension reduces the duration of protracted diarrhea by 3.3 days (after Henker, J. et al., 2008). 40 Fig. 14: Chronic constipation, Mutaflor vs. laculose, 12 weeks, N =

18 Clinical effects of Mutaflor in cases of inflammatory and functional intestinal disease Ulcerative colitis The influence of Mutaflor on the remission maintenance in patients with ulcerative colitis was examined in three randomized double-blind studies. When administered in a dose of two capsules daily, Mutaflor was proved to be equally effective as mesalazine in this indication. Patients 50 Mutaflor 53 Mesalazine Design/ Duration randomized, double-blind, multicentric, 12 weeks Author/Year Kruis et al., In another study 44, 116 ulcerative colitis patients had already been included in the study during an acute attack and treated with gentamicin 3 x 80 mg/d (Day 1 79) and prednisolone in slowly decreasing doses. The patients received either 2 x 2 capsules of Mutaflor /d or mesalazine 3 x 800 mg/d as test medication. When a state of remission was reached, maintenance therapy proceeded with 2 x 1 capsule of Mutaflor /d or 3 x 400 mg/d mesalazine over a period of 12 months. The median duration of remission amounted to 185 ( Mutaflor ) and/or 175 days, respectively. 57 Mutaflor 59 Mesalazine 162 Mutaflor 165 Mesalazine 52 Mutaflor 11 Mesalazine 46 Mutaflor 11 Placebo randomized, double-blind, 1 year randomized, double-blind, multicentric, 1 year open, 1 year randomized, double-blind, 8 weeks Rembacken et al., Kruis et al., Henker et al., Matthes et al., Probability for maintaining remission (%) Time to relapse (days) Mutaflor Mesalazine Table 6: Clinical studies with Mutaflor on remission maintenance in patients with ulcerative colitis which proved the equivalence of Mutaflor and mesalazine. In the first of these studies, Kruis et al. 43 compared the remission behavior in 103 patients with ulcerative colitis under therapy with two capsules Mutaflor daily versus 3 x 500 mg mesalazine over a period of 12 weeks. No significant differences were determined between both groups with regard to the parameter Clinical Activity Index (CAI), Relapse Rate (16.0 % with Mutaflor versus 11.3 % with mesalazine) and Time-to-Remission (106 ± 5 days with Mutaflor versus 103 ± 4 days with mesalazine). Fig. 15: Mutaflor versus mesalazine in remission maintenance in cases of ulcerative colitis. Kaplan-Meier plot of remission curves within a period of 12 months (after Rembacken et al., 1999). 44 The rectal instillation of E. coli strain Nissle 1917 produces a dose-dependent improvement of the clinical, endoscopic and histological findings of an active mild to moderate distal ulcerative colitis (DAI 4-9 (Disease Activity Index after Sutherland)). This is the result of a per-protocol analysis with 57 patients who received various volumes (10 ml, 20 ml or 40 ml) of a placebo or E. coli strain Nissle 1917 (10 8 CFU/ml) rectally over a maximum of eight weeks. Remission (DAI of 2) was reached in the placebo-group 2/11 (18.2 %), the 18

19 10-ml-E. coli strain Nissle 1917 group 3/11 (27.3 %), the 20-ml-E. coli strain Nissle 1917-group 8/18 (44.4 %) and the 40-ml-E. coli strain Nissle 1917-group 9/17 (52.9%). There were no significant differences between placebo and EcN as far as tolerance is concerned. 47 % 60 Ranking test p = % % for the duration of one year. 222 patients (122 patients with mesalazine, 110 patients with Mutaflor ) completed the study in conformity with the protocol. 38/112 patients in the measalzine group (33.9 %) and 40/110 patients in the Mutaflor group (36.4 %) revealed a relapse [Fig. 17]. The equivalence of both pharmaceuticals was corroborated with a statistical significance of p = 0.003, hence both products were equally effective % 27.3 % 60 Significant equivalence p = in % 0 Placebo 10 ml EcN-Klysma 20 ml EcN-Klysma 40 ml EcN-Klysma % 339 % Fig. 16: Remission rate in cases of distal ulcerative colitis after rectal application of E. coli strain Nissle 1917 or placebo. Per-protocol analysis (N = 57) (modified after Matthes et al., 2010). 47 In the third major study 45, which was carried out in ten European countries, 327 patients with ulcerative colitis in remission could be included and received either 1.5 g mesalazine or 1 x 2 capsules of Mutaflor daily, 20 0 Mutaflor Mesalazine Fig. 17: Relapse rate among patient with ulcerative colitis within one year. Per-protocol analysis (N = 222), (after Kruis et al., 2004) 45 The guideline of the European Crohn's & Colitis Organization (ECCO): E. coli strain Nissle 1917 was acknowledged as an evidence-based medicinal substance belonging to the group of probiotics for the remission maintenance of ulcerative colitis. In addition, the equivalence of Mutaflor and mesalazine was determined with regard to remission maintenance

20 Clinical effects of Mutaflor in cases of inflammatory and functional intestinal diseases Crohn's disease Malchow treated 28 patients with active Crohn's disease of the colon over one year with prednisolone and with an adjuvant therapy of Mutaflor versus placebo. 49 After remission had been reached, less relapses occurred in the patient group treated with Mutaflor than in the control group (33.3 % versus 63.6 %). In addition, the adjuvant administration of Mutaflor elicited a steroid sparing effect. Prednisolone (mg/d) Average prednisolone dose Stratum I (Week 1 53) Mutaflor Placebo N = 28 Irritable bowel syndrome In a multicentric study, 41 patients were treated with Mutaflor over a period of four weeks. 50 Mutaflor therapy produced an improvement of the irritable symptoms in a majority of the patients. In particular, meteorism and the pain symptoms associated with it could be improved and the quality of life thus increased. 78 % of the patients rated the tolerance of Mutaflor therapy as good to very good. Patients with irritable bowel syndrome who had suffered from infection or had been treated with antibiotics before the onset of their disease benefited significantly from a Mutaflor therapy which had lasted for several weeks. 29 Probiotics such as E. coli strain Nissle 1917 are expressly recommended as a therapy option in cases of irritable bowel syndrome by the Guideline of the German Society of Digestive and Metabolic Diseases (DGVS) Duration of therapy (weeks) Intensity Duration Frequency Prednisolone (mg/d) Average prednisolone dose Stratum I (Week 11 53) Mutaflor Placebo Pain (VAS*, mm) p = p = p = Week of therapy Duration of therapy (weeks) Fig. 19: Pain rating (intensity, duration and frequency) of patients in diaries, represented as weekly mean values (after Keller et al., 2010). 50 *VAS = Visual analogue scale. Fig. 18: Mutaflor versus placebo in cases of Crohn's disease Stratum I = colon disease (after Malchow, HA., 1997)

21 Chronic recurrent diarrhea Irritable bowel syndrome Chronic constipation Protracted diarrhea Chronic inflammatory bowel diseases Extraintestinal indications Antibiotic-associated pseudomembranous colitis Intestinal mycoses Diverticulosis Other (e. g. dietary intolerances/malabsorption) Non-ulcer dyspepsia Fig 20: Therapeutic experiences with E. coli strain Nissle 1917 based on results obtained by Krammer et al., Mutaflor in other diseases A prospective post-marketing surveillance study including patients revealed the extraordinary width of the empirical-based therapeutic spectrum and actions of E. coli strain Nissle 1917 ( Mutaflor ). 52 Apart from diarrhea and the irritable bowel syndrome, EcN has also been applied in cases of extraintestinal diseases such as neurodermitis or urinary tract infections [Fig. 20.] The study results obtained with Mutaflor are not applicable to other probiotics because of the strain-specific properties of E. coli strain Nissle

22 VI Application and Dosage Recommendations Indications and therapeutic-medical experiences made with Mutaflor According to current marketing authorizations ( Mutaflor, Mutaflor Suspension) Ulcerative colitis in the stage of remission Chronic obstipation Colonization prophylaxis in premature and full-term neonates Enhancement of postnatal immune competence in premature and fullterm neonates Diarrhea in newborns, infants and children Diarrhea in newborns, infants and children under gastric tube feeding According to clinical studies Crohn's disease Pouchitis Collagenous colitis Antibiotic-associated colitis/ pseudo-membranous colitis Irritable bowel syndrome Diverticulosis Polymorphous photodermatosis According to experiential reports/ casuistics Non-ulcer dyspepsia Dietary intolerance/malabsorption Halitosis Susceptibility to infection Mycoses of the gastrointestinal tract Other, e. g. neurodermitis, reactive arthritis, radiation enteritis/radiation colitis, recurrent urinary tract infections Table 7 Overview of the pertinent studies 39; 40 Diarrhea Henker et al. 2007, 2008 Irritable bowel syndrome Krammer et al , Plassmann , Keller et al , Kruis et al Antibiotic-associated colitis/pseudomembranous colitis Goerg u. Schlörer , Goerg et al Ulcerative colitis Kruis et al , Rembacken et al , Kruis et al , Henker et al , Matthes et al Crohn's disease Malchow Pouchitis Kuzela et al Collagenous colitis Tromm et al Chronic obstipation Bruckschen et al , Möllenbrink et al Diverticulosis of the colon Frič et al Colonization prophylaxis and enhancement of immune competence in full-term and premature neonates Lodinová-Žádníková et al , Lodinová-Žádníková und Sonnenborn , Schröder , Cukrowska et al Intestinal-dependent halitosis Henker et al Polymorphous photodermatosis Wurzel Table 8 22

23 E. coli strain Nissle 1917 is antibiotic-sensitive As a bacterial isolate from the pre-antibiotic era, E. coli strain Nissle 1917 (Mutaflor ) is sensitive to all antibiotics which are predominately directed against Gram-negative bacteria, e. g. coli bacteria. A certain loss of Mutaflor efficacy must be taken into account. Nevertheless, a protective Mutaflor therapy should be initiated in addition to antibiotic therapy. Antibiotic sensitivity of E. coli strain Nissle 1917 Amikacin Gentamicin Amoxicillin/Clavulanic acid Imipenem Ampicillin Latamoxef Azlocillin Mezlocilin Cefaclor Nitrofurantoin Cefazolin Norfloxacin Cefoperazone Pipemidic acid Cefotaxime Piperacillin Ceftriaxone Tetracycline Cephalotin Ticarcillin Chloramphenicol Tobramycin Ciprofloxacin Trimethoprim Doxycycline Sulfamethoxazole Table 9 Combination with antibiotics Mutaflor is not sensitive to antibiotics which are predominately directed against Gram-positive bacteria. That is why EcN may be combined with these antibiotics. Dosage recommendation for Mutaflor One hard-capsule Mutaflor contains Escherichia coli strain Nissle 1917 as its active pharmaceutical ingredient (API), equivalent to x 10 9 viable cells (CFU). Adolescents and adults take 1 capsule Mutaflor daily from day 1 to 4, then 2 capsules daily. In case of persistent constipation, a daily intake of up to 4 capsules Mutaflor might be reasonable. Patients should take Mutaflor as a long-term therapy in case of ulcerative colitis to maintain remission. The standard dose should be taken unchewed and with plenty of liquid during a meal, preferably breakfast. In Germany (country of Origin): Reimbursable for treatment of ulcerative colitis in the phase of remission in case of mesalazine intolerance (BAnZ. No. 77 (p. 8905) from Apr. 23, 2004) Potentially occurring flatulence is invariably a sign of a too high dose application and will mostly disappear when the dose is reduced. The daily dose of several capsules must not be taken at one time, but may be distributed over the day and taken with regular meals. Natural antibiotic resistance of E. coli strain Nissle 1917 Cefsulodin Quinupristin/Dalfopristin Clindamycin Rifampin Erythromycin Teicoplanin Metronidazole Vancomycin Penicillin G Table 10 23

24 Application and Dosage Recommendations Applications and dose recommendations Pharmaceutical form of Mutaflor Mutaflor is available as capsules. These capsules possess an enteric coating. They only open in the terminal ileum or at the beginning of the colon [Fig. 21] where they release the active substance. Dose recommendation for Mutaflor Suspension 1 ml Mutaflor Suspension contains: bacterial culture of Escherichia coli strain Nissle 1917 equivalent to 10 8 viable cells (CFU). For colonization prophylaxis and enhancement of immune competence, Mutaflor should be applied as near as possible to the date of birth, for example, in the scope of a U1 examination. On the following days, application is recommended in the morning before first drinking. Proximal ileum: closed capsule Pack sizes Mutaflor 20 hard capsules 50 hard capsules 100 hard capsules Lyophilized viable Escherichia coli strain Nissle 1917 Hard-gelatin capsule Enteric coating Terminal ileum: open capsule Fig. 21: X-ray images of an enteric coated Mutaflor capsule filled with iron spherules Mutaflor Suspension is in general dosed as follows Diarrhea: Newborns, infants and children: 1 3 x 1 ml Mutaflor Suspension per day. Duration of application: Up to 5 days in cases of acute diarrhea; up to 15 days in cases of longer persisting diarrhea. Diarrhea under tube-feeding: Newborns, infants and children: 1 x 1 5 ml Mutaflor Suspension per day. Duration of application: up to 5 days per period of diarrhea Therapy should be continued for several days after initial treatment success. Colonization prophylaxis: Premature and full-term neonates: 1 x 1 ml per day Enhancement of postnatal immune competence: Premature and full-term neonates: First week of life: 1 x 1 ml per day; In Germany (country of Origin): Reimbursable for treatment of diarrhea in newborns and infants in addition to rehydration measures (BAnZ. No. 36 (p. 927) from Mar. 4, 2011) Second to third week of life: 1 ml per day, 3-times a week 24

25 Pharmaceutical Form of Mutaflor Suspension Mutaflor Suspension is bottled in single-dose containers which must be shaken prior to use. As opposed to the capsules, the suspension possesses no enteric coating. For this reason, application to newborns shall proceeds before the first drinking and to infants during meals. Mutaflor Suspension can be dripped directly into the mouth. Mutaflor Suspension may also be applied by a gastric or intestinal tube. Pack sizes 5 x 1 ml suspension 10 x 1 ml suspension 25 x 1 ml suspension 5 x 5 ml suspension 25 x 5 ml suspension Storage information The probiotic medicinal product Mutaflor contains viable bacteria in a high concentration. For this reason, it should be stored in a refrigerator at a temperature ranging between +2 C and +8 C. An interruption of chilling for a few days will have normally no influence on the number of viable cells. In case of a prolonged heat exposure, however, the bacterial cells will to some extent lose their viability. No toxic substances will be develop. 25

26 VII Medicinal Product Safety A probiotic which is registered as a medicinal product must comply with high standards. As opposed to dietary supplements or balanced diets, evidence of clinical efficacy must be submitted for defined medical indications and documents on efficacy and action mechanisms are required. In addition, the compliance with special product quality and safety criteria must be guaranteed (e. g. microbiological purity and defined viable cell counts as well as the non-pathogenicity and genetic stability of the therapeutically applied microorganism). 11 Mutaflor fulfills all modern-day safety requirements of a microbial medicinal product comprehensively and may therefore be applied even in high doses to patients of any age group and to pregnant and breast-feeding women as well. E. coli stain Nissle 1917 is deposited in the German Collection of Microorganisms and Cell Cultures in Brunswick under the designation of E. coli DMS 6601 and it is the probably best investigated thera peutically applied E. coli strain worldwide. It non-pathogenicity has been elucidated down to the level of molecular genetics. 61; 62 The progress made in deciphering the genome of EcN has resulted in the development of a specific DNA-based PCR identification method, allowing a direct determination of Mutaflor in patient stools samples. 63 The genome 64 of EcN has been completely sequenced by now. The comparative sequence analysis indicates which DNA segments encode the known properties of the strain and defines where in the genome yet other genes responsible for hitherto unknown characteristics are localized. is not invasive. The pathogenicity features typical of pathogenic E. coli-bacteria do not exist in the EcN strain. The Central Committee for Biological Safety (ZKBS) therefore assigned EcN to risk group 1 (safe microorganisms). Strain characteristics of E. coli strain Nissle 1917 Serological O6 : K5 : H1 features Biochemical/ Multiple fermentation tube technique microbiological (21 strain-specific metabolic features reactions) Cellular protein spectrum Membrane protein profile Structure of lipopolysaccharides Molecular genetic Genome and plasmid analyses, features Specific PCR detection Table 11 Safety aspects pertaining to E. coli strain Nissle 1917 Genetic stability production of enterotoxins production of cytotoxins production of hemolysins pathogenic adhesion factors invasiveness No immunotoxicity serum resistance (no hazard of sepsis) E. coli strain Nissle 1917 is non-pathogenic The comprehensive research results prove that E. coli strain Nissle 1917 in Mutaflor does not have any pathogenic properties. It produces neither hemolysin nor other toxins, displays no mannose-resistant hemagglutination; it is not serum-resistant, possesses no typical features of uropathogenicity and Table 12 uropathogenicity toxicity in germ-free and conventionally kept animals 26

27 Unambiguous molecular biological identification EcN can be identified by an analysis of its strain-specific metabolic reactions, its cell protein patterns or cell wall structures, by a differential molecular analysis of the bacterial DNA, or by means of strain-specific DNA probes and subsequent polymerase chain reaction (PCR) [Fig. 22]. 63 Fig. 22: Strain-specific PCR analysis of EcN. Lane 1: DNA length standard, Lanes 2 7: EcN cultures. E. coli strain Nissle 1917 does not possess any antibiotic resistance plasmids What is very important nowadays: As an isolate of the pre-antibiotic era, E. coli strain Nissle 1917 does not possess any antibiotic-resistance plasmids. Furthermore, EcN is genetically stable and a poor recipient of foreign DNA in the form of antibiotic resistance and other promiscuous plasmids. 27

28 VIII References 1) Escherich T. Die Darmbacterien des Neugeborenen und Säuglings. Fortschr Med 1885;3: , ) Escherich T. Die Darmbakterien des Säuglings und ihre Beziehung zur Physiologie der Verdauung. Stuttgart: Verlag Ferdinand Enke, ) Sonnenborn U, Stobernack HP, Proppert Y. Die Entwicklung der aeroben Darmflora bei Neugeborenen. Fortschr Med 1990;108: ) Schröder H. Entwicklung der aeroben Darmflora bei Neugeborenen nach Kolonisierung mit dem E. coli-stamm Nissle Der Kinderarzt 1992; 23(10): ) Hoogkamp-Korstanje JA et al. Composition and ecology of the human intestinal flora. Antonie Van Leeuwenhoek 1979; 45: ) Schulze J et al. Probiotika Mikroökologie, Mikrobiologie, Qualität, Sicherheit und gesundheitliche Effekte. Hippokrates, Stuttgart, ISBN , ) Bischoff SC. (Hrsg). Probiotika, Präbiotika und Synbiotika. Georg Thieme Verlag, Stuttgart, ISBN , ) Rasche C et al. Differential immunomodulating effects of inactivated probiotic bacteria on the allergic immune response. Acta Derm Venereol 2007; 87: ) Blum G et al. Properties of Escherichia coli strains of serotype O 6. Infection 1995; 23: ) Sonnenborn U, Schulze J. The non-pathogenic Escherichia coli strain Nissle 1917 features of a versatile probiotic. Microbial Ecol Health Dis 2009; 21: ) Schiemann M et al. 125 Jahre E. coli Bedeutung in Forschung und Medizin. Alfred-Nissle -Gesellschaft (Hrsg.) Hagen, ISBN , ) Troge A et al. More than a marine propeller the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus. Int J Med Microbiol 2012; 302: ) Schlee M et al. Induction of human beta-defensin 2 by the probiotic Escherichia coli Nissle 1917 is mediated through flagellin.infect Immun 2007; 75(5): ) Patzer S I et al. The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN. Microbiology 2003; 149: ) Oelschlaeger TA et al. Inhibition of Salmonella typhimurium invasion into intestinal cells by the probiotic E. coli strain Nissle Gastroenterology 2001; 120: A ) Boudeau J et al. Inhibitory effect of probiotic Escherichia coli strain Nissle 1917 on adhesion to and invasion of intestinal epithelial cells by adherent-invasive E. coli strains isolated from patients with Crohn s disease. Aliment Pharmacol Ther 2003; 18: ) Altenhoefer A et al. The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens. FEMS Immunol Med Microbiol 2004; 40: ) Leatham MP et al. Precolonized human commensal Escherichia coli strains serve as a barrier to E. coli O157:H7 growth in the streptomycin-treated mouse intestine. Infect Immun 2009; 77: ) Reissbrodt R et al. Inhibition of growth of Shiga toxin-producing Escherichia coli by non pathogenic Escherichia coli. FEMS Microbiol Lett 2009; 290: ) Kleta S et al. Role of F1C fimbriae, flagella, and secreted bacterial components in the inhibitory effect of probiotic Escherichia coli Nissle 1917 on atypical enteropathogenic E. coli infection. Infect Immun 2014; 82(5): ) Hacker J, Heesemann J. Molekulare Infektionsbiologie: Interaktionen zwischen Mikroorganismen und Zellen. Berlin: Spektrum Akademischer Verlag, ) Cichon C et al. DNA-Microarray-based comparison of cellular responses in polarized T84 epithelial cells triggered by probiotics: E. coli Nissle 1917 (EcN) and Lactobacillus aci dophilus PZ1041. Gastroenterology 2004; 126(4): A ) Schultz M et al. Preventive effects of Escherichia coli strain Nissle 1917 on acute and chronic intestinal inflammation in two different murine models of colitis. Clin Diagn Lab Immunol 2004; 11(2): ) Wehkamp J et al. NF-kappaB- and AP-1-mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium. Infect Immun 2004; 72(10): ) Lata J et al. The effect of probiotics on gut flora, level of endotoxin and Child-Pugh score in cirrhotic patients: results of a double-blind randomized study. Eur J Gastroenterol Hepatol 2007; 19: ) Zyrek AA et al. Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCz redistribution resulting in tight junction and epithelial barrier repair. Cell Microbiol 2007; 9: ) Ukena SN et al. Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity. PLOS ONE 2007; 12: e1308; ) Rund SA et al. Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7. Int J Med Microbiol 2013; 303: ) Kruis W et al., A double-blind placebo-controlled trial to study therapeutic effects of probiotic Escherichia coli Nissle 1917 in subgroups of patients with irritable bowel syndrome. Int J Colorectal Dis 2012; 27(4): ) Bär F et al. Cell-free supernatants of Escherichia coli Nissle 1917 modulate human colonic motility: evidence from an in vitro organ bath study. Neurogastroenterol Motil 2009; 21: 559-e17. 31) Sonnenborn U et al. Antimutagenic activity of the probiotic E. coli strain Nissle J Crohns Colitis 2009; 3: S ) Otte JM et al. Probiotics regulate the expression of COX-2 in intestinal epithelial cells. Nutr Cancer 2009; 61(1): ) Hockertz S. Immunmodulierende Wirkung von abgetöteten apathogenen Escherichia coli Stamm Nissle 1917, auf das Makrophagensystem. Arzneim-Forsch/Drug Res 1991;41: ) Hockertz S. Steigerung der körpereigenen Abwehr gegen Bakterien- und Pilz infektionen bei Mäusen nach Vorbehandlung mit dem apathogenen Escherichia coli-stamm Nissle Arzneim.-Forsch./Drug Res. 1997;47(I)(6): ) Lodinová-Žádníková R et al. Local and serum antibody response in fullterm and prema ture infants after artificial colonization of the intestine with E. coli strain Nissle 1917 ( Mutaflor). Pediatr Allergy Immunol 1992; 3: ) Lodinová-Žádníková R, Sonnenborn U. Effect of preventive administration of a nonpathogenic Escherichia coli strain on the colonization of the intestine with microbial pathogens in newborn infants. Biol Neonate 1997; 71:

29 37) Cukrowska B et al. Specific proliferative and antibody responses of premature infants to intestinal colonization with nonpathogenic probiotic E. coli strain Nissle Scand J Immunol 2002; 55: ) Lodinová-Žádníková 1991, persönliche Mitteilung 39) Henker J et al. The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhoea in infants and toddlers. Eur J Pediatr 2007; 166(4): ) Henker J et al. Probiotic Escherichia coli Nissle 1917 versus placebo for treating diarrhea of greater than 4 days duration in infants and toddlers. Pediatr Infect Dis J 2008; 27(6): ) Möllenbrink M, Bruckschen E. Behandlung der chronischen Obstipation mit physiolo gischen Esche richia-coli-bakterien. Med Klin 1994; 89: ) Bruckschen E, Horosiewicz H. Chronische Obstipation, Vergleich von mikrobiologischer Therapie und Lactulose. Münch Med Wochenschr 1994;16: ) Kruis W et al. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 1997; 11: ) Rembacken BJ et al. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 1999; 354: ) Kruis W, et al. Maintaining remission of ulcerative colitis with the pro biotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 2004; 53: ) Henker J et al. Probiotic Escherichia coli Nissle 1917 (EcN) for successful remission maintenance of ulcerative Colitis in children and adolescents: an open-label pilot study. Z Gastroenterol 2008; 46: ) Matthes H et al. Clinical trial: probiotic treatment of acute distal ulcerative colitis with rectally administered Escherichia coli Nissle 1917 (EcN). BMC Complement Altern Med 2010;10:13. 48) Travis SPL et al. European evidence-based Consensus on the management of ulcerative colitis: Current management. Journal of Crohn s and Colitis 2008; 2(1): ) Malchow HA. Crohn s Disease and E. coli. J Clin Gastro enterol 1997; 25: ) Keller J et al. Reizdarm-Patienten mit Meteorismus als dominantem Symptom profitieren von einer Therapie mit E. coli Nissle 1917 Ergebnisse einer Pilotstudie. Z Gastroenterol 2010; 48: ) Layer P. S3-Leitlinie Reizdarmsyndrom: Definition, Pathophysiologie, Diagnostik und Therapie. Z Gastroenterol 2011; 49: ) Krammer H et al. Probiotische Arzneimitteltherapie mit E. coli Stamm Nissle 1917 (EcN): Ergebnisse einer prospektiven Datenerhebung mit 3807 Patienten. Z Gastroenterol 2006; 44: ) Plassmann D, Schulte-Witte H. Treatment of irritable bowel syndrome with Escherichia coli strain Nissle 1917 (EcN): a retrospective survey. Med Klin (Munich) 2007; 102(11): ) Goerg KJ, Schlörer E. Probiotic therapy of pseudomembranous colitis. Combination of intestinal lavage and oral administration of Escherichia coli. Dtsch Med Wochenschr 1998;123(43): ) Goerg KJ et al. A new approach in pseudomembranous colitis: probiotic Escherichia coli Nissle 1917 after intestinal lavage. Eur J Gastroenterol Hepatol 2008; 20(2): ) Kuzela L et al. Induction and maintenance of remission with nonpathogenic Escherichia coli in patients with pouchitis. Am J Gastroenterol 2001; 96: ) Tromm A et al. The probiotic E. coli strain Nissle 1917 for the treatment of collagenous colitis: First results of an open-labelled trial Z. Gastroenterol 2004; ) Fricˇ P et al. The effect of non-pathogenic Escherichia coli in symptomatic uncomplicated diverticular disease of the colon. Eur J Gastroenterol Hepatol 2003, 15; ) Henker J et al. Successful treatment of gut-caused halitosis with a suspension of living non-pathogenic Escherichia coli bacteria-a case report. Eur J Pediatr 2001; 160: ) Wurzel R. Prophylaxe der polymorphen Lichtdermatose. Akt Dermatol 1999; 25: ) Grozdanov L et al. A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle J Bacteriol 2002; 184: ) Grozdanov L et al. Analysis of the Genome Structure of the Nonpathogenic Probiotic Escherichia coli Strain Nissle 1917 J Bacteriol. 2004; 186: ) Blum-Oehler G et al. Development of strain-specific PCR reactions for the detection of the probiotic Escherichia coli strain Nissle 1917 in fecal samples. Res Microbiol 2003; 154: ) Reister M et al. Complete genome sequence of the Gram-negative probiotic Escherichia coli strain Nissle J Biotechnol 2014;187: Further reading: In addition to the sources mentioned, there are numerous publications, especially with reference to preclinical trials. If you wish to learn more about a particular subject please let us know: office@ardeypharm.de 29

30 Mutaflor and Mutaflor Suspension comprehensively fulfill all microbiological and molecular biological safety requirements. Efficacy Efficacy in defined indications evidenced by controlled clinical studies Mechanism of action Suppression of growth and colonization of undesired microorganisms Contribution to optimizing the intestinal milieu and luminal metabolism Communication with the intestinal epithelium Colonization ability Product quality Microbiological purity Defined number of viable cells Exact identification and differentiation from closely related microorganisms Viability in the gastrointestinal tract Safety Non-pathogenicity Genetic stability 30

31 Mutaflor Active Pharmaceutical Ingredient: Escherichia coli strain Nissle 1917 Qualitative and quantitative composition: 1 enteric coated hard capsule Mutaflor contains Escherichia coli strain Nissle 1917 as the pharmaceutically active substance, equivalent to x 10 9 viable cells (CFU). Excipients: maltodextrin, talcum powder, polymethyl acrylic acid co-methyl methacrylate copolymer (1:1), Macrogol, (4000), triethyl citrate, glycerol 85%, titanium oxide, iron-(iii) oxide, gelatin, bleached wax, carnauba wax, shellac, purified water. Therapeutic indications: Ulcerative colitis in the emission stage, chronic obstipation. Contraindications: Hypersensitivity to any constituent of the product. Side effects: Initial flatulence is of often occurrence. Alterations in the consistency or frequency of stools, abdominal pain, stomach rumble, meteorism, nausea or vomiting were rarely observed. Skin efflorescences, erythemas or skin scaling were observed very rarely. Headaches were very rarely observed. Warning: Store at 2 to 8 C in the refrigerator! Mutaflor Suspension Active Pharmaceutical Ingredient: Escherichia coli strain Nissle 1917 Qualitative and quantitative composition: 1 ml suspension contains: bacterial culture with Escherichia coli strain Nissle 1917, equivalent to 108 viable cells (CFU). Excipients: purified water, sodium chloride, potassium chloride, magnesium sulfate heptahydrate, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium hydroxide solution 32%. Therapeutic indications: Diarrhea in newborns, infants and children. Diarrhea in newborns, infants and children under tube feeding, colonization prophylaxis in premature and full-term neonates, enhancement of postnatal immune competence in premature and full-term neonates. Contraindications: Hypersensitivity to any constituent of the product. Side effects: Initial flatulence has been observed in very rare cases and was invariably a sign of a too high dose. It disappears when the dose is reduced. Furthermore, diarrhea, vomiting or abdominal pain were rarely observed. Very rare cases of urticaria or allergic reactions were observed. Single cases of sepsis were reported to have occurred in very premature neonates. The incidence rate cannot be estimated on account of the available data. Warning: Store at 2 to 8 C in the refrigerator! Applicable after breaking open the 5 ml-ampoule: 5 days. 31