The non-pathogenic Escherichia coli strain Nissle 1917 features of a versatile probiotic

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1 Microbial Ecology in Health and Disease ISSN: (Print) (Online) Journal homepage: The non-pathogenic Escherichia coli strain Nissle 1917 features of a versatile probiotic Ulrich Sonnenborn & Jürgen Schulze To cite this article: Ulrich Sonnenborn & Jürgen Schulze (29) The non-pathogenic Escherichia coli strain Nissle 1917 features of a versatile probiotic, Microbial Ecology in Health and Disease, 21:3-4, , DOI: 1.319/ To link to this article: Informa UK Ltd Published online: 26 Dec 29. Submit your article to this journal Article views: 111 View related articles Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 2 January 218, At: 7:17

2 Microbial Ecology in Health and Disease. 29; 21: REVIEW ARTICLE The non-pathogenic Escherichia coli strain Nissle 1917 features of a versatile probiotic ULRICH SONNENBORN 1 & JÜRGEN SCHULZE 2 1 Department of Biological Research and 2 Department of Medicine, Ardeypharm GmbH, Herdecke, Germany Abstract Probiotics are non-pathogenic living microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Probiotic microorganisms most often are constituents of special foodstuffs, such as fermented milk products or food supplements. They are also employed as feed additives in livestock breeding and as active components of medical remedies in human and veterinary medicine. In the food industry, probiotics traditionally are lactic acid bacteria (LAB), especially lactobacilli and lactococci, although bifidobacteria and strepcococci (enterococci) are also used. In livestock breeding and medicine, besides lactic acid bacteria other non-pathogenic microorganisms with health-promoting characteristics, e.g. certain strains of yeast ( Saccharomyces boulardii ) and Escherichia coli (E. coli Nissle 1917), are also applied. This review focuses on the probiotic E. coli strain Nissle 1917 (EcN), its origin and medical history, microbiology, genetics, biological activities, safety, and toxicological aspects. Furthermore, clinical trials performed with this special E. coli strain will be summarized. The EcN strain was detected and isolated by Alfred Nissle in 1917 because of its antagonistic activity against some pathogenic enterobacteria. With respect to its metabolic capacities, EcN is a typical E. coli strain. It is a non-pathogenic member of the Escherichia coli family, because it does does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic, and is rapidly killed by non-specific defense factors of blood serum. Besides this, EcN has some special characteristics that distinguish it from other pathogenic and non-pathogenic E. coli strains and are thought to be important for its probiotic activities. Thus, EcN carries so-called genomic islands (GEIs), integrated into its chromosome. On these GEIs, gene clusters coding for several fitness factors are present, e.g. genes for the production of microcins which inhibit the growth of other enterobacteria. Another strain-specific feature of EcN is a special lipopolysaccharide (LPS) in its outer cell membrane, being responsible for the fact that EcN exhibits immunomodulating properties without showing immunotoxic effects. The immunomodulating properties mainly focus on anti-inflammatory activities that are important for the clinical efficacy in remission maintenance of ulcerative colitis. Also of importance is the bacterial epithelial crosstalk between EcN and the intestinal epithelial cells, leading to a strengthening of the epithelial barrier and the curing of leaky gut phenomena. The restoration of a disturbed gut barrier by EcN is thought to be due to the stimulation of epithelial defensin production as well as to a sealing effect on the tight junctions of the enterocytes. EcN has also been shown to induce the development of the gut immune system in animal models and human newborns. In addition, it has been found that products of EcN metabolism, probably acetic acid, promote colonic motility that might be helpful for therapeutic application in chronically constipated patients. Randomized controlled clinical trials (RCTs) have shown EcN to be therapeutically effective in rather diverse indications, such as ulcerative colitis, chronic constipation, and acute and protracted diarrhea. Key words: EcN, medical history, microbiology, genetics, biological activities, safety, toxicological aspects, clinical trials Introduction Microbial ecology of the gut The intestinal ecosystem has been described as a precarious alliance among epithelium, immunity and microbiota (1) and, under normal conditions, the commensal microorganisms indeed fulfill a wide array of beneficial tasks for the host, including protection from pathogenic microorganisms and modulatory effects on the development and functions of the gastrointestinal tract and the gut immune system (2 7). Important functions of the indigenous intestinal microbiota are given in Table I. Present address: Alice-Bloch-Str. 7, D Nuthetal, Germany. JuR.Schulze@t-online.de Correspondence: Dr Ulrich Sonnenborn, Department of Biological Research, Ardeypharm GmbH, Loerfeldstr. 2, D Herdecke, Germany. Fax: sonnenborn@ardeypharm.de (Received 2 September 29; accepted 22 October 29) ISSN 891-6X print/issn online 29 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS) DOI: 1.319/

3 Table I. Important functions of the intestinal microbiota. Barrier function ( colonization resistance ) Modulation of the local immune system (GALT) Intraluminal metabolism Support of energy metabolism and stimulation of blood flow of the colonic mucosa Stimulation of gut motility Preparation and maintenance of the specific colonic milieu Adjustment of bacterial translocation Support of vitamin supply GALT, gut-associated lymphoid tissue. A breakdown of the microecological balance in the gut may be caused by different exogenous and endogenous factors and will result in changes of composition, numbers, and activities of indigenous microbial communities. This may be followed by destruction of the physiological barriers of the body, enhanced epithelial irritation, inflammation and permeability, and bad consequences for the health of the host organism (1,3,8 13). Intestinal dysfunctions and diseases that are associated with or caused by disturbances of the indigenous microbiota are summarized in Table II. For a long period of time, roughly for the second half of the last century, the use of antibiotics has generally been recommended in human medicine for changing an unwelcome microbial colonization of distinct sites of the body, i.e. in cases of diseases caused by infectious microorganisms. However, the increasing recognition of adverse effects and the development and spread of resistance to a wide range of antibiotics by a growing number of Table II. Gastrointestinal diseases associated with disturbances of the intestinal microbiota. Diseases Infectious diseases Functional diseases Inflammatory bowel diseases (IBD) Diseases associated with morphological or anatomical changes Diseases caused by immunological disturbances Iatrogenic diseases Examples Cholera, salmonellosis, shigellosis, infections by Campylobacter, pathogenic E. coli (ETEC, EPEC, EHEC, etc.) Irritable bowel syndrome (IBS), non-ulcer dyspepsia, habitual constipation Ulcerative colitis, Crohn s disease, collagenous colitis, Whipple s disease Diverticulosis (cave: diverticulitis), strictures, blind loops, carcinoma Celiac disease, food allergy Diarrhea/functional diseases after treatment with antibiotics, chemotherapeutic agents or radiation ETEC, enterotoxigenic E. coli; EPEC, enteropathogenic E. coli; EHEC, enterohemorrhagic E. coli. E. coli strain Nissle 1917 as probiotic 123 pathogens (14 18) has led to the renaissance of a therapeutic concept from the pre-antibiotic era: the prevention or treatment of specific diseases with living non-pathogenic microorganisms, i.e. probiotics. In recent years our knowledge concerning the regulatory processes within the gut ecosystem in health and disease has grown enormously (1,3 5,7,13,19). The knowledge of particular characteristics and activities of members of the indigenous intestinal microbiota and their interactions with the host is fundamental to the application of living microorganisms as health-promoting or therapeutic agents (12,19 25). The term probiotics development of definitions Today, probiotics are defined as non-pathogenic living microorganisms which, when administered in adequate amounts, confer a health benefit on the host. This definition was coined by an expert committee of a joint FAO/WHO meeting on probiotics in 21 (26). The word probiotic has been derived from the Greek language and can roughly be translated into for life. According to Vergin (1954) (27), in Germany the term Probiotika was originally coined by the physician and dietician Werner Kollath shortly after the Second World War, meaning food ingredients with health-promoting characteristics beyond their nutritional value. Later and independently, the term probiotic was introduced into the international literature by Lilly and Stillwell (1965) (28), saying that a probiotic is a microbial substance that is produced by one microorganism and has a growth-promoting effect on another microorganism, thus being the exact opposite of an antibiotic. However, this definition did not gain wide acceptance and failed to survive. Since that time, the meaning of the term probiotic has changed considerably. Parker (1974) (29) was the first to use the word probiotic in a sense that is very similar to today s use. He adopted the term to describe organisms and substances which contribute to intestinal microbial balance. This definition associated the use of probiotics with an effect on gut microecology. Since Parker s definition would also include antibiotics ( substances ), Fuller in 1989 redefined a probiotic as a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance (3). In 1995, a workshop hosted by the Lactic Acid Bacteria Industrial Platform (LABIP) and sponsored by the European Community issued a consensus definition of the term probiotics, saying oral probiotics are living micro-organisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition (31). Accordingly, probiotics may be either consumed as

4 124 U. Sonnenborn et al. food components or applied as non-food preparations. The latest definition of the FAO/WHO expert committee (see above) broadened the meaning by omitting the relation to solely oral administration and intestinal effects as well as to nutrition. According to this definition, also microbial preparations for medical use and for extraintestinal, e.g. vaginal, applications can be termed probiotics, provided that they contain living microorganisms and confer a health benefit on the host. For microorganisms with specific therapeutic properties the term biotherapeutic agent (instead of probiotic) has been proposed by McFarland and colleagues (32,33). This may be helpful to differentiate between food and feed probiotics and pharmaceutical probiotics, but has not gained broader acceptance in the scientific and medical literature, mainly because this term is close to the terms biologicals or biological agents, which include native and recombinant biological preparations, such as vaccines, immunomodulators, interleukins, growth hormones, specific antibodies, and others (34 37). The growing scientific and medical interest in probiotics is reflected by the steadily increasing number of publications on this topic, as shown in Figure 1. Application of probiotics Probiotic microorganisms are often found as food ingredients, e.g. in fermented milk products and food supplements, but are also employed as feed additives Year in livestock breeding and as active components of medical remedies in human and veterinary medicine [19,38 42]. Usually, especially in the food industry, probiotics mostly are lactic acid bacteria (LAB), such as lactobacilli, lactococci, bifidobacteria, and streptococci (enterococci) (43). However, other nonpathogenic microorganisms, e.g. certain yeast strains ( Saccharomyces boulardii ) and Escherichia coli strains, like E. coli Nissle 1917 (EcN), are also used, not in food processing, but rather in human and veterinary medicine (19,21,33,44 47). Origin and medical history of E. coli strain Nissle 1917 E. coli strain Nissle 1917 (EcN) is the active component of the pharmaceutical preparation Mutaflor, a microbial drug licensed for use in human medicine in Germany and some other European countries today. This drug has been traditionally used since 1917 (48,49) to treat various diseases and dysfunctions of the intestinal tract (19,21,47,5 52). The Mutaflor story began in the early years of the last century when the physician and bacteriologist Alfred Nissle (Figure 2) of Freiburg, Germany, screened human intestinal E. coli strains for growthinhibiting (antagonistic) activity against Salmonella, Shigella, and other enteropathogens. Searching for a novel therapeutic approach to combat virulent enterobacteria, Nissle recognized that some fecal E. coli isolates showed antagonistic action against these Search term "probiotic" Search term "probiotic" limited to article type "clinical trials" Number of publications Figure 1. The increase in basic scientific and clinical publications on probiotics during the years , indicating the growing interest of the scientific and the medical community in this topic. The black bars (and the corresponding numbers on top of the bars) represent the total numbers of scientific and clinical publications in the respective year. The inserted open bars (and the corresponding numbers) represent the numbers of published clinical trials with probiotics. Source: National Library of Medicine Database MEDLINE; search and graphics performed by M. Schiemann, Herdecke, Germany.

5 E. coli strain Nissle 1917 as probiotic 125 deposit rules at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) (DSMZ, Braunschweig) as E. coli DSM 661.] In the pre-antibiotic era, it was first used as an anti-infective agent against infectious diarrhea and its clinical consequences, later on, non-infectious gastrointestinal disturbances and diseases, such as chronic constipation or inflammatory bowel diseases, have also been treated. Great effort has been made in the past two decades to elucidate in detail the microbiological characteristics and the molecular genetic background of EcN. At the same time its toxicological profile and its pharmacological and immunological modes of action, as well as its clinical efficacy for the treatment of specific intestinal diseases and dysfunctions have been studied extensively. Figure 2. Photographic portrait of Professor Alfred Nissle MD ( ), discoverer of the antagonistic action of certain commensal E. coli strains against enteropathogens. In 1917, Alfred Nissle isolated the specific E. coli strain, now called E. coli Nissle 1917 (EcN), from the feces of a healthy young man (48,49). Photograph courtesy of Alfred-Nissle-Gesellschaft, Hagen, Germany. pathogens in vitro, while others failed to inhibit their growth. This led Nissle to the idea that these antagonistically active E. coli strains might be useful as therapeutics in diarrheal diseases. Nissle isolated the EcN strain in 1917 during the First World War from the feces of a member of the armed services who took part in the military campaign on the Balkan peninsula. In contrast to his companions, this soldier did not develop infectious diarrhea when staying in the region of Dobrudja, which was heavily contaminated with enteropathogens at that time (49). Nissle assumed that this soldier harbored a strong, antagonistically active E. coli strain in his gut that had protected him from infection. This was in fact the case, and Nissle after performing laboratory tests and some self-experiments with this E. coli isolate introduced the EcN strain into medical practice. From the beginning, the Mutaflor preparation contained (and still contains) the Nissle strain in viable form. [Note that, today, E. coli Nissle 1917 (EcN) is deposited as an industrially used strain under patent Microbiological properties and phenotypic strain characteristics General properties The E. coli strain Nissle 1917 has been thoroughly analyzed by means of microbiological, biochemical, and molecular genetic methods (53 56). Its basic characteristics are shown in Table III. The strain does not exhibit any virulence factors (see below), but has gene clusters located on genomic islands (GEIs) on its chromosome responsible for the synthesis of several so-called fitness factors, which contribute to the strain s probiotic nature. Serologically, EcN belongs to the E. coli O6 group and is of serotype O6:K5:H1 (54,55). EcN is a typical gram-negative enterobacterium containing lipopolysaccharide (LPS) as a structural component of its outer cell membrane. The O6 surface antigen represents the outer part of the strain s LPS and exhibits some peculiar features. The molecular structure of the LPS of EcN has been completely resolved (55). The LPS of EcN differs in several molecular aspects from all other LPS types of E. coli (57). For instance, the O6 polysaccharide side-chain is very short, consisting of only one single repeating unit of the oligosaccharide building block typical of the O6 antigen, giving the strain a so-called semi-rough phenotypic appearance when grown on solid nutrient media. The reason for this is a point mutation introducing a stop codon in the gene for the O6 antigen polymerase (55). Also, modifications previously unknown for E. coli LPS were found in the oligosaccharide core segment of the molecule. The specific features of the LPS of EcN are likely to explain the phenomenon whereby the strain exhibits immunomodulating properties without showing immunotoxic effects (55).

6 126 U. Sonnenborn et al. Table III. Basic microbiological and molecular genetic characteristics of E. coli strain Nissle 1917 (EcN, serotype O6:K5:H1). Characteristics Gene products/ biosynthesis results Genes/DNA probes Genotype Presence in EcN Phenotype Adhesion Common type I fim + + fimbria (F1A) F1C fimbria foc + + Curli fimbria csga + + S-type fimbria sfa P-type fimbria pap M-type fimbria NA ND CFA I/II (colonization cfa I/II ND factor antigens I/II) Toxins α-hemolysin hly CNF I (cytotoxic cnfi necrotizing factor I) H-LT (heat-labile etx enterotoxin) H-ST (heat-stable est enterotoxin) Shiga toxin stx Iron acquisition (siderophores) Enterobactin ent ND + Salmochelin iro + + Aerobactin iuc/aer + + Yersiniabactin ybt + + Hemin uptake system chu + + Citrate uptake system cit + ND Further characteristics Capsule (K5-type) kps + + Flagella (H1-type) fla + + Arginine dihydrolase NA ND + Cellulose biosynthesis bcs + + Microcin H47 mch + + Microcin M mcm + + Colibactin synthesis clb/pks + + LPS core wa + + LPS O6 repeating unit wb + + O6 antigen polymerase, wzy(rfb) + + non-functional Serum resistance NA ND Extrachromosomal elements: two cryptic, non-transferable plasmids pmut1 (3173 bp) a Muta 5/6 + ND pmut2 (5526 bp) a Muta 7/8, + ND Muta 9/1 + ND +, present;, not present; ND, not determined; NA, no specific DNA probe available. EHEC, typical enterotoxins. Present in 1% of E. coli isolates. Present in 7% of E. coli isolates. wzy gene truncated because of integrated stop codon. No carriage of antibiotic resistance genes or virulence genes. a Data extracted from G. Blum-Oehler et al. (53). As indicated by its serotype (O6:K5:H1), EcN is able to form an extracellular capsule of the K5 serotype. This kind of capsule is present in only about 1% of E. coli isolates. The gene loci on the chromosomal DNA responsible for the synthesis of the K5 capsule have been detected by using a gene probe specific for the K5 capsule gene cluster (58). In many extraintestinal pathogenic E. coli (ExPEC) strains the existence of a capsule protects the pathogen from attacks by non-specific defense components of blood serum (e.g. complement), thus making the bacterium serum-resistant (59). Serum resistance lengthens the time of survival in the bloodstream and increases the virulence of a pathogen (59). Interestingly, this is not the case with EcN (55). Despite its capability to form a capsule, in the classic serum resistance test (6) the

7 EcN strain is nevertheless serum-sensitive and is rapidly killed in the presence of human serum or sera of other mammalian species (bovine and porcine sera). EcN possesses flagella of serotype H1 and is thus quite mobile. The possession of flagella enables the microbe to actively move within the viscous intestinal mucus layer, e.g. in the direction of the intestinal mucosa, which serves as an oxygen source, useful for aerobic catabolism of substrates by E. coli. Besides their function as driving apparatus, the flagella are important in bacterial crosstalk with the epithelium (see below) and have also been described as bacterial sensors for humidity (61,62). EcN possesses three different types of fimbriae: F1A and F1C fimbriae (54,63,64), as well as so-called curli fimbriae (56,65,66), which mediate adhesion to intestinal epithelial cells in cell culture experiments or to the mucus layer of the intestinal wall in vivo (see below), facilitating colonization of the gut. Fitness factors Pathogenic bacteria are able to produce so-called virulence factors, which enhance the infectious capacity of the respective strains. Virulence factors are often encoded by genes representing special DNA sequences located on the bacterial chromosome, probably obtained by horizontal gene transfer in the evolution of the pathogen (67). These DNA sequences are called pathogenicity islands (PAIs) (68). Non-pathogenic (avirulent) commensal bacteria may also produce specific factors that enable them to compete with other strains in the ecological system of the gut and to effectively communicate with the host organism. These factors are designated fitness factors, which are also often encoded by special DNA sequences (genomic islands, GEIs) on the bacterial chromosome (69,7). Different fitness factors have been detected in the EcN strain in recent years. Siderophores are iron-chelating substances needed for bacterial iron uptake. EcN produces an unexpectedly wide array of siderophores: aerobactin, enterobactin, salmochelin, and yersiniabactin, as well as a hemin- and a citrate-dependent iron acquisition system. Besides the siderophore ferric iron uptake systems, EcN also possesses an elemental ferrous iron uptake system (EfeU) (71). Regarding the siderophores aerobactin and yersiniabactin as well as the hemin and the citrate system of EcN, the chromosomal DNA of the strain has been checked for the existence of the corresponding genes by using specific DNA probes (72 75). Positive signals for all four iron acquisition systems could be obtained on its genomic DNA. E. coli strain Nissle 1917 as probiotic 127 EcN was detected by Nissle in 1917 because of its growth-inhibiting (antagonistic) activities against enteropathogenic microorganisms. The antagonistic actions of EcN are due, at least in part, to the formation of microcins against which the producer strain itself is immune. One of these microcins (microcin H47) had already been described before for another E. coli strain (76 78), whereas the second microcin was unknown so far. The latter has been termed microcin M (M from Mutaflor) by Klaus Hantke and colleagues (79). In addition to the production of microcins, EcN belongs to that phylogenetic subgroup of E. coli bacteria (group B2) that contains strains able to synthesize peptide/polyketide hybrids (8,81). These substances belong to a heterogeneous group of molecules with antimicrobial and antitumor activities. To date it is not known whether the peptide/polyketide hybrides of EcN contribute to the antagonistic action against other bacteria. Metabolic properties EcN has been characterized biochemically by analysis of its strain-specific metabolic capacities and fermentation properties, using commercially available identification kits for Enterobacteriaceae in which a range of biochemical tests are carried out simultaneously. Table IV. Taxonomically important enzymes present in and biochemical reactions performed by E. coli strain Nissle 1917 (EcN); comparison with the corresponding characteristics typical of the enterobacterial species Escherichia coli in general. Activity/enzyme EcN E. coli in general (+/ /v) (% positive) Oxidase Dextrose metabolism Voges Proskauer reaction β-galactosidase (ONPG test) H 2 S production 1 Lysine decarboxylase Arginine dihydrolase + 7 Ornithine decarboxylase Urease Citrate metabolism 1 Malonate metabolism Tryptophan deaminase Indole production Lactose metabolism Saccharose metabolism v 44 Arabinose metabolism Raffinose metabolism 27 Rhamnose metabolism Sorbitol metabolism Inositol metabolism 2 Ribitol metabolism 2 +, positive reaction;, negative reaction; v, variable. Tests performed with commercially available test kits: MHK/ID/GNI system (Biotest) and API 2E system (BioMérieux).

8 128 U. Sonnenborn et al. As far as its metabolic capacities are concerned, EcN is a typical E. coli strain, with the exception that it is capable of metabolizing arginine (Table IV). This property is seen in just about 7% of all E. coli isolates. Gas chromatographic analysis of spent culture supernatants (82) has revealed that EcN produces short-chain fatty acids as end products of carbohydrate metabolism under aerobic and anaerobic growth conditions as well. These are mainly acetic acid and formic acid, as well as minor amounts of propionic and butyric acid (G. Sollorz and U. Sonnenborn, unpublished results). Molecular genetic properties Genome structure The genome structure of E. coli Nissle 1917 has been elucidated by three different approaches: (1) DNA sequence analysis of the gene loci for trna genes as bcs chu wa iuc, sat, iha, sap, kps GEI II EcN (13 kb) fim leux leuv pheu aspvthrw glyy thrt argu prom glnx selc lysq prok leuu mety Chromosome of E. coli Nissle Mb phev glyu metv argq iley ssra alax lysv argw prol sat potential integration sites for DNA sequences (GEIs) obtained by horizontal gene transfer during evolution; (2) DNA sequence analysis of the detected genomic islands; and (3) comparison of the EcN genome with those of other E. coli strains by DNA DNA hybridization (56). PCR-based screening of 324 pathogenic and non-pathogenic E. coli strains of different origin revealed distinct chromosomal regions occurring in non-pathogenic E. coli and in intestinal and extraintestinal pathogenic E. coli as well. In EcN, five large genomic islands and some smaller genomic islets have been detected so far, carrying gene clusters for different known fitness factors of this special E. coli strain (Figure 3). These insertions into the chromosome are thought to originate from horizontal gene transfer (56). In a dissertation by Schmidt, additional DNA sequences were found on the chromosome of the Nissle strain that also might have been acquired by horizontal gene transfer during the evolution of EcN (83). Comparison of the genome wb leuz seru asnt asnw asnu asnv valw ybt serw sert wrba serx tyrv GEI IV EcN (3 kb) mch/mcm, foc, iro csg GEI I EcN (97 kb) GEI III EcN (38 kb) clb/pks GEI V EcN (55 kb) Figure 3. Functional genomic map of E. coli Nissle 1917 (EcN) (56,8). Five large genetic islands (GEI I to GEI V) and some smaller genetic islets coding for different so-called fitness factors are inserted at distinct sites (mainly next to trna-encoding sequences) into the chromosome of the EcN strain. The bars on the chromosome circle mark the positions of trna genes (in gray: trna genes with sequence contexts identical to those of the completely sequenced non-pathogenic E. coli K-12 strain MG1655; in black: trna genes with sequence contexts identical to the completely sequenced uropathogenic E. coli O6 strain CFT73). The following genes and gene clusters of the EcN strain have been identified and sequenced: fim encoding F1A fimbriae ( common type-1 fimbriae ); csg encoding curli fimbriae; mch/mcm encoding microcin H47 and microcin M synthesis; foc encoding F1C fimbriae; iro/ybt/iuc encoding the siderophores salmochelin, yersiniabactin, and aerobactin; clb/pks (colibactin gene cluster) encoding non-ribosomal peptide synthetases, polyketide synthases and accessory proteins; sat encoding Sat protease; iha encoding Iha adhesion; sap encoding Sap-like autotransporter; kps encoding capsule synthesis; chu encoding hemin iron uptake system; bcs encoding cellulose biosynthesis; wa /wb encoding LPS biosynthesis.

9 structure of EcN with those of the E. coli K-12 strain MG1655 and the uropathogenic E. coli (UPEC) O6 strains 536 and CFT73 revealed similarities in gene structure and organization between the different E. coli strains, especially between the isolates of serotype O6. With respect to the genome size of the EcN strain, with 5.1 Mb its genome is larger than that of E. coli K-12 strain MG1655 (4.7 Mb) and UPEC strain 536 (5. Mb), but a bit smaller than that of UPEC strain CFT73 (5.23 Mb). The lack of gene clusters coding for classical pathogenicity traits of ExPEC, e.g. α-hemolysin, P-fimbriae, S-fimbriae, combined with the existence of genes coding for fitness factors (e.g. iron acquisition systems, microcins) and the genetically fixed synthesis of a semi-rough LPS may together be of importance for the probiotic nature of EcN (84). First results of the annotation of DNA sequences obtained after shotgun sequencing of the whole EcN genome have been published, focusing on genes representing the metabolic capacities of the Nissle strain (85) and on the search for genes coding for suspected pathogenicity or fitness traits (83). Plasmid characterization EcN possesses two small cryptic plasmids, termed pmut1 and pmut2 (53). Both plasmids, with 2 bp Marker (1 bp ladder) EcN 1 2 [cfu/ml] 427 bp 361 bp 313 bp Figure 4. Strain-specific multiplex PCR for identification of E. coli Nissle 1917 (EcN). PCR and agarose gel electrophoresis were performed as described (53). The PCR yields three distinct DNA products of M r 313, 361, and 427 bp length. Using this test system, the detection limit of EcN is between 1 2 and 1 3 bacterial cells/ml. Photograph: K. Eiteljörge, Herdecke. E. coli strain Nissle 1917 as probiotic 129 molecular masses of M r 2.1 and 3.7 Md (megadaltons), respectively, are genetically stable and are not transferable to other E. coli strains. The smaller plasmid (pmut1) consists of 3173 bp (base pairs), the larger one (pmut2) consists of 5526 bp. The circular DNAs of these two strain-specific plasmids have been completely sequenced (53). Strainspecific DNA sequences from both plasmids, which have not been found so far in other E. coli strains, can be used in a multiplex PCR method developed to specifically identify the EcN strain (53) (Figure 4). The function of these cryptic plasmids in EcN is not known as yet, since plasmid-free clones of EcN do not show morphological, cultural, or metabolic differences compared to the original strain carrying pmut1 and pmut2. There is, however, some evidence that cryptic plasmids per se may protect the carrier bacterium from attacks by mobile genetic elements such as bacteriophages, thus having an influence on the genetic stability of the strain (86). Modes of action Antagonistic activities against other microorganisms E. coli Nissle 1917 has been tested for its antagonistic activities (antimicrobial effects) against other microorganisms, in particular against microbial pathogens, both in vitro and in vivo. In vitro, antagonistic effects of EcN have been determined by means of co-cultivation with different enteropathogenic, uropathogenic, and other E. coli strains, as well as with individual strains of the species Proteus vulgaris, Salmonella enteritidis, Shigella dysenteriae, Yersinia enterocolitica, Vibrio cholerae, and Candida albicans (87,88). Co-cultivation experiments with EcN and the respective test strain have been performed either in suitable liquid media, where the number of viable bacteria of each strain has been determined after aerobic growth at 37 C, or on solid media by means of an agar diffusion test (similar to the classic test for antibiotic sensitivity), where EcN produced an inhibition zone against the test strain (in vitro tests according to Halbert (89) and Mayr-Harting et al. (9), Figure 5). Co-cultivation of EcN with Shiga toxin-producing E. coli (STEC) in liquid cultures resulted in a dose-dependent decrease of Shiga toxin levels (87). The antagonistic action of EcN was also confirmed in vivo by means of co-association of EcN with other microorganisms (pathogenic and non-pathogenic E. coli, Salmonella typhimurium, Candida albicans, Lactobacillus johnsonii) in germ-free or gnotobiotic animals (rats, mice, piglets) (91 94) (Figure 6).

10 13 U. Sonnenborn et al. Figure 5. Determination of antagonistic activity of E. coli Nissle 1917 (EcN) against another E. coli strain (DSM 423) in vitro. A suspension of E. coli DSM 423 was homogeneously spread across the surface of an agar plate (PAG minimal medium). Small cylinders of agar were punched out using a punching tool and the resulting cavities were filled with 1, 2 or 5 μl of an overnight culture of EcN (2 1 9 cfu/ml). Tests were performed in duplicate. The picture was taken after 24 h of aerobic incubation at 37 C. Antagonistic activity becomes visible by the development of inhibition zones (halos) around the cavities containing the EcN suspension. Photograph: U. Sonnenborn, Herdecke. Viable cell counts (cfu log 1 /g faecal mass, wet wt) Successive diassociation Germ-free animals have been inoculated per os either with EcN and the particular pathogenic strain simultaneously, or the test strain has been administered first and EcN several days later. The excrements of the animals have been regularly analyzed microbiologically, whereby the viable cell counts of the administered bacteria have been determined. In gnotobiotic animal models, a decrease or even complete eradication of the pathogenic test strains from the intestines has been achieved by oral administration of EcN, and symptoms of disease occurring due to inoculation with the pathogenic microorganisms disappeared (92 94). In conventional streptomycin-treated CD-1 mice, precolonization of the intestine with EcN limited the growth of E. coli EDL933, an O157:H7 Shiga toxin-producing enterohemorrhagic E. coli (EHEC) strain, when the latter was subsequently administered (95). By contrast, the E. coli K-12 laboratory strain did not inhibit growth of the EHEC strain. The inhibitory effects of EcN on facultative and obligate pathogens have also been demonstrated in humans by preventive oral administration of the strain to neonates (colonization prophylaxis) (96,97). The stools of the neonates were analyzed microbiologically to confirm colonization with EcN. In addition, the effect of colonization with this E. coli strain on the spectrum of other aerobic gut bacteria has been determined. All newborns that received EcN E. coli strain 542/88 E. coli strain Nissle Days Figure 6. Antagonistic action of E. coli Nissle 1917 (EcN) against enteropathogenic E. coli 542/88 in vivo in gnotobiotic piglets (94). Four 7-day-old germ-free piglets were infected orally with 1 8 cfu of E. coli 542/88 (arrow). The enteropathogenic E. coli strain quickly colonized the gut (dotted black line) and reached stable bacterial counts (about 1 1 cfu/g contents). Shortly after infection the animals showed signs of diarrheal illness. At day 6 of the experiment, when the piglets were visibly ill, they received an EcN suspension p.o. (2 1 8 cfu) (double arrow). Although the pathogenic E. coli strain had already settled in the gut, EcN also colonized the intestine fairly well (straight black curve). Six days later, the pathogen was completely expelled from the gut, whereas EcN continuously showed a high population level, and the piglets recovered.

11 E. coli strain Nissle 1917 as probiotic 131 Infants with pathogens (%) Colonized infants (+ EcN) Placebo infants ( EcN) 1 Day 1 Day 2 Day 3 Day 5 Day 21 Month 6 Study period Figure 7. Antagonistic activity of E. coli Nissle 1917 (EcN) in humans, as shown by the effect of preventive administration of EcN on the colonization of the newborn s gut with true and potential microbial pathogens (96). Using a double-blind study design, 54 full-term newborns were randomly assigned to two treatment groups and received orally either 1 ml of an EcN suspension (1 8 cfu, black bars) or 1 ml of a placebo suspension (open bars) once a day for the first 5 days of life. During the hospital stay (on days 1, 2, 3, and 5) and thereafter (on day 21 and during the 6th month), colonization of the gut with true and potential microbial pathogens was determined in fecal samples and is presented as the percentage of children carrying pathogenic and potentially pathogenic microorganisms. Regarding the colonization with pathogens, differences between the EcN group and the placebo group were recognized first on day 2 and were significant on day 3 (15% vs 57%, p.3), day 5 (15% vs 62%, p.1), and after 6 months (28% vs 85%, p.2). On day 21, the difference amounted to 33% vs 47%, but this was not significant. Significantly different colonization between EcN and placebo groups. were colonized by the administered strain, most of them showing long-term colonization for several months. The establishment of unwanted microorganisms, i.e. facultative and obligate pathogens, in the children s gut was significantly reduced by intentional colonization with EcN (Figure 7, see also next section). In patients with ulcerative colitis in remission, mucosal biopsies have been obtained during sigmoidoscopies, both before and after a 6-week oral treatment with EcN. The samples were analyzed for their content of mucosa-associated aerobic and anaerobic intestinal bacteria using classic microbiological cultural methods and quantitative PCR. Treatment with EcN resulted in a significant decrease of bacterial counts of aerobic and anaerobic mucosa-associated bacteria in a majority of the patients (98). As stated above, the antagonistic actions of EcN are mainly due to the formation of microcins (microcin H47, microcin M). The genetic organization of the two chromosomally localized gene clusters responsible for synthesis, transport, and secretion of both microcins discovered to date in EcN as well as for the immunity of the strain against these antimicrobial substances, has been examined in detail (79). The DNA sequences of the microcin genes have been determined (79) (Figure 8). Like colicin V, both microcins of EcN are secreted with the help of a protein export machinery of the RND type containing the membrane protein TolC. Inhibition of the invasion of epithelial cells by enteroinvasive bacteria In cell culture experiments using the human embryonic intestinal cell line INT47, EcN inhibits the invasion of epithelial cells by different pathogenic invasive bacteria, such as Salmonella, Shigella, enteroinvasive E. coli, Listeria, Yersinia, and Legionella strains (99,1), as well as adherent-invasive E. coli strains from patients with Crohn s disease (11). Interestingly, this inhibitory effect is not due to the production of microcins, since the isogenic, microcinnegative EcN mutant H5445 in this test system exhibits nearly the same inhibitory efficacy as the original strain (Figure 9). In addition, it has been shown that no physical contact is necessary for the anti-invasive effect, neither between EcN and the epithelial cells nor between EcN and the invasive bacterial pathogens. Moreover, the anti-invasive activity of EcN is not plasmid-coded and is not dependent on the presence of adhesins (F1A fimbriae, F1C fimbriae), since isogenic, Fim- and Focnegative deletion mutants of EcN have been shown to be as effective as the original strain. With the help of a so-called transwell filter system introduced into the cell culture, by which EcN can be separated from

12 132 U. Sonnenborn et al aa 516 aa 15 aa 424 aa 698 aa I 73 aa 92 aa I 228 aa mchx mchi mchb Open reading frame (mchc?) mcmd mcma mcmb mcmi mcmc mcmp Microcin H47 gene cluster: mchb Structural gene of microcin H47 Microcin M gene cluster: mcmc Structural gene of microcin M mchc? Modification (?) mcmd Modification gene mchx Regulatory gene mcmp Regulatory gene mchi I Immunity gene mcmi I Immunity gene mcmb mcma Export genes Figure 8. Structural organization of the chromosomal gene clusters located on GEI I, encoding the components necessary for synthesis, export, and regulation of microcins H47 and M of E. coli Nissle 1917 (EcN) (79). The number of amino acids (aa) of the individual gene products of the corresponding genes are given on top of the figure. The microcin M gene cluster ( M from Mutaflor ) was discovered for the first time in the EcN strain. both the epithelial cells and the invasive bacteria, it has been demonstrated that the invasion-inhibiting principle is diffusible and is secreted by EcN (99,1). It might be that this soluble factor inhibits the manipulation of the host cell cytoskeleton by the pathogenic microorganisms. (a) Invasive activity (%) E. coli K-12 EcN S. Typhimurium C17 S. Typhimurium LT2 Immunomodulatory and anti-inflammatory effects In vitro and in vivo experiments have demonstrated EcN to possess remarkable immunomodulatory activities, which address both the innate and the (b) Invasive activity of S.Typhimurium (%) Co-incubation E. coli K-12 + S. T. C17 EcN + S. T. C17 E. coli K-12 + S. T. LT2 EcN + S. T. LT2 (c) 45 Transwell unit EcN S. Typhimurium Intestinal epithelial cells INT47 % Invasive activity of S. Typhimurium (%) S. T. C17 EcN + S. T. C17 Microcin-negative mutant of EcN (H5445) + S. T. C17 Figure 9. Inhibition of Salmonella typhimurium (S.T.) invasion into INT47 intestinal epithelial cells by E. coli Nissle 1917 (EcN) (99,1). (a) Cell cultures with INT47 cells were separately incubated with either E. coli K-12 or EcN, or with the S. typhimurium strains C17 or LT2. While both non-pathogenic E. coli strains were also non-invasive, the Salmonella typhimurium strains showed differently strong invasive activity. (b) Co-incubation of INT47 cells with E. coli K-12 and S. typhimurium C17, EcN and S. typhimurium C17, E. coli K-12 and S. typhimurium LT2, and EcN and S. typhimurium LT2. While co-incubation with E. coli K-12 had no effect on the invasive activity of the S. typhimurium strains, co-incubation with EcN markedly inhibited the invasive activity of both S. typhimurium strains. (c) As shown by cell culture experiments using a transwell system (left), the anti-invasive activity of EcN is not due to direct interaction between EcN and S. typhimurium nor to occupation of receptor sites by EcN at the epithelial surface, since the effect was still present after separating EcN from S. typhimurium and from the intestinal cells as well (right).

13 adaptive immune system. With respect to adaptive immunity, EcN affects the humoral as well as the cellular branch of the immune system. However, in vivo significant immunomodulating activities are only reliably detected in gnotobiotic experimental animals, in human newborns, or in diseased animals and patients. In healthy human adults and animals, the immunomodulating activities of EcN are only very weakly expressed or not detectable at all (12,13) (R. Gruber, unpublished results). In vitro experiments. A significant dose-dependent improvement of secretory performance of mouse macrophages after direct contact with formalin-killed cells of EcN has been observed in vitro (14). The production of interleukin-6 (IL-6), tumor necrosis factor (TNF), and oxygen radicals increased significantly. Further experiments revealed an augmentation of tumor cell lysis and parasite lysis (leishmaniae) by macrophages prestimulated with EcN. On the other hand, the efficiency of phagocytosis of the macrophages was raised only slightly. However, these in vitro effects could only partially be confirmed in vivo, following oral administration of EcN to conventionally kept mice. In vivo, for instance, no induction of TNF synthesis has been observed. Experiments with epithelial cell lines and isolated lymphocytes have shown EcN to exhibit different immunomodulating activities, and these strongly depend on the cell types/cell lines employed and the parameters and read-out systems used (15 115). One example is the dose-dependent inhibition of the TNF-α-induced IL-8 secretion by EcN in HCT-15 intestinal epithelial cells (18,116). The inhibition is reflected by the reduced expression level of IL-8 mrna in the epithelial cells, as shown by real-time PCR. The inhibition of IL-8 synthesis is caused by secreted soluble factors that have not been identified so far. Interestingly, the EcN-mediated inhibition of TNFα-induced IL-8 transcription is independent of the AP-1- and nuclear factor κb (NF-κB)-regulated gene expression. The latter, however, is important for the induction of human β-defensin-2 (HBD-2) synthesis in gut epithelial cells (Caco-2 cells) by EcN (114). In the mouse monocyte/macrophage cell line J774A.1, addition of EcN resulted in an increase of IL-12, TNF-α, and IL-1 secretion (15). By contrast, IL-18 and TGF-β synthesis were not affected. Compared with the effects of viable EcN cells, addition of killed bacterial cells resulted in a much less pronounced induction of cytokine synthesis. In confluent Caco-2 epithelial cells, contact with EcN resulted in an up-regulation of gene expression of monocyte chemoattractant protein-i ligand 2 (MCP-I) and an increase of MCP-I secretion (113). MCP-I is thought to be involved in the inflammatory E. coli strain Nissle 1917 as probiotic 133 immune reaction. On the other hand, Otte et al. (117) found that contact of intestinal epithelial cells (Colo32 and SW48 cells) with EcN led to inhibition of the inflammatory response by down-regulation of TNF-α- or gastrin-induced cyclooxygenase-2 (COX-2) activity. This was followed by reduced secretion of proinflammatory prostaglandin E 2 (PGE 2 ). The immunomodulating action of EcN on peripheral as well as on gut mucosal lamina propria T lymphocytes has also been studied in vitro (112). Anti-CD3-stimulated peripheral and lamina propria T cells have been exposed to heat-inactivated cells of EcN and to EcN-conditioned medium (spent culture supernatants), and the effects of these treatments on lymphocyte cell cycle and on markers of apoptosis have been examined on the molecular level. EcNconditioned medium dose-dependently inhibited cell cycle progression and T-cell expansion of lymphocytes from peripheral blood, but not of lymphocytes from the gut mucosa. This selective action on T cells from peripheral blood could also be shown by using bacterial lipoproteins. In contrast to this, heat-inactivated cells of EcN, its purified LPS, or CpG-DNA had no effect. In peripheral T cells, addition of EcNconditioned medium reduced expression of cyclin D2, B1, and retinoblastoma protein, which are involved in T-cell proliferation. Interaction of EcNconditioned medium with peripheral T cells significantly inhibited the expression of IL-2, TNF-α, and IFN-γ, whereas the production of IL-1 was enhanced (Figure 1). The differential effects of EcN on circulating and intestinal T cells is supposed to be an important aspect of its efficacy in inflammatory bowel diseases (112,118). By down-regulation of the expansion of newly recruited T cells into the gut mucosa, intestinal inflammation will be limited. Compared with this, activated T cells already residing in the gut wall are not influenced by EcN and thus are available for the continuous fight against pathogenic microorganisms (112). In the T-cell subgroup of γδ T cells, EcN increased activation, cell cycling, and cell expansion, but thereafter induced apoptosis via caspase- and FasL-dependent signaling pathways (119). These effects of EcN were targetspecific, since they were only seen with γδ T cells, but not with αβ T cells. Using human peripheral blood mononuclear cells, Helwig et al. (17) compared the immunomodulating activities of gram-positive Lactobacillus and Bifidobacterium strains with those of the gramnegative EcN strain. These experiments revealed that gram-positive and gram-negative bacteria exerted partly different, but also partly similar immunomodulatory activities on the blood mononuclear cells (Figure 11). With respect to some special immune parameters, different responses were seen also

14 134 U. Sonnenborn et al. Cytokines (ng/ml) IL-2 TNF-α IFN-γ IL-1 1% Control with EcN-conditioned medium 25% Figure 1. Anti-inflammatory effects of E. coli Nissle 1917 (EcN) on human peripheral blood T cells (PBT) in vitro, as shown by cytokine expression profiles induced by EcN-conditioned medium (EcN-CM) (112). EcN-CM was obtained after 2 h incubation of EcN bacteria in T-cell medium (RPMI with 1% FCS, 1.5% HEPES) at 37 C and 5% CO 2. Thereafter, bacteria were removed by centrifugation. The resulting supernatant was sterile-filtered through a filter of.22 μm pore size. The sterile-filtered supernatant (EcN-CM) was added to freshly isolated PBT (1 5 cells) in different concentrations (open bars, controls no addition of EcN-CM; hatched bars, 1% v/v EcN-CM; black bars, 25% v/v EcN-CM). T cells were then stimulated by adding anti-cd3-mab and cultured for 3 days in RPMI. Culture supernatants were collected and assayed for cytokines. EcN-CM dose-dependently and significantly inhibited IL-2, TNF-α, and IFN-γ synthesis, while IL-1 production was markedly up-regulated. p.5 vs CD3 controls. between the bifidobacteria and the lactobacilli, leading to the assumption that the immunomodulatory action of probiotics might be rather strain-specific and not genus- or species-specific. Interestingly, in this test system the EcN strain provoked a high output of the anti-inflammatory signal molecule IL-1. Fink and Frokiaer (16) could show that interaction IL-1 [pg/ml] area under the curve Bifidobacteria Lactobacilli EcN of different bacteria including EcN resulted in different maturation responses of dendritic cells from different locations of the body (Peyer s patches, mesenteric lymph nodes, and spleen). Recently, in vitro experiments with isolated blood lymphocytes from healthy and allergic persons (111) have shown that the EcN strain induced strong antiallergic responses. These are interesting new findings that may lead to new indications for EcN in the future. The investigations with mononuclear cells from peripheral blood, obtained from allergic persons, have shown that contact of the lymphocytes with EcN induced a shift from a T-helper-2 (Th2)- dominated, allergy-specific immune response to a Th1-dominated, non-allergic response (111). In vivo experiments. In young gnotobiotic piglets, colonization with EcN resulted in a rapid development of the gut-associated immune system. This was reflected by a significant increase in the numbers of IgA, IgM, and IgG antibody-producing lymphocytes in the lamina propria of intestinal villi and in the intestinal lymph follicles as well as by an increase in SLA-D+(MHC II+) cells and CD4-positive cells in all mucosal and submucosal tissues, without any signs of an inflammatory reaction (infiltration of granulocytes) (93,12). Investigations on human newborns have revealed that both full-term neonates and premature infants show a significant rise in the levels of immunoglobulins (IgA and IgM) in blood serum and of secretory IgA and IgM in stool filtrates within a few days after intentional colonization of the gut with EcN (121) (Figure 12). On the other hand, there was no influence on the IgG level in blood serum. Besides these immunomodulatory effects on the development of TNf-α [pg/ml] area under the curve Bifidobacteria Lactobacilli EcN Figure 11. Immunomodulatory activities of gram-positive (bifidobacteria, lactobacilli) and gram-negative (E. coli Nissle 1917, EcN) bacteria on human peripheral blood mononuclear cells (PBMNCs) in vitro (17). Supernatant concentrations of the cytokines IL-1 and TNF-α after co-incubation of PBMNCs with cell debris of bacteria from different genera and species are shown. With regard to bifidobacteria (open bars), results are pooled from the data obtained by testing B. breve, B. infantis, and B. longum. With regard to lactobacilli (hatched bars), results are pooled from the data obtained by testing L. acidophilus, L. bulgaricus, L. casei, L. plantarum, and L. rhamnosus strain GG (LGG). Data from experiments with E. coli Nissle 1917 (EcN) are presented by the black bars. Concentrations of IL-1 and TNF-α are shown in pg/ml as area under the curve (AUC, mean SE). Significantly different, p.5.

15 E. coli strain Nissle 1917 as probiotic FC PC 2 1 P before Days IgA IgM % of reference before the humoral branch of the immune system, stimulation of cell-mediated immune responses and of nonspecific natural immunity after oral administration of EcN has also been detected in a controlled clinical study with preterm infants (122). In established animal models of inflammatory bowel diseases (IBD), such as the IL-2 knockout mouse, EcN shows no proinflammatory effects (123,124). On the contrary, using different experimental animal models for IBD (T-lymphocyte transfer model in SCID mice, IL-1 knockout mice, dextran sodium sulfate (DSS)-induced or trinitrobenzenesulfonic acid (TNBS)-induced colitis), anti-inflammatory effects of EcN have been consistently observed (118,125 13). In one model of acute intestinal inflammation, oral administration of EcN to mice FC PC P Days Full-term children colonized with EcN n = 22 (FC) Pre-term children, colonized with EcN n = 9 (PC) Pre-term controls n = 7 (P) Figure 12. Effect of intentional colonization of the gut of newborns by E. coli Nissle 1917 (EcN) on the levels of secretory IgA and IgM (121). One ml of EcN suspension (1 8 cfu/ml) was administered p.o. once a day for 5 days, starting from the first day of life. IgA and IgM levels against EcN antigen were measured in stool filtrates after colonization of full-term and preterm infants with EcN and compared to the IgA and IgM levels in stool filtrates of non-colonized preterm infants (controls). Antibody levels are expressed as percentage of the reference sample (mean SE). In stool filtrates of full-term infants IgA and IgM levels were significantly higher (from day 3 onwards) than the levels detected before colonization (p.5). IgA levels were significantly higher in full-term than in preterm infants on day 5 and day 14 (p.1), and IgM levels were higher on all days after colonization (p.5 p.1). Preterm colonized infants had higher fecal IgA and IgM titers than non-colonized preterm infants (controls) on all days after colonization (p.5 p.1). pretreated with 2% DSS led to a decrease of the proinflammatory cytokines IFN-γ and IL-6, without showing a significant effect on mucosal inflammation (129). In another model of acute gut inflammation, oral application of EcN to mice pretreated with 1.3% DSS significantly ameliorated body weight loss, disease activity index, as well as macroscopic and microscopic damage of the intestinal mucosa (127). Interestingly, administration of heat-killed EcN as well as of its genomic DNA also showed some anti-inflammatory actions in DSS colitis mice (reduced colon weight, lower histological score), although the effects on the disease activity index (DAI) were not as marked as those achieved by viable bacterial cells (127) (Table V). In IL-1 knockout mice (IL-1 / mice), an animal model of chronic intestinal inflammation, per os Table V. Effects of live and heat-killed E. coli Nissle 1917 (EcN) and of EcN-DNA on DSS-induced colitis in specific pathogen-free C57BL/6 mice. Treatment n DAI (day 7) Colon weight (mg/cm) Histological score PBS Live EcN Heat-killed EcN EcN-DNA DAI, disease activity index; DSS, dextran sodium sulfate; PBS, phosphate-buffered saline. Data are mean SEM. p.5 and p.1, significant difference compared with PBS group (N. Kamada et al. (127)).

16 136 U. Sonnenborn et al. administration of EcN led to a decrease of tissue lesions and a decrease of the infiltrations of the colonic mucosa by mononuclear cells and polymorphonuclear cells, as judged by macroscopic and microscopic examinations (127). Moreover, EcN treatment significantly reduced the number of mice with rectal prolapse (a characteristic symptom of severe rectal inflammation), lowered the concentrations of the inflammatory marker protein serum amyloid A (SAA) in blood serum, and reduced the expression of proinflammatory cytokines and chemokines in colonic Lamina propria mononuclear cells (LPMCs) of colitis mice (significant for IFN-γ and MIP-2). In the immunologically mediated chronic colitis model of T-lymphocyte transfer in SCID mice, oral application of EcN significantly ameliorated intestinal inflammation, as judged by histologic examinations, and reduced secretion of proinflammatory cytokines IFN-γ, IL-5, and IL-6, without affecting the level of the anti-inflammatory cytokine IL-1 (129) (Figure 13). (a) Clinical Score (b) Histological Score (c) CFU/g MLN ,5 2 1,5 1, PBS-treated healthy mice + EcN PBS-treated healthy mice + EcN IFN-γ (pg/ml) IL-6 (pg/ml) T cell transfer mice PBS-treated T cell transfer mice EcN + EcN healthy mice EcN + EcN + EcN T cell transfer mice EcN + EcN T cell transfer mice EcN + EcN (d) (e) (f) IL-5 (pg/ml) PBS-treated healthy mice + EcN PBS-treated healthy mice + EcN T cell transfer mice EcN + EcN T cell transfer mice EcN + EcN Figure 13. Anti-inflammatory action of E. coli Nissle 1917 (EcN) in immunodeficient C.B.-17 SCID mice with chronic colitis (129). Chronic colitis was induced by adoptive T-cell transfer, using purified CD4 + CD62L + splenic T lymphocytes from healthy BALB/c mice, which were introduced intraperitoneally into recipient SCID mice. After T-cell transfer, mice were divided into two groups. The first group received 2 μl of EcN suspension (5 1 1 cfu/ml) by gastric gavage twice per week from week 1 post-transfer to week 8 at the end of the experiment ( T-cell transfer mice + EcN, black bars). The second group of SCID mice was treated in the same way and for the same period of time, but received plain water by gastric gavage instead of EcN ( T-cell transfer mice EcN, open bars). This second group served as positive control (colitis mice). Healthy BALB/c mice were treated intraperitoneally with PBS (phosphate-buffered saline) instead of splenic T cells, and thereafter received EcN by the oral route ( PBS-treated healthy mice + EcN, gray bars). These mice served as negative controls. (a) Clinical score of CD4 + CD62L + SCID mice treated or not treated with EcN p.o., in comparison to the score of the healthy control group injected i.p. with PBS and treated with EcN p.o. p.1 vs CD62L. (b) Histological score of the colonic inflammation in mice following the induction of colitis by transfer of naïve CD4 + CD62L + T lymphocytes and p.o. administration of EcN, compared to that of colitis mice not treated with EcN, and to the healthy control group which received an i.p. injection of PBS and was treated with EcN p.o. p.2 vs CD62L. (c) Evidence of bacterial translocation into mesenteric lymph nodes (MLNs). Total concentration of E. coli-like colonies in MLNs of mice from the transfer model following oral administration of EcN for 8 weeks compared to that of colitis mice not treated with EcN. EcN was identified by REP-PCR. p.1 vs CD62L. (d, e, f) Proinflammatory cytokine secretion from MLNs following the induction of colitis in SCID mice by transfer of naïve CD4 + CD62L + T lymphocytes. Anti-inflammatory effect of the p.o. administration of EcN, compared to the results obtained in colitis mice not treated with EcN, and to the healthy control group which received an i.p. injection of PBS and was treated with EcN p.o. Values given are means SEM (pg/ml). (d) IFN-γ, p.3 vs CD62L; (e) IL-6, p.2 vs CD62L; (f) IL-5, p.2 vs CD62L.

17 In both a rat model of TNBS-induced colitis and a mouse model of LPS-induced sepsis, orally administered EcN exerted local as well as systemic antiinflammatory effects (125). TNF-α level was reduced in the gut of colitic rats, while TNF-α and T-cell cytokines were reduced in blood plasma and lungs of sepsis mice, and the IgG release from splenocytederived B cells was down-regulated. Using Toll-like receptor 2 (TLR-2) and Toll-like receptor 4 (TLR-4) knockout mice, it has been demonstrated that the inhibition of T-cell proliferation and the amelioration of DSS-induced colitis by EcN is mediated via TLR-2- and TLR-4-dependent pathways (112,118). In an experimental allergy study conducted by Bickert and colleagues (131), it was found that EcN inhibits the allergen-induced Th2-dominated immune response in the airways of C57BL/6 mice that had been hypersensitized by i.p. and i.v. administration of ovalbumin. However, this anti-allergic effect could only be demonstrated, if EcN was given together with the allergen. Infection-preventing effects In in vivo experiments using conventionally housed C3H/HeN mice, single oral doses of EcN inhibited proliferation of subsequently intravenously inoculated Candida albicans and Listeria monocytogenes infections in a dose-dependent manner (132) (Figure 14). In a placebo-controlled veterinary clinical trial on 335 newborn calves it was shown that administration of EcN significantly (p.1) reduced the incidence of neonatal calf diarrhea from 63% (placebo group) to 12% (EcN group) (133). Communication with intestinal epithelial cells In the last few years, it has become obvious that many probiotic effects of orally administered microbial preparations are due to interactions between the bacteria, the gut epithelial cells, and the gut immune system (115,13,134,135). In particular, the communication with the epithelial cell lining seems to be important, leading to a strengthening of the epithelial barrier function (115,13,136). Thus, co-incubation of EcN with human intestinal epithelial cells (polarized T 84 cells) resulted in a significant modulation (up- and down-regulation) of the activity of about 3 different genes in the epithelial cells, as demonstrated by transcriptome analysis using the microarray technique with DNA chips (Affimetrix) (115,137). Moreover, after infection of T 84 cells with an enteropathogenic E. coli strain (EPEC E2348/69), treatment with EcN eliminated the negative impact of EPEC infection on the L. monocytogenes C. albicans (log 1 cfu/organ) E. coli strain Nissle 1917 as probiotic 137 SPLEEN LIVER KIDNEY Control IFNγ EcN cfu epithelial barrier function, i.e. EPEC-induced disruption of epithelial cell tight junctions (Figure 15). This action of EcN seems to be mediated via the protein kinase C signaling pathway in the epithelial cells. In another cell culture study, it has been observed that co-incubation of EcN with human epithelial cell lines T 84 and HT-29 resulted in increased IL-8 secretion. Interestingly, the presence of EcN prevented cell death of the epithelial cells, induced by exposure to Salmonella dublin (11). Examinations of cell cultures of human intestinal epithelial cells (Caco-2 cells, HT-29 cells) have demonstrated EcN to stimulate the synthesis of inducible antimicrobially acting defensins (HBD-2, HBD-3) (114,138) (Figure 16). Induction of HBD-2 gene expression by EcN has been shown to exceed several Figure 14. Augmentation of host defense against systemic bacterial and fungal infections in mice by oral pretreatment with E. coli Nissle 1917 (EcN) (132). Four groups of mice were pretreated once with 1 6, 1 7, 1 8, or 1 9 viable cells of EcN by oral administration, before they were challenged 24 h later by intravenous infection with Listeria monocytogenes (6 1 3 cfu) or Candida albicans (5 1 5 cfu). For each infection model, further groups of mice served as controls. These were either pretreated with placebo (negative controls, open bars) or with murine IFN-γ (positive controls, gray bars). Three days after infection with L. monocytogenes and 1 day after infection with C. albicans, mice were sacrificed and the parasite burden of the respective main target organs was determined. Compared with placebo, the pretreatment with IFN-γ resulted in a significant decrease of parasite load in spleen and liver of mice infected with L. monocytogenes, and a significant decrease of C. albicans counts in the kidneys. Likewise, pretreatment with EcN via the oral route significantly and dosedependently reduced the parasite burden in spleen, liver, and kidneys. p.5 vs negative controls.

18 138 U. Sonnenborn et al. Control without bacteria EPEC 12 min A B C EcN 12 min Separate incubation EPEC + Medium 6 min EPEC + EcN 6 min EPEC + EcN 12 min D E F Co-incubation Figure 15. Effects of the non-pathogenic E. coli strain Nissle 1917 (EcN) and the enteropathogenic E. coli (EPEC) strain E2348/69 on the distribution of the tight junction protein zonula occludens-2 (ZO-2) in T 84 epithelial cells (115). T 84 monolayers were incubated with bacteria for different periods of time and stained for ZO-2 using a fluorescent anti-zo-2 antibody. (A) Control, T 84 epithelial cells without bacteria. (B) T 84 cells incubated with EPEC for 12 min. (C) T 84 cells incubated with EcN for 12 min. (D) T 84 cells incubated with EPEC for 6 min, then washed and further incubated with regular medium for another 6 min. (E) T 84 cells incubated with EPEC for 6 min, then washed and further incubated with EcN for another 6 min. (F) T 84 cells co-incubated with EcN plus EPEC for 12 min. Bacteria were added in a 1:1 ratio. The EcN strain had no negative influence on the distribution of the tight junction protein ZO-2, but abolished the negative impact of EPEC on ZO-2 distribution. times the extent of induction mediated by 4 other E. coli strains tested. By contrast, gene expression of constitutively synthesized HBD-1 defensin was not influenced by EcN. Stimulation of HBD-2 synthesis by EcN is mediated in the epithelial cell nucleus by transcription factors NF-κB and AP-1 (114). The factor responsible for HBD-2 synthesis induction by EcN is the structural flagellum component flagellin Relative transcript level of HBD nonstimulated EcN EPEC E. coli K-12 UPEC E. coli strains PZ # (139). The flagellin signal might be transmitted by TLR-5, which is present in Caco-2 epithelial cells, but other signaling pathways might also be involved (139). Here, further investigations are required. In mice with DSS-induced colitis, the interaction between EcN and the intestinal mucosa resulted in the restoration of the impaired permeability barrier ( leaky gut ) (Figure 17) (13). The contact Mucosal permeability to Evans Blue (absorbance 62 nm [abs./cm]),12,1,8,6,4,2 Figure 16. Stimulation of human β-defensin-2 (HBD-2) gene transcription in Caco-2 intestinal epithelial cells by pathogenic (EPEC E2348/69, UPEC 536) and non-pathogenic E. coli strains (E. coli K-12 DSM 498, EcN), and by E. coli strains with unknown pathogenicity (fecal isolates from healthy persons (PZ ) and colitis ulcerosa patients (PZ 83, 835) (114,138). Caco-2 cells were stimulated for 4.5 h with heat-inactivated bacteria/ml. Transcription of the HBD-2 gene was analyzed by real-time PCR. HBD-2 gene expression is shown after stimulation by the different E. coli strains, including probiotic E. coli Nissle 1917 (EcN). Data represent the means SEM normalized to the basal expression of controls (set at 1) from one to six separate experiments run in triplicate. p.6; p.1. Control DSS DSS + EcN Figure 17. Inhibition of leaky gut phenomena by oral administration of E. coli Nissle 1917 (EcN) to mice with dextran sodium sulfate (DSS)-induced colitis (13). Intestinal permeability to Evans Blue was determined in healthy mice (control, open bar), mice with DSS-induced colitis (hatched bar), and mice treated with DSS plus EcN (black bar). Compared with healthy control mice, a significant increase in the uptake of Evans Blue by the colonic mucosa of DSS-treated mice was observed. This increase was strongly reduced in the group of mice treated with DSS plus EcN to almost normal values. p.1.

19 of EcN with the intestinal epithelial cells induced the de novo synthesis and the redistribution of tight junction proteins, such as zonula occludens protein-1 (ZO-1), from the cytosol to the cell membrane. This effect is important for the cohesiveness of the epithelial cell layer. Concomitantly, DSS-induced disturbances of epithelial transport functions, such as the strongly decreased activity of the membrane-bound sodium pump, were restored (13). For bacterial epithelial interactions, the presence of the K5 capsule in the EcN strain also seems to be important (14). Similar mechanisms of action of EcN are responsible for the recently detected prevention of the development of gastric mucosal lesions in mice, induced by acute stress (141). The authors showed EcN to exhibit anti-inflammatory and blood flowstimulating activities. Concomitantly, in the epithelial cells an increased synthesis of stress-protective proteins, such as heat-shock protein 7 (Hsp 7), could be demonstrated. The stress-induced enhanced gene expression of mucosal inflammatory mediators and the activation of transcription factor NF-κB was down-regulated by EcN. Calprotectin is a calcium- and zinc-binding protein, the synthesis of which is stimulated by LPS (142,143). Calprotectin exerts antimicrobial activity by binding zinc and inhibiting the adhesion of bacteria to epithelial cells of the gut mucosa (144). In 8-day-old germ-free piglets, oral administration of EcN led to an increase of calprotectin synthesis in E. coli Nissle 1917 (EcN) E. coli strain Nissle 1917 as probiotic 139 the small intestine (145). Another non-pathogenic E. coli strain (E. coli O86) and an enteropathogenic isolate (E. coli O55) did not show this effect in the gut. In contrast to EcN and to E. coli O86, oral administration of the enteropathogenic O55 strain provoked an increase of calprotectin level in blood plasma and, in addition, a pronounced increase of calprotectin concentration in bronchoalveolar lavage that was coincident with the development of septicemia. All these favorable effects induced by EcN are thought to be due to communication between the probiotic and the epithelial cells by so-called bacterial epithelial crosstalk (19, ), mediated via specific signaling molecules, most of them being unknown up to now (Figure 18). Effects on visceral hypersensitivity and constipation The irritable bowel syndrome (IBS) belongs to those functional bowel diseases that in about 25% of patients have developed some time after an episode of acute infectious diarrhea (15,151). This post-infectious IBS is characterized by a hypersensitivity of the visceral sensory system of the gut (so-called visceral hyperalgesia). In an established rat model of TNBS-induced visceral hyperalgesia, oral administration of EcN led to a significant reduction of the visceromotor reflexes (VMR) of the gut muscles, which were enhanced before as a consequence of the short-term inflammation induced by TNBS (152). Direct antagonistic activity Inhibition of growth / killing of pathogenic bacteria and fungi Production of defensins, cathelicidin, calprotectin Inhibition of invasion (Salmonella, EIEC, AIEC, Shigella, Yersinia, Listeria, Candida) Anti inflammatory effects (inhibition of IL-5, IL-6, IFN- γ, TNFα effect on IL-8) Immunomodulation, e.g. IL-1 induction Gut epithelium Restorative effect on the epithelial permeability barrier Prevention of leaky gut symptoms in case of disturbed barrier function Improvement of ion transport activity of epithelial cells disturbed by intestinal inflammation Figure 18. Schematic summary of pharmacodynamic activities of probiotic E. coli strain Nissle 1917 (EcN) in the gut lumen (antagonistic activity), and at the intestinal epithelium and beyond (host cell signaling).

20 14 U. Sonnenborn et al. Another functional bowel disease is chronic constipation, where colonic motility is reduced and, accordingly, intestinal transit slows down. In this context, it is important that cell-free spent supernatants of EcN cultures positively modulated colonic motility in an in vitro organ bath study with human colonic circular smooth muscle strips (153). This effect may be due, at least in part, to EcN s ability to produce acetic acid, the main metabolic end product of its carbohydrate metabolism. Antimutagenic activity Microsatellite instability (MSI) in chronically inflamed colonic tissue has been suggested to be associated with the increased risk of ulcerative colitis patients developing colorectal carcinoma. Substances with anti-inflammatory activities, such as mesalazine (5-aminosalicylic acid) or EcN, might improve MSI. Therefore, Goel and co-workers (154) performed an ex vivo analysis of MSI in 156 biopsies from 39 colitis ulcerosa patients who had been treated with either mesalazine or EcN in a randomized, double-blind clinical trial (155). Overall, 2% (31/156) of biopsies displayed MSI. After 1 year on medication, 6/2 patients showed MSI improvement (change to microsatellite stability (MSS): 3 on mesalazine and 3 on EcN). Worsening of MSI (change from MSS to MSI) was also observed, but only in patients treated with mesalazine (6/2). Using standard mutagenicity tests (Ames-test, Comet-assay) (156,157), it has been shown recently that EcN also exhibits antimutagenic activity against some well-known mutagens, such as 4-nitroquinoline 1-oxide (NQO), benzo(a)pyrene, and H2O2 (158). Colonization capability Following oral administration of E. coli Nissle 1917, the strain exhibits good colonization capability in the gut of gnotobiotic rats (92) and piglets (93). In general, oral administration of EcN to different conventionally kept mice and rats resulted in a longer-term persistence or even a true colonization of the gut. This has been shown for the original strain and for recombinant and transformed EcN derivatives as well (63,95,159,16). In NMRI mice, the good colonization capability of EcN is dependent on the presence of the regulatory protein RfaH, as has been shown by colonization experiments comparing the original EcN strain with a RfaH-negative mutant (161). In this context, RfaH does not seem to be important for bacterial adhesion to epithelial cells, but seems to enhance resistance of the EcN strain to bile acids. In vivo, the higher resistance to bile provides better chances of survival during transit of EcN through the gastrointestinal tract (161). Colonization is thought to be facilitated by the capability of EcN to produce and secrete cellulose, a feature important for biofilm formation (162). Interestingly, EcN is able to form biofilms at 37 C (Figure 19). In this respect the strain is different from other E. coli strains, which are only able to produce biofilms at lower temperatures ( 3 C). It is thought that this characteristic of EcN might also be important for its persistence in the gut after oral inoculation (63,162). Curli fimbriae of E. coli are important factors mediating the formation of microcolonies, which represent the first step in biofilm formation (163). Besides curli fimbriae, EcN expresses F1A and F1C fimbriae (54,64). The corresponding gene clusters for the Figure 19. Biofilm formation by E. coli Nissle 1917 (EcN) in vitro as demonstrated by laser scanning microscopy. (A) Computer-assisted height measurement; (B) density measurement. Pictures taken from: J. Schulze, M. Schiemann, U. Sonnenborn. 12 years of E. coli its importance in research and medicine. Alfred-Nissle-Gesellschaft, editor. Hagen, p 33, 26; with kind permission. Scanning micrographs courtesy of U. Dobrindt and H. Merkert, University of Wuerzburg, Germany.