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Microbial Ecology in Health and Disease

ISSN: (Print) 1651-2235 (Online) Journal homepage: http://www.tandfonline.com/loi/zmeh20

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 (2009) The non-pathogenic Escherichia
coli strain Nissle 1917 – features of a versatile probiotic, Microbial Ecology in Health and Disease,
21:3-4, 122-158, DOI: 10.3109/08910600903444267

To link to this article: https://doi.org/10.3109/08910600903444267

© Informa UK Ltd

Published online: 26 Dec 2009.

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Microbial Ecology in Health and Disease. 2009; 21: 122–158

REVIEW ARTICLE

The non-pathogenic Escherichia coli strain Nissle 1917 – features


of a versatile probiotic

ULRICH SONNENBORN1 & JÜRGEN SCHULZE2∗


1Department of Biological Research and 2Department of Medicine, Ardeypharm GmbH, Herdecke, Germany

Abstract
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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 rem-
edies 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 entero-
bacteria. 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 chromo-
some. 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 fulfill a wide array of beneficial tasks for the host,


including protection from pathogenic microorgan-
Microbial ecology of the gut
isms and modulatory effects on the development
The intestinal ecosystem has been described as ‘a and functions of the gastrointestinal tract and the
precarious alliance among epithelium, immunity gut immune system (2–7). Important functions of
and microbiota’ (1) and, under normal condi- the indigenous intestinal microbiota are given in
tions, the commensal microorganisms indeed Table I.

∗Presentaddress: Alice-Bloch-Str. 7, D-14558 Nuthetal, Germany. E-mail: JuR.Schulze@t-online.de


Correspondence: Dr Ulrich Sonnenborn, Department of Biological Research, Ardeypharm GmbH, Loerfeldstr. 20, D-58313 Herdecke, Germany.
Fax: +49 2330 977697. E-mail: sonnenborn@ardeypharm.de
(Received 2 September 2009; accepted 22 October 2009)
ISSN 0891-060X print/ISSN 1651-2235 online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.3109/08910600903444267
E. coli strain Nissle 1917 as probiotic 123
Table I. Important functions of the intestinal microbiota. pathogens (14–18) has led to the renaissance of a
Barrier function (‘colonization resistance’) therapeutic concept from the pre-antibiotic era: the
Modulation of the local immune system (GALT) prevention or treatment of specific diseases with liv-
Intraluminal metabolism ing non-pathogenic microorganisms, i.e. probiotics.
Support of energy metabolism and stimulation of blood flow of In recent years our knowledge concerning the regu-
the colonic mucosa
Stimulation of gut motility
latory processes within the gut ecosystem in health
Preparation and maintenance of the specific colonic milieu and disease has grown enormously (1,3–5,7,13,19).
Adjustment of bacterial translocation The knowledge of particular characteristics and
Support of vitamin supply activities of members of the indigenous intestinal
GALT, gut-associated lymphoid tissue. microbiota and their interactions with the host is
fundamental to the application of living microor-
ganisms as health-promoting or therapeutic agents
A breakdown of the microecological balance in (12,19–25).
the gut may be caused by different exogenous and
endogenous factors and will result in changes of
composition, numbers, and activities of indigenous The term ‘probiotics’ – development of definitions
microbial communities. This may be followed by Today, probiotics are defined as non-pathogenic liv-
destruction of the physiological barriers of the body, ing microorganisms which, when administered in
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enhanced epithelial irritation, inflammation and per- adequate amounts, confer a health benefit on the
meability, and bad consequences for the health of the host. This definition was coined by an expert com-
host organism (1,3,8–13). Intestinal dysfunctions mittee of a joint FAO/WHO meeting on probiotics
and diseases that are associated with or caused by in 2001 (26). The word ‘probiotic’ has been derived
disturbances of the indigenous microbiota are sum- from the Greek language and can roughly be trans-
marized in Table II. lated into ‘for life’. According to Vergin (1954) (27),
For a long period of time, roughly for the second in Germany the term ‘Probiotika’ was originally
half of the last century, the use of antibiotics has coined by the physician and dietician Werner Kollath
generally been recommended in human medicine shortly after the Second World War, meaning ‘food
for changing an unwelcome microbial colonization ingredients with health-promoting characteristics
of distinct sites of the body, i.e. in cases of diseases beyond their nutritional value’. Later and indepen-
caused by infectious microorganisms. However, dently, the term ‘probiotic’ was introduced into the
the increasing recognition of adverse effects and the international literature by Lilly and Stillwell (1965)
development and spread of resistance to a (28), saying that a probiotic is a microbial substance
wide range of antibiotics by a growing number of that is produced by one microorganism and has a
growth-promoting effect on another microorganism,
Table II. Gastrointestinal diseases associated with disturbances of
the intestinal microbiota.
thus being the exact opposite of an antibiotic. How-
ever, this definition did not gain wide acceptance and
Diseases Examples failed to survive. Since that time, the meaning of the
Infectious diseases Cholera, salmonellosis, term ‘probiotic’ has changed considerably. Parker
shigellosis, infections by (1974) (29) was the first to use the word ‘probiotic’
Campylobacter, pathogenic in a sense that is very similar to today’s use. He
E. coli (ETEC, EPEC, adopted the term to describe ‘organisms and sub-
EHEC, etc.)
Functional diseases Irritable bowel syndrome
stances which contribute to intestinal microbial bal-
(IBS), non-ulcer dyspepsia, ance’. This definition associated the use of probiotics
habitual constipation with an effect on gut microecology. Since Parker’s
Inflammatory bowel diseases Ulcerative colitis, Crohn’s definition would also include antibiotics (‘sub-
(IBD) disease, collagenous colitis, stances’), Fuller in 1989 redefined a probiotic as
Whipple’s disease
Diseases associated with Diverticulosis (cave:
‘a live microbial feed supplement which beneficially
morphological or anatomical diverticulitis), strictures, affects the host animal by improving its intestinal
changes blind loops, carcinoma microbial balance’ (30). In 1995, a workshop hosted
Diseases caused by Celiac disease, food allergy by the Lactic Acid Bacteria Industrial Platform
immunological disturbances (LABIP) and sponsored by the European Community
Iatrogenic diseases Diarrhea/functional diseases
after treatment with
issued a consensus definition of the term probiotics,
antibiotics, chemotherapeutic saying ‘oral probiotics are living micro-organisms,
agents or radiation which upon ingestion in certain numbers, exert
ETEC, enterotoxigenic E. coli; EPEC, enteropathogenic E. coli;
health benefits beyond inherent basic nutrition’ (31).
EHEC, enterohemorrhagic E. coli. Accordingly, probiotics may be either consumed as
124 U. Sonnenborn et al.
food components or applied as non-food prepara- in livestock breeding and as active components of
tions. The latest definition of the FAO/WHO expert medical remedies in human and veterinary medicine
committee (see above) broadened the meaning by [19,38–42]. Usually, especially in the food industry,
omitting the relation to solely oral administration probiotics mostly are lactic acid bacteria (LAB),
and intestinal effects as well as to nutrition. Accord- such as lactobacilli, lactococci, bifidobacteria, and
ing to this definition, also microbial preparations for streptococci (enterococci) (43). However, other non-
medical use and for extraintestinal, e.g. vaginal, pathogenic microorganisms, e.g. certain yeast strains
applications can be termed probiotics, provided that (‘Saccharomyces boulardii’) and Escherichia coli strains,
they contain living microorganisms and confer a like E. coli Nissle 1917 (EcN), are also used, not in
health benefit on the host. For microorganisms with food processing, but rather in human and veterinary
specific therapeutic properties the term ‘biothera- medicine (19,21,33,44–47).
peutic agent’ (instead of probiotic) has been pro-
posed by McFarland and colleagues (32,33). This
Origin and medical history of E. coli strain
may be helpful to differentiate between food and feed
Nissle 1917
probiotics and pharmaceutical probiotics, but has
not gained broader acceptance in the scientific and E. coli strain Nissle 1917 (EcN) is the active compo-
medical literature, mainly because this term is close nent of the pharmaceutical preparation Mutaflor®,
to the terms ‘biologicals’ or ‘biological agents’, which a microbial drug licensed for use in human medicine
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include native and recombinant biological prepara- in Germany and some other European countries
tions, such as vaccines, immunomodulators, interleu- today. This drug has been traditionally used since
kins, growth hormones, specific antibodies, and 1917 (48,49) to treat various diseases and dysfunc-
others (34–37). The growing scientific and medical tions of the intestinal tract (19,21,47,50–52).
interest in probiotics is reflected by the steadily The Mutaflor® story began in the early years of
increasing number of publications on this topic, as the last century when the physician and bacteriolo-
shown in Figure 1. gist Alfred Nissle (Figure 2) of Freiburg, Germany,
screened human intestinal E. coli strains for growth-
inhibiting (antagonistic) activity against Salmonella,
Application of probiotics
Shigella, and other enteropathogens. Searching for a
Probiotic microorganisms are often found as food novel therapeutic approach to combat virulent enter-
ingredients, e.g. in fermented milk products and food obacteria, Nissle recognized that some fecal E. coli
supplements, but are also employed as feed additives isolates showed antagonistic action against these

2008 140 1059


159 926
2006 127 775
101 700
2004 60 530
42 438
2002 40 416
22 299
2000 24 206
Year

14 169
1998 7 119
8 60
Search term "probiotic*"
1996 4 31
23 Search term "probiotic*" limited to article type "clinical trials"
1994 1 12
2 16
1992 8
7
1990 6
0 200 400 600 800 1000 1200
Number of publications

Figure 1. The increase in basic scientific and clinical publications on probiotics during the years 1990–2008, 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.
E. coli strain Nissle 1917 as probiotic 125
deposit’ rules at the Deutsche Sammlung von Mikro-
organismen und Zellkulturen (German Collection
of Microorganisms and Cell Cultures) (DSMZ, Braun-
schweig) as E. coli DSM 6601.] 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.
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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
Figure 2. Photographic portrait of Professor Alfred Nissle MD gene clusters located on genomic islands (GEIs) on
(1874–1965), discoverer of the antagonistic action of certain its chromosome responsible for the synthesis of sev-
commensal E. coli strains against enteropathogens. In 1917, Alfred
Nissle isolated the specific E. coli strain, now called E. coli Nissle
eral so-called ‘fitness factors’, which contribute to
1917 (EcN), from the feces of a healthy young man (48,49). the strain’s probiotic nature. Serologically, EcN
Photograph courtesy of Alfred-Nissle-Gesellschaft, Hagen, belongs to the E. coli O6 group and is of serotype
Germany. O6:K5:H1 (54,55). EcN is a typical gram-negative
enterobacterium containing lipopolysaccharide
pathogens in vitro, while others failed to inhibit their (LPS) as a structural component of its outer cell
growth. This led Nissle to the idea that these antag- membrane. The O6 surface antigen represents the
onistically active E. coli strains might be useful as outer part of the strain’s LPS and exhibits some
therapeutics in diarrheal diseases. Nissle isolated the peculiar features.
EcN strain in 1917 during the First World War from The molecular structure of the LPS of EcN has
the feces of a member of the armed services who took been completely resolved (55). The LPS of EcN dif-
part in the military campaign on the Balkan peninsula. fers in several molecular aspects from all other LPS
In contrast to his companions, this soldier did not types of E. coli (57). For instance, the O6 polysac-
develop infectious diarrhea when staying in the charide side-chain is very short, consisting of only
region of Dobrudja, which was heavily contaminated one single ‘repeating unit’ of the oligosaccharide
with enteropathogens at that time (49). Nissle building block typical of the O6 antigen, giving the
assumed that this soldier harbored a ‘strong, antago- strain a so-called ‘semi-rough’ phenotypic appear-
nistically active’ E. coli strain in his gut that had pro- ance when grown on solid nutrient media. The rea-
tected him from infection. This was in fact the case, son for this is a point mutation introducing a stop
and Nissle – after performing laboratory tests and codon in the gene for the O6 antigen polymerase
some self-experiments with this E. coli isolate – intro- (55). Also, modifications previously unknown for
duced the EcN strain into medical practice. From E. coli LPS were found in the oligosaccharide core
the beginning, the Mutaflor® preparation contained segment of the molecule. The specific features of the
(and still contains) the Nissle strain in viable form. LPS of EcN are likely to explain the phenomenon
[Note that, today, E. coli Nissle 1917 (EcN) is depos- whereby the strain exhibits immunomodulating prop-
ited as an industrially used strain under ‘patent erties without showing immunotoxic effects (55).
126 U. Sonnenborn et al.
Table III. Basic microbiological and molecular genetic characteristics of E. coli strain Nissle 1917 (EcN, serotype O6:K5:H1).

Presence in EcN
Gene products/
Characteristics biosynthesis results Genes/DNA probes Genotype 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)
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Shiga toxin† stx – –


Iron acquisition Enterobactin ent ND +
(siderophores) 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 pMUT1 (3173 bp)a Muta 5/6 + ND
elements: pMUT2 (5526 bp)a Muta 7/8, + ND
two cryptic, Muta 9/10 + ND
non-transferable
plasmids∗∗

+, 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.
aData extracted from G. Blum-Oehler et al. (53).

As indicated by its serotype (O6:K5:H1), EcN is existence of a capsule protects the pathogen from
able to form an extracellular capsule of the K5 sero- attacks by non-specific defense components of blood
type. This kind of capsule is present in only about serum (e.g. complement), thus making the bacterium
1% of E. coli isolates. The gene loci on the chromo- serum-resistant (59). Serum resistance lengthens the
somal DNA responsible for the synthesis of the K5 time of survival in the bloodstream and increases the
capsule have been detected by using a gene probe virulence of a pathogen (59). Interestingly, this is not
specific for the K5 capsule gene cluster (58). In many the case with EcN (55). Despite its capability to form
extraintestinal pathogenic E. coli (ExPEC) strains the a capsule, in the classic serum resistance test (60) the
E. coli strain Nissle 1917 as probiotic 127
EcN strain is nevertheless serum-sensitive and is EcN was detected by Nissle in 1917 because of
rapidly killed in the presence of human serum or its growth-inhibiting (antagonistic) activities against
sera of other mammalian species (bovine and porcine enteropathogenic microorganisms. The antagonistic
sera). actions of EcN are due, at least in part, to the forma-
EcN possesses flagella of serotype H1 and is thus tion of microcins against which the producer strain
quite mobile. The possession of flagella enables the itself is immune. One of these microcins (microcin
microbe to actively move within the viscous intestinal H47) had already been described before for another
mucus layer, e.g. in the direction of the intestinal E. coli strain (76–78), whereas the second microcin
mucosa, which serves as an oxygen source, useful for was unknown so far. The latter has been termed
aerobic catabolism of substrates by E. coli. Besides ‘microcin M’ (M from Mutaflor) by Klaus Hantke
their function as driving apparatus, the flagella are and colleagues (79).
important in bacterial crosstalk with the epithelium In addition to the production of microcins, EcN
(see below) and have also been described as bacterial belongs to that phylogenetic subgroup of E. coli
sensors for humidity (61,62). bacteria (group B2) that contains strains able to
EcN possesses three different types of fimbriae: synthesize peptide/polyketide hybrids (80,81). These
F1A and F1C fimbriae (54,63,64), as well as so-called substances belong to a heterogeneous group of mol-
‘curli’ fimbriae (56,65,66), which mediate adhesion ecules with antimicrobial and antitumor activities. To
to intestinal epithelial cells in cell culture experiments date it is not known whether the peptide/polyketide
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or to the mucus layer of the intestinal wall in vivo (see hybrides of EcN contribute to the antagonistic action
below), facilitating colonization of the gut. against other bacteria.

Fitness factors Metabolic properties

Pathogenic bacteria are able to produce so-called EcN has been characterized biochemically by analysis
virulence factors, which enhance the infectious of its strain-specific metabolic capacities and fermen-
capacity of the respective strains. Virulence factors tation properties, using commercially available identi-
are often encoded by genes representing special fication kits for Enterobacteriaceae in which a range of
DNA sequences located on the bacterial chromo- biochemical tests are carried out simultaneously.
some, probably obtained by horizontal gene transfer
in the evolution of the pathogen (67). These DNA Table IV. Taxonomically important enzymes present in and
biochemical reactions performed by E. coli strain Nissle 1917
sequences are called pathogenicity islands (PAIs) (EcN); comparison with the corresponding characteristics typical
(68). Non-pathogenic (avirulent) commensal bacte- of the enterobacterial species Escherichia coli in general.
ria may also produce specific factors that enable
E. coli in general
them to compete with other strains in the ecological
system of the gut and to effectively communicate Activity/enzyme EcN (+/–/v) (% positive)
with the host organism. These factors are designated Oxidase – – 0
fitness factors, which are also often encoded by spe- Dextrose metabolism + + 100
cial DNA sequences (genomic islands, GEIs) on the Voges–Proskauer reaction – – 0
bacterial chromosome (69,70). Different fitness fac- β-Galactosidase (ONPG test) + + 95
tors have been detected in the EcN strain in recent H2S production – – 1
Lysine decarboxylase + + 87
years.
Arginine dihydrolase + – 7
Siderophores are iron-chelating substances Ornithine decarboxylase + + 72
needed for bacterial iron uptake. EcN produces an Urease – – 0
unexpectedly wide array of siderophores: aerobactin, Citrate metabolism – – 1
enterobactin, salmochelin, and yersiniabactin, as well Malonate metabolism – – 0
Tryptophan deaminase – – 0
as a hemin- and a citrate-dependent iron acquisition
Indole production + + 98
system. Besides the siderophore ferric iron uptake Lactose metabolism + + 91
systems, EcN also possesses an elemental ferrous Saccharose metabolism – v 44
iron uptake system (EfeU) (71). Regarding the Arabinose metabolism + + 99
siderophores aerobactin and yersiniabactin as well as Raffinose metabolism – – 27
the hemin and the citrate system of EcN, the chro- Rhamnose metabolism + + 87
Sorbitol metabolism + + 94
mosomal DNA of the strain has been checked for the Inositol metabolism – – 2
existence of the corresponding genes by using spe- Ribitol metabolism – – 2
cific DNA probes (72–75). Positive signals for all +, positive reaction; –, negative reaction; v, variable.Tests performed
four iron acquisition systems could be obtained on with commercially available test kits: MHK/ID/GNI system
its genomic DNA. (Biotest) and API 20E system (BioMérieux).
128 U. Sonnenborn et al.
As far as its metabolic capacities are concerned, EcN potential integration sites for DNA sequences (GEIs)
is a typical E. coli strain, with the exception that it is obtained by horizontal gene transfer during evolu-
capable of metabolizing arginine (Table IV). This prop- tion; (2) DNA sequence analysis of the detected
erty is seen in just about 7% of all E. coli isolates. genomic islands; and (3) comparison of the EcN
Gas chromatographic analysis of spent culture genome with those of other E. coli strains by DNA–
supernatants (82) has revealed that EcN produces DNA hybridization (56). PCR-based screening of
short-chain fatty acids as end products of carbohy- 324 pathogenic and non-pathogenic E. coli strains of
drate metabolism under aerobic and anaerobic different origin revealed distinct chromosomal regions
growth conditions as well. These are mainly acetic occurring in non-pathogenic E. coli and in intestinal
acid and formic acid, as well as minor amounts and extraintestinal pathogenic E. coli as well. In EcN,
of propionic and butyric acid (G. Sollorz and five large genomic islands and some smaller genomic
U. Sonnenborn, unpublished results). 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
Molecular genetic properties chromosome are thought to originate from horizontal
gene transfer (56). In a dissertation by Schmidt, addi-
Genome structure
tional DNA sequences were found on the chromo-
The genome structure of E. coli Nissle 1917 has been some of the Nissle strain that also might have been
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elucidated by three different approaches: (1) DNA acquired by horizontal gene transfer during the
sequence analysis of the gene loci for tRNA genes as evolution of EcN (83). Comparison of the genome
fim

leuX leuV
pheU aspV
glyY thrW
thrT
argU
proM csg
wa* glnX
bcs
selC lysQ

proK serW

mch/mcm, foc, iro


chu
Chromosome of serT
wrbA
E. coli Nissle 1917 serX
5.1 Mb
leuU
metY tyrV
iuc

GEI IEcN
, sa

pheV
leuZ (97 kb)
t , ih

glyU serU
metV asnT
a, s

argQ asnW valW


ap,

ileY asnU
kps

ssrA alaX
asnV
lysV argW proL
GEI IIEcN
(103 kb) GEI IVEcN
ybt

(30 kb)
sat w b*

GEI IIIEcN clb/pks


(38 kb)
GEI VEcN
(55 kb)

Figure 3. Functional genomic map of E. coli Nissle 1917 (EcN) (56,80). 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 CFT073). 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.
E. coli strain Nissle 1917 as probiotic 129
structure of EcN with those of the E. coli K-12 strain molecular masses of Mr  2.1 and 3.7 Md (mega-
MG1655 and the uropathogenic E. coli (UPEC) O6 daltons), respectively, are genetically stable and are
strains 536 and CFT073 revealed similarities in gene not transferable to other E. coli strains. The smaller
structure and organization between the different plasmid (pMUT1) consists of 3173 bp (base pairs),
E. coli strains, especially between the isolates of sero- the larger one (pMUT2) consists of 5526 bp. The
type O6. With respect to the genome size of the EcN circular DNAs of these two strain-specific plasmids
strain, with 5.1 Mb its genome is larger than that of have been completely sequenced (53). Strain-
E. coli K-12 strain MG1655 (4.7 Mb) and UPEC specific DNA sequences from both plasmids, which
strain 536 (5.0 Mb), but a bit smaller than that of have not been found so far in other E. coli strains,
UPEC strain CFT073 (5.23 Mb). can be used in a multiplex PCR method developed
The lack of gene clusters coding for classical to specifically identify the EcN strain (53) (Figure
pathogenicity traits of ExPEC, e.g. α-hemolysin, 4). The function of these cryptic plasmids in EcN
P-fimbriae, S-fimbriae, combined with the existence is not known as yet, since plasmid-free clones of
of genes coding for fitness factors (e.g. iron acquisi- EcN do not show morphological, cultural, or met-
tion systems, microcins) and the genetically fixed abolic differences compared to the original strain
synthesis of a semi-rough LPS may together be of carrying pMUT1 and pMUT2. There is, however,
importance for the probiotic nature of EcN (84). some evidence that cryptic plasmids per se may
First results of the annotation of DNA sequences protect the carrier bacterium from attacks by
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obtained after shotgun sequencing of the whole EcN mobile genetic elements such as bacteriophages,
genome have been published, focusing on genes rep- thus having an influence on the genetic stability of
resenting the metabolic capacities of the Nissle strain the strain (86).
(85) and on the search for genes coding for suspected
pathogenicity or fitness traits (83).
Modes of action
Plasmid characterization
Antagonistic activities against other
EcN possesses two small cryptic plasmids, termed microorganisms
pMUT1 and pMUT2 (53). Both plasmids, with
E. coli Nissle 1917 has been tested for its antagonis-
tic activities (antimicrobial effects) against other
microorganisms, in particular against microbial
pathogens, both in vitro and in vivo. In vitro, antago-
nistic 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
427 bp in suitable liquid media, where the number of viable
361 bp bacteria of each strain has been determined after
313 bp
aerobic growth at 37°C, or on solid media by means
200 bp of an agar diffusion test (similar to the classic test for
antibiotic sensitivity), where EcN produced an inhi-
bition zone against the test strain (in vitro tests
according to Halbert (89) and Mayr-Harting et al.
(90), Figure 5). Co-cultivation of EcN with Shiga
108 107 106 105 104 103 102 [cfu/ml] toxin-producing E. coli (STEC) in liquid cultures
resulted in a dose-dependent decrease of Shiga toxin
Marker EcN
(100 bp ladder) levels (87).
The antagonistic action of EcN was also confirmed
Figure 4. Strain-specific multiplex PCR for identification of E. coli in vivo by means of co-association of EcN with other
Nissle 1917 (EcN). PCR and agarose gel electrophoresis were microorganisms (pathogenic and non-pathogenic
performed as described (53). The PCR yields three distinct DNA
products of Mr 313, 361, and 427 bp length. Using this test
E. coli, Salmonella typhimurium, Candida albicans,
system, the detection limit of EcN is between 102 and 103 bacterial Lactobacillus johnsonii) in germ-free or gnotobiotic
cells/ml. Photograph: K. Eiteljörge, Herdecke. animals (rats, mice, piglets) (91–94) (Figure 6).
130 U. Sonnenborn et al.
Germ-free animals have been inoculated per os either
with EcN and the particular pathogenic strain simulta-
neously, or the test strain has been administered first
and EcN several days later. The excrements of the ani-
mals have been regularly analyzed microbiologically,
whereby the viable cell counts of the administered bac-
teria 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 patho-
genic microorganisms disappeared (92–94). In conven-
tional streptomycin-treated CD-1 mice, precolonization
of the intestine with EcN limited the growth of E. coli
EDL933, an O157:H7 Shiga toxin-producing entero-
hemorrhagic 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
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Figure 5. Determination of antagonistic activity of E. coli Nissle EHEC strain.


1917 (EcN) against another E. coli strain (DSM 423) in vitro. The inhibitory effects of EcN on facultative and
A suspension of E. coli DSM 423 was homogeneously spread obligate pathogens have also been demonstrated in
across the surface of an agar plate (PAG minimal medium). humans by preventive oral administration of the
Small cylinders of agar were punched out using a punching tool
strain to neonates (colonization prophylaxis) (96,97).
and the resulting cavities were filled with 10, 20 or 50 μl of an
overnight culture of EcN (2  109 cfu/ml). Tests were performed The stools of the neonates were analyzed microbio-
in duplicate. The picture was taken after 24 h of aerobic logically to confirm colonization with EcN. In addi-
incubation at 37°C. Antagonistic activity becomes visible by the tion, the effect of colonization with this E. coli strain
development of inhibition zones (halos) around the cavities on the spectrum of other aerobic gut bacteria has
containing the EcN suspension. Photograph: U. Sonnenborn,
been determined. All newborns that received EcN
Herdecke.

Successive diassociation
Viable cell counts (cfu log10/g faecal mass, wet wt)

11
10

9
8
E. coli strain 542/88
7
E. coli strain Nissle 1917
6
5
4
3
2
1
0
2 4 6 8 10 12 18 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 108 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 1010 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  108 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.
E. coli strain Nissle 1917 as probiotic 131
100
Colonized infants (+ EcN) *
90
Placebo infants (– EcN)
80
Infants with pathogens (%) 70 *
*
60

50
40

30

20

10

0
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
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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 (108 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  0.003), day 5 (15% vs 62%, p  0.001), and after 6 months (28% vs 85%, p  0.002). 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 protein export machinery of the RND type contain-
them showing long-term colonization for several ing the membrane protein TolC.
months. The establishment of unwanted microor-
ganisms, i.e. facultative and obligate pathogens, in
Inhibition of the invasion of epithelial cells by
the children’s gut was significantly reduced by inten-
enteroinvasive bacteria
tional colonization with EcN (Figure 7, see also next
section). In cell culture experiments using the human embry-
In patients with ulcerative colitis in remission, onic intestinal cell line INT407, EcN inhibits the
mucosal biopsies have been obtained during sig- invasion of epithelial cells by different pathogenic
moidoscopies, both before and after a 6-week oral invasive bacteria, such as Salmonella, Shigella, entero-
treatment with EcN. The samples were analyzed for invasive E. coli, Listeria, Yersinia, and Legionella strains
their content of mucosa-associated aerobic and (99,100), as well as adherent-invasive E. coli
anaerobic intestinal bacteria using classic micro- strains from patients with Crohn’s disease (101).
biological cultural methods and quantitative PCR. Interestingly, this inhibitory effect is not due to the
Treatment with EcN resulted in a significant production of microcins, since the isogenic, microcin-
decrease of bacterial counts of aerobic and anaero- negative EcN mutant H5445 in this test system
bic mucosa-associated bacteria in a majority of the exhibits nearly the same inhibitory efficacy as the
patients (98). original strain (Figure 9). In addition, it has been
As stated above, the antagonistic actions of EcN shown that no physical contact is necessary for
are mainly due to the formation of microcins (micro- the anti-invasive effect, neither between EcN and
cin H47, microcin M). The genetic organization of the epithelial cells nor between EcN and the inva-
the two chromosomally localized gene clusters sive bacterial pathogens. Moreover, the anti-invasive
responsible for synthesis, transport, and secretion of activity of EcN is not plasmid-coded and is not
both microcins discovered to date in EcN as well as dependent on the presence of adhesins (F1A fim-
for the immunity of the strain against these antimi- briae, F1C fimbriae), since isogenic, Fim- and Foc-
crobial substances, has been examined in detail (79). negative deletion mutants of EcN have been shown
The DNA sequences of the microcin genes have to be as effective as the original strain. With the help
been determined (79) (Figure 8). Like colicin V, both of a so-called ‘transwell’ filter system introduced into
microcins of EcN are secreted with the help of a the cell culture, by which EcN can be separated from
132 U. Sonnenborn et al.
73 aa
39 69 75 aa 516 aa 150 aa 424 aa 698 aa 92 aa 228 aa

I I

mchX Open reading mcmI


mchI frame mcmD mcmA mcmB mcmC mcmP
mchB (mchC ?)

Microcin H47 gene cluster: Microcin M gene cluster:


mchB Structural gene of microcin H47 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 Export genes


mcmA

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
Downloaded by [201.75.65.196] at 11:37 25 December 2017

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 Immunomodulatory and anti-inflammatory
has been demonstrated that the invasion-inhibiting effects
principle is diffusible and is secreted by EcN (99,100).
It might be that this soluble factor inhibits the In vitro and in vivo experiments have demonstrated
manipulation of the host cell cytoskeleton by the EcN to possess remarkable immunomodulatory
pathogenic microorganisms. activities, which address both the innate and the

(a) 45 (b) 45
Co-incubation

E. coli K-12 + S. T. LT2


40 40
of S.Typhimurium (%)
S. Typhimurium LT2

35 35
Invasive activity (%)

Invasive activity

EcN + S. T. C17
E. coli K-12 + S. T. C17

30 30
S. Typhimurium C17

25 25
20 20 EcN + S. T. LT2
E. coli K-12

15 15
10 10
EcN

5 5
0 0

(c) 45
40
of S. Typhimurium (%)

Microcin-negative mutant of
% Invasive activity

35
EcN (H5445) + S. T. C17

Transwell
unit 30
EcN 25
S. Typhimurium
EcN + S. T. C17

20
Intestinal epithelial
cells INT407 15
S. T. C17

10
5
0

Figure 9. Inhibition of Salmonella typhimurium (S.T.) invasion into INT407 intestinal epithelial cells by E. coli Nissle 1917 (EcN) (99,100).
(a) Cell cultures with INT407 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 INT407 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).
E. coli strain Nissle 1917 as probiotic 133

adaptive immune system. With respect to adaptive immune reaction. On the other hand, Otte et al.
immunity, EcN affects the humoral as well as the (117) found that contact of intestinal epithelial cells
cellular branch of the immune system. However, in (Colo320 and SW480 cells) with EcN led to inhibi-
vivo significant immunomodulating activities are tion of the inflammatory response by down-regulation
only reliably detected in gnotobiotic experimental of TNF-α- or gastrin-induced cyclooxygenase-2
animals, in human newborns, or in diseased animals (COX-2) activity. This was followed by reduced secre-
and patients. In healthy human adults and animals, tion of proinflammatory prostaglandin E2 (PGE2).
the immunomodulating activities of EcN are only The immunomodulating action of EcN on periph-
very weakly expressed or not detectable at all eral as well as on gut mucosal lamina propria T lym-
(102,103) (R. Gruber, unpublished results). phocytes has also been studied in vitro (112).
Anti-CD3-stimulated peripheral and lamina propria
In vitro experiments. A significant dose-dependent T cells have been exposed to heat-inactivated cells of
improvement of secretory performance of mouse EcN and to EcN-conditioned medium (spent culture
macrophages after direct contact with formalin-killed supernatants), and the effects of these treatments on
cells of EcN has been observed in vitro (104). The lymphocyte cell cycle and on markers of apoptosis
production of interleukin-6 (IL-6), tumor necrosis have been examined on the molecular level. EcN-
factor (TNF), and oxygen radicals increased signifi- conditioned medium dose-dependently inhibited cell
cantly. Further experiments revealed an augmenta- cycle progression and T-cell expansion of lympho-
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tion of tumor cell lysis and parasite lysis (leishmaniae) cytes from peripheral blood, but not of lymphocytes
by macrophages prestimulated with EcN. On the from the gut mucosa. This selective action on T cells
other hand, the efficiency of phagocytosis of the mac- from peripheral blood could also be shown by using
rophages was raised only slightly. However, these in bacterial lipoproteins. In contrast to this, heat-inac-
vitro effects could only partially be confirmed in vivo, tivated cells of EcN, its purified LPS, or CpG-DNA
following oral administration of EcN to convention- had no effect. In peripheral T cells, addition of EcN-
ally kept mice. In vivo, for instance, no induction of conditioned medium reduced expression of cyclin
TNF synthesis has been observed. D2, B1, and retinoblastoma protein, which are
Experiments with epithelial cell lines and isolated involved in T-cell proliferation. Interaction of EcN-
lymphocytes have shown EcN to exhibit different conditioned medium with peripheral T cells signifi-
immunomodulating activities, and these strongly cantly inhibited the expression of IL-2, TNF-α, and
depend on the cell types/cell lines employed and the IFN-γ, whereas the production of IL-10 was enhanced
parameters and read-out systems used (105–115). (Figure 10). The differential effects of EcN on circu-
One example is the dose-dependent inhibition of the lating and intestinal T cells is supposed to be an
TNF-α-induced IL-8 secretion by EcN in HCT-15 important aspect of its efficacy in inflammatory
intestinal epithelial cells (108,116). The inhibition is bowel diseases (112,118). By down-regulation of the
reflected by the reduced expression level of IL-8 mRNA expansion of newly recruited T cells into the gut
in the epithelial cells, as shown by real-time PCR. mucosa, intestinal inflammation will be limited.
The inhibition of IL-8 synthesis is caused by secreted Compared with this, activated T cells already resid-
soluble factors that have not been identified so far. ing in the gut wall are not influenced by EcN and
Interestingly, the EcN-mediated inhibition of TNF- thus are available for the continuous fight against
α-induced IL-8 transcription is independent of the pathogenic microorganisms (112). In the T-cell sub-
AP-1- and nuclear factor κB (NF-κB)-regulated group of γδ T cells, EcN increased activation, cell
gene expression. The latter, however, is important for cycling, and cell expansion, but thereafter induced
the induction of human β-defensin-2 (HBD-2) synthe- apoptosis via caspase- and FasL-dependent signaling
sis in gut epithelial cells (Caco-2 cells) by EcN (114). pathways (119). These effects of EcN were target-
In the mouse monocyte/macrophage cell line specific, since they were only seen with γδ T cells, but
J774A.1, addition of EcN resulted in an increase of not with αβ T cells.
IL-12, TNF-α, and IL-10 secretion (105). By con- Using human peripheral blood mononuclear
trast, IL-18 and TGF-β synthesis were not affected. cells, Helwig et al. (107) compared the immuno-
Compared with the effects of viable EcN cells, addi- modulating activities of gram-positive Lactobacillus
tion of killed bacterial cells resulted in a much less and Bifidobacterium strains with those of the gram-
pronounced induction of cytokine synthesis. In con- negative EcN strain. These experiments revealed that
fluent Caco-2 epithelial cells, contact with EcN gram-positive and gram-negative bacteria exerted
resulted in an up-regulation of gene expression of partly different, but also partly similar immunomod-
monocyte chemoattractant protein-I ligand 2 ulatory activities on the blood mononuclear cells
(MCP-I) and an increase of MCP-I secretion (113). (Figure 11). With respect to some special immune
MCP-I is thought to be involved in the inflammatory parameters, different responses were seen also
134 U. Sonnenborn et al.

*
of different bacteria including EcN resulted in differ-
14
* ent maturation responses of dendritic cells from dif-
12 ferent locations of the body (Peyer’s patches,
mesenteric lymph nodes, and spleen).
Cytokines (ng/ml)

10
* Recently, in vitro experiments with isolated blood
8 *
lymphocytes from healthy and allergic persons (111)
6
* have shown that the EcN strain induced strong anti-
*
4 * allergic responses. These are interesting new findings
2 that may lead to new indications for EcN in the
0
future. The investigations with mononuclear cells
IL-2 TNF-α IFN-γ IL-10 from peripheral blood, obtained from allergic per-
Control with
10%
EcN-conditioned medium
sons, have shown that contact of the lymphocytes
25% with EcN induced a shift from a T-helper-2 (Th2)-
Figure 10. Anti-inflammatory effects of E. coli Nissle 1917 (EcN) dominated, allergy-specific immune response to a
on human peripheral blood T cells (PBT) in vitro, as shown by Th1-dominated, non-allergic response (111).
cytokine expression profiles induced by EcN-conditioned medium
(EcN-CM) (112). EcN-CM was obtained after 2 h incubation of In vivo experiments. In young gnotobiotic piglets,
EcN bacteria in T-cell medium (RPMI with 10% FCS, 1.5% colonization with EcN resulted in a rapid develop-
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HEPES) at 37°C and 5% CO2. Thereafter, bacteria were removed ment of the gut-associated immune system. This was
by centrifugation. The resulting supernatant was sterile-filtered reflected by a significant increase in the numbers of
through a filter of 0.22 μm pore size.The sterile-filtered supernatant
(EcN-CM) was added to freshly isolated PBT (105 cells) in
IgA, IgM, and IgG antibody-producing lymphocytes
different concentrations (open bars, controls  no addition of in the lamina propria of intestinal villi and in the
EcN-CM; hatched bars, 10% v/v EcN-CM; black bars, 25% v/v intestinal lymph follicles as well as by an increase in
EcN-CM). T cells were then stimulated by adding anti-CD3-mAb SLA-D+(MHC II+) cells and CD4-positive cells in
and cultured for 3 days in RPMI. Culture supernatants were all mucosal and submucosal tissues, without any
collected and assayed for cytokines. EcN-CM dose-dependently
and significantly inhibited IL-2, TNF-α, and IFN-γ synthesis,
signs of an inflammatory reaction (infiltration of
while IL-10 production was markedly up-regulated. ∗p  0.05 vs granulocytes) (93,120).
CD3 controls. Investigations on human newborns have revealed
that both full-term neonates and premature infants
between the bifidobacteria and the lactobacilli, lead- show a significant rise in the levels of immunoglobu-
ing to the assumption that the immunomodulatory lins (IgA and IgM) in blood serum and of secretory
action of probiotics might be rather strain-specific IgA and IgM in stool filtrates within a few days after
and not genus- or species-specific. Interestingly, in intentional colonization of the gut with EcN (121)
this test system the EcN strain provoked a high out- (Figure 12). On the other hand, there was no influ-
put of the anti-inflammatory signal molecule IL-10. ence on the IgG level in blood serum. Besides these
Fink and Frokiaer (106) could show that interaction immunomodulatory effects on the development of

* * *
9000
1600 *
TNf-α [pg/ml] area under the curve
IL-10 [pg/ml] area under the curve

8000
1400
7000
1200
6000
1000
5000
800
4000
600 3000
400 2000
200 1000
0 0
Bifidobacteria Lactobacilli EcN 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 (107). Supernatant concentrations of the cytokines IL-10 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-10 and TNF-α are
shown in pg/ml as area under the curve (AUC, mean  SE). ∗Significantly different, p  0.05.
E. coli strain Nissle 1917 as probiotic 135

% of reference
70
60
50
FC
Full-term children

IgA
40 colonized with EcN
30 PC n = 22 (FC)
20
10 P
0 Pre-term children,
before 3 5 14 21 Days colonized with EcN
n = 9 (PC)
20
18 FC
16 Pre-term controls
14 n = 7 (P)
12
IgM

10
8
6 PC
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4
2 P
0
before 3 5 14 21 Days

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 (108 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  0.05). IgA levels were significantly higher in full-term than in preterm infants on day 5
and day 14 (p  0.01), and IgM levels were higher on all days after colonization (p  0.05 … p  0.01). Preterm colonized infants had
higher fecal IgA and IgM titers than non-colonized preterm infants (controls) on all days after colonization (p  0.05 … p  0.01).

the humoral branch of the immune system, stimula- pretreated with 2% DSS led to a decrease of the
tion of cell-mediated immune responses and of non- proinflammatory cytokines IFN-γ and IL-6, without
specific natural immunity after oral administration of showing a significant effect on mucosal inflammation
EcN has also been detected in a controlled clinical (129). In another model of acute gut inflammation,
study with preterm infants (122). oral application of EcN to mice pretreated with 1.3%
In established animal models of inflammatory DSS significantly ameliorated body weight loss, dis-
bowel diseases (IBD), such as the IL-2 knockout ease activity index, as well as macroscopic and micro-
mouse, EcN shows no proinflammatory effects scopic damage of the intestinal mucosa (127).
(123,124). On the contrary, using different experi- Interestingly, administration of heat-killed EcN as well
mental animal models for IBD (T-lymphocyte transfer as of its genomic DNA also showed some anti-inflam-
model in SCID mice, IL-10 knockout mice, dextran matory actions in DSS colitis mice (reduced colon
sodium sulfate (DSS)-induced or trinitrobenzenesul- weight, lower histological score), although the effects on
fonic acid (TNBS)-induced colitis), anti-inflammatory the disease activity index (DAI) were not as marked as
effects of EcN have been consistently observed those achieved by viable bacterial cells (127) (Table V).
(118,125–130). In one model of acute intestinal In IL-10 knockout mice (IL-10–/– mice), an animal
inflammation, oral administration of EcN to mice 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 19 2.35  0.17 51.68  0.20 10.26  0.55


Live EcN 18 1.61  0.18∗ 45.43  1.01† 8.89  0.37†
Heat-killed EcN 16 1.83  0.12 46.40  1.25∗ 8.63  0.43†
EcN-DNA 17 1.94  0.20 45.26  1.16† 8.53  0.32†

DAI, disease activity index; DSS, dextran sodium sulfate; PBS, phosphate-buffered saline. Data are mean  SEM.
∗p  0.05 and †p  0.01, significant difference compared with PBS group (N. Kamada et al. (127)).
136 U. Sonnenborn et al.
administration of EcN led to a decrease of tissue proinflammatory cytokines and chemokines in colonic
lesions and a decrease of the infiltrations of the colonic Lamina propria mononuclear cells (LPMCs) of colitis
mucosa by mononuclear cells and polymorphonuclear mice (significant for IFN-γ and MIP-2).
cells, as judged by macroscopic and microscopic In the immunologically mediated chronic colitis
examinations (127). Moreover, EcN treatment sig- model of T-lymphocyte transfer in SCID mice, oral
nificantly reduced the number of mice with rectal application of EcN significantly ameliorated intestinal
prolapse (a characteristic symptom of severe rectal inflammation, as judged by histologic examinations,
inflammation), lowered the concentrations of the and reduced secretion of proinflammatory cytokines
inflammatory marker protein serum amyloid A (SAA) IFN-γ, IL-5, and IL-6, without affecting the level of the
in blood serum, and reduced the expression of anti-inflammatory cytokine IL-10 (129) (Figure 13).

(a) (d) 300000


5 250000

IFN-γ (pg/ml)
Clinical Score

4 200000
3 150000
* *
2 100000
1 50000
0 0
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PBS-treated T cell transfer mice PBS-treated T cell transfer mice


healthy mice – EcN + EcN healthy mice – EcN + EcN
+ EcN + EcN
(b) (e)
2,5 1800
Histological Score

* 1600
2 *
IL-6 (pg/ml)

1400
1200
1,5 1000
1 800
600
0,5 400
200
0 0
PBS-treated T cell transfer mice PBS-treated T cell transfer mice
healthy mice – EcN + EcN healthy mice – EcN + EcN
+ EcN + EcN

(c) (f)
250 60
* 50
CFU/g MLN

200
IL-5 (pg/ml)

40
150 *
30
100
20
50 10
0 0
T cell transfer mice PBS-treated T cell transfer mice
– EcN + EcN healthy mice – EcN + 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 200 μl of EcN suspension (5  1010 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  0.001 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  0.02 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  0.0001 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  0.03 vs
CD62L; (e) IL-6, ∗p  0.02 vs CD62L; (f) IL-5, ∗p  0.02 vs CD62L.
E. coli strain Nissle 1917 as probiotic 137
In both a rat model of TNBS-induced colitis and 7.3
a mouse model of LPS-induced sepsis, orally admin- 7.1
istered EcN exerted local as well as systemic anti- 6.9 * SPLEEN
inflammatory effects (125). TNF-α level was reduced 6.7 *
in the gut of colitic rats, while TNF-α and T-cell

L. monocytogenes
6.5 *
cytokines were reduced in blood plasma and lungs 6.3
*
of sepsis mice, and the IgG release from splenocyte- 6.1 *

derived B cells was down-regulated.


8.1 LIVER
Using Toll-like receptor 2 (TLR-2) and Toll-like

(log10 cfu/organ)
receptor 4 (TLR-4) knockout mice, it has been dem- * *
7.6
onstrated that the inhibition of T-cell proliferation *

and the amelioration of DSS-induced colitis by EcN *


7.1
is mediated via TLR-2- and TLR-4-dependent path- *
6.6
ways (112,118). 8.7
In an experimental allergy study conducted by 8.5
* KIDNEY
Bickert and colleagues (131), it was found that EcN 8.3 *

C. albicans
8.1 *
inhibits the allergen-inducedTh2-dominated immune 7.9 *
response in the airways of C57BL/6 mice that had
Downloaded by [201.75.65.196] at 11:37 25 December 2017

7.7
been hypersensitized by i.p. and i.v. administration 7.5 *
7.3
of ovalbumin. However, this anti-allergic effect could
only be demonstrated, if EcN was given together Control IFNγ EcN
with the allergen. 106 107 108 109 cfu

Figure 14. Augmentation of host defense against systemic bacterial


Infection-preventing effects and fungal infections in mice by oral pretreatment with E. coli
Nissle 1917 (EcN) (132). Four groups of mice were pretreated
In in vivo experiments using conventionally housed once with 106, 107, 108, or 109 viable cells of EcN by oral
C3H/HeN mice, single oral doses of EcN inhibited administration, before they were challenged 24 h later by
proliferation of subsequently intravenously inocu- intravenous infection with Listeria monocytogenes (6  103 cfu) or
Candida albicans (5  105 cfu). For each infection model, further
lated Candida albicans and Listeria monocytogenes
groups of mice served as controls. These were either pretreated
infections in a dose-dependent manner (132) (Figure with placebo (negative controls, open bars) or with murine IFN-γ
14). In a placebo-controlled veterinary clinical trial (positive controls, gray bars). Three days after infection with L.
on 335 newborn calves it was shown that admini- monocytogenes and 1 day after infection with C. albicans, mice were
stration of EcN significantly (p  0.001) reduced sacrificed and the parasite burden of the respective main target
organs was determined. Compared with placebo, the pretreatment
the incidence of neonatal calf diarrhea from 63%
with IFN-γ resulted in a significant decrease of parasite load in
(placebo group) to 12% (EcN group) (133). 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 dose-
Communication with intestinal epithelial cells dependently reduced the parasite burden in spleen, liver, and
In the last few years, it has become obvious that kidneys. ∗p  0.05 vs negative controls.
many probiotic effects of orally administered micro-
bial preparations are due to interactions between the epithelial barrier function, i.e. EPEC-induced dis-
bacteria, the gut epithelial cells, and the gut immune ruption of epithelial cell tight junctions (Figure 15).
system (115,130,134,135). In particular, the com- This action of EcN seems to be mediated via the pro-
munication with the epithelial cell lining seems to be tein kinase C signaling pathway in the epithelial cells.
important, leading to a strengthening of the epithelial In another cell culture study, it has been observed
barrier function (115,130,136). that co-incubation of EcN with human epithelial cell
Thus, co-incubation of EcN with human intesti- lines T84 and HT-29 resulted in increased IL-8 secre-
nal epithelial cells (polarized T84 cells) resulted in a tion. Interestingly, the presence of EcN prevented
significant modulation (up- and down-regulation) of cell death of the epithelial cells, induced by exposure
the activity of about 300 different genes in the epi- to Salmonella dublin (110).
thelial cells, as demonstrated by transcriptome anal- Examinations of cell cultures of human intestinal
ysis using the microarray technique with DNA chips epithelial cells (Caco-2 cells, HT-29 cells) have dem-
(Affimetrix) (115,137). Moreover, after infection of onstrated EcN to stimulate the synthesis of inducible
T84 cells with an enteropathogenic E. coli strain antimicrobially acting defensins (HBD-2, HBD-3)
(EPEC E2348/69), treatment with EcN eliminated (114,138) (Figure 16). Induction of HBD-2 gene
the negative impact of EPEC infection on the expression by EcN has been shown to exceed several
138 U. Sonnenborn et al.

Control without bacteria EPEC 120 min EcN 120 min


A B C

Separate
incubation

EPEC + Medium 60 min EPEC + EcN 60 min EPEC + EcN 120 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 T84 epithelial cells (115). T84 monolayers were incubated with
Downloaded by [201.75.65.196] at 11:37 25 December 2017

bacteria for different periods of time and stained for ZO-2 using a fluorescent anti-ZO-2 antibody. (A) Control, T84 epithelial cells without
bacteria. (B) T84 cells incubated with EPEC for 120 min. (C) T84 cells incubated with EcN for 120 min. (D) T84 cells incubated with
EPEC for 60 min, then washed and further incubated with regular medium for another 60 min. (E) T84 cells incubated with EPEC for
60 min, then washed and further incubated with EcN for another 60 min. (F) T84 cells co-incubated with EcN plus EPEC for 120 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 40 other (139). The flagellin signal might be transmitted by
E. coli strains tested. By contrast, gene expression of TLR-5, which is present in Caco-2 epithelial cells,
constitutively synthesized HBD-1 defensin was not but other signaling pathways might also be involved
influenced by EcN. Stimulation of HBD-2 synthesis (139). Here, further investigations are required.
by EcN is mediated in the epithelial cell nucleus by In mice with DSS-induced colitis, the interac-
transcription factors NF-κB and AP-1 (114). The tion between EcN and the intestinal mucosa resulted
factor responsible for HBD-2 synthesis induction by in the restoration of the impaired permeability
EcN is the structural flagellum component flagellin barrier (‘leaky gut’) (Figure 17) (130). The contact
Relative transcript level of HBD-2

25 **
0,12 ***
20 * ***
***
Mucosal permeability to Evans Blue

0,10
15
(absorbance 620 nm [abs./cm])

10 0,08

5 0,06
0
830
860

915
861
864

863
867
835
865

862
866

868
870
ed

EC
E. c PEC
-12
EcN

0,04
ulat

UP
oli K
E
stim

E. coli strains PZ #
0,02
non

Figure 16. Stimulation of human β-defensin-2 (HBD-2) gene 0


transcription in Caco-2 intestinal epithelial cells by pathogenic Control DSS DSS + EcN
(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 Figure 17. Inhibition of ‘leaky gut’ phenomena by oral
pathogenicity (fecal isolates from healthy persons (PZ 860–915) administration of E. coli Nissle 1917 (EcN) to mice with dextran
and colitis ulcerosa patients (PZ 830, 835) (114,138). Caco-2 sodium sulfate (DSS)-induced colitis (130). Intestinal permeability
cells were stimulated for 4.5 h with 3  108 heat-inactivated to Evans Blue was determined in healthy mice (control, open bar),
bacteria/ml. Transcription of the HBD-2 gene was analyzed by mice with DSS-induced colitis (hatched bar), and mice treated
real-time PCR. HBD-2 gene expression is shown after stimulation with DSS plus EcN (black bar). Compared with healthy control
by the different E. coli strains, including probiotic E. coli Nissle mice, a significant increase in the uptake of Evans Blue by the
1917 (EcN). Data represent the means  SEM normalized to the colonic mucosa of DSS-treated mice was observed. This increase
basal expression of controls (set at 1) from one to six separate was strongly reduced in the group of mice treated with DSS plus
experiments run in triplicate. ∗p  0.0006; ∗∗p  0.0001. EcN to almost normal values. ∗∗∗p  0.001.
E. coli strain Nissle 1917 as probiotic 139
of EcN with the intestinal epithelial cells induced the small intestine (145). Another non-pathogenic E.
the de novo synthesis and the redistribution of tight coli strain (E. coli O86) and an enteropathogenic iso-
junction proteins, such as zonula occludens late (E. coli O55) did not show this effect in the gut.
protein-1 (ZO-1), from the cytosol to the cell In contrast to EcN and to E. coli O86, oral adminis-
membrane. This effect is important for the cohesive- tration of the enteropathogenic O55 strain provoked
ness of the epithelial cell layer. Concomitantly, an increase of calprotectin level in blood plasma and,
DSS-induced disturbances of epithelial transport in addition, a pronounced increase of calprotectin
functions, such as the strongly decreased activity of concentration in bronchoalveolar lavage that was
the membrane-bound sodium pump, were restored coincident with the development of septicemia.
(130). For bacterial–epithelial interactions, the pres- All these favorable effects induced by EcN are
ence of the K5 capsule in the EcN strain also seems thought to be due to communication between
to be important (140). the probiotic and the epithelial cells by so-called
Similar mechanisms of action of EcN are respon- bacterial–epithelial crosstalk (19,146–149), medi-
sible for the recently detected prevention of the ated via specific signaling molecules, most of them
development of gastric mucosal lesions in mice, being unknown up to now (Figure 18).
induced by acute stress (141). The authors showed
EcN to exhibit anti-inflammatory and blood flow-
Effects on visceral hypersensitivity and constipation
stimulating activities. Concomitantly, in the epithe-
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lial cells an increased synthesis of stress-protective The irritable bowel syndrome (IBS) belongs to
proteins, such as heat-shock protein 70 (Hsp 70), those functional bowel diseases that in about 25%
could be demonstrated. The stress-induced enhanced of patients have developed some time after an
gene expression of mucosal inflammatory mediators episode of acute infectious diarrhea (150,151).
and the activation of transcription factor NF-κB was This post-infectious IBS is characterized by a hyper-
down-regulated by EcN. sensitivity of the visceral sensory system of the gut
Calprotectin is a calcium- and zinc-binding pro- (so-called visceral hyperalgesia). In an established
tein, the synthesis of which is stimulated by LPS rat model of TNBS-induced visceral hyperalgesia,
(142,143). Calprotectin exerts antimicrobial activ- oral administration of EcN led to a significant
ity by binding zinc and inhibiting the adhesion of reduction of the visceromotor reflexes (VMR) of the
bacteria to epithelial cells of the gut mucosa (144). gut muscles, which were enhanced before as a con-
In 8-day-old germ-free piglets, oral administration of sequence of the short-term inflammation induced
EcN led to an increase of calprotectin synthesis in by TNBS (152).

E. coli Nissle 1917 Direct antagonistic


(EcN) activity
• Inhibition of growth /
• killing
of pathogenic bacteria
and fungi

• Production of • Anti • Restorative effect


defensins, inflammatory on the epithelial
cathelicidin, effects permeability barrier
calprotectin (inhibition of
IL-5, IL-6, IFN-γ, • Prevention of leaky
TNFα effect gut symptoms in
• Inhibition of on IL-8) case of disturbed
invasion barrier function
(Salmonella, EIEC, • Immuno-
AIEC, Shigella, modulation,
Yersinia, Listeria,
• Improvement of ion
e.g. IL-10 transport activity of
Candida) induction epithelial cells disturbed
by intestinal inflammation

Gut epithelium

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).
140 U. Sonnenborn et al.
Another functional bowel disease is chronic con- Colonization capability
stipation, where colonic motility is reduced and,
Following oral administration of E. coli Nissle 1917,
accordingly, intestinal transit slows down. In this
the strain exhibits good colonization capability in the
context, it is important that cell-free spent superna-
gut of gnotobiotic rats (92) and piglets (93). In gen-
tants of EcN cultures positively modulated colonic
eral, oral administration of EcN to different conven-
motility in an in vitro organ bath study with human
tionally kept mice and rats resulted in a longer-term
colonic circular smooth muscle strips (153). This
persistence or even a true colonization of the gut.
effect may be due, at least in part, to EcN’s ability
This has been shown for the original strain and for
to produce acetic acid, the main metabolic end prod-
recombinant and transformed EcN derivatives as
uct of its carbohydrate metabolism.
well (63,95,159,160). In NMRI mice, the good col-
onization capability of EcN is dependent on the pres-
Antimutagenic activity
ence of the regulatory protein RfaH, as has been
Microsatellite instability (MSI) in chronically shown by colonization experiments comparing the
inflamed colonic tissue has been suggested to be original EcN strain with a RfaH-negative mutant
associated with the increased risk of ulcerative colitis (161). In this context, RfaH does not seem to be
patients developing colorectal carcinoma. Substances important for bacterial adhesion to epithelial cells,
with anti-inflammatory activities, such as mesalazine but seems to enhance resistance of the EcN strain to
Downloaded by [201.75.65.196] at 11:37 25 December 2017

(5-aminosalicylic acid) or EcN, might improve MSI. bile acids. In vivo, the higher resistance to bile pro-
Therefore, Goel and co-workers (154) performed an vides better chances of survival during transit of EcN
ex vivo analysis of MSI in 156 biopsies from 39 coli- through the gastrointestinal tract (161).
tis ulcerosa patients who had been treated with either Colonization is thought to be facilitated by the
mesalazine or EcN in a randomized, double-blind capability of EcN to produce and secrete cellulose, a
clinical trial (155). Overall, 20% (31/156) of biopsies feature important for biofilm formation (162). Interest-
displayed MSI. After 1 year on medication, 6/20 ingly, EcN is able to form biofilms at 37°C (Figure 19).
patients showed MSI improvement (change to mic- In this respect the strain is different from other E.
rosatellite stability (MSS): 3 on mesalazine and 3 on coli strains, which are only able to produce biofilms
EcN). Worsening of MSI (change from MSS to MSI) at lower temperatures (30°C). It is thought that this
was also observed, but only in patients treated with characteristic of EcN might also be important for its
mesalazine (6/20). persistence in the gut after oral inoculation (63,162).
Using standard mutagenicity tests (Ames-test, Curli fimbriae of E. coli are important factors mediat-
Comet-assay) (156,157), it has been shown recently ing the formation of microcolonies, which represent
that EcN also exhibits antimutagenic activity against the first step in biofilm formation (163). Besides curli
some well-known mutagens, such as 4-nitroquinoline fimbriae, EcN expresses F1A and F1C fimbriae
1-oxide (NQO), benzo(a)pyrene, and H2O2 (158). (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. 120 years of E. coli – its
importance in research and medicine. Alfred-Nissle-Gesellschaft, editor. Hagen, p 33, 2006; with kind permission. Scanning micrographs
courtesy of U. Dobrindt and H. Merkert, University of Wuerzburg, Germany.
E. coli strain Nissle 1917 as probiotic 141
individual fimbriae types of EcN are localized on the However, these are special circumstances: in gnoto-
bacterial chromosome. Their existence on the chro- biotic animals and human neonates no interfering
mosomal DNA has been shown by using specific intestinal microbiota is present, which could prevent
DNA probes (164–167). The structural genes coding colonization by the ‘intruder’ EcN. In adults pre-
for the F1A fimbriae (fimA) and the F1C fimbriae treated with antibiotics plus lavage, the endogenous
(focA) have been sequenced and partially differ from microbiota is heavily disturbed, facilitating the settle-
corresponding fimbrial DNA sequences found in ment of the newly introduced probiotic strain.
other E. coli strains (53). In contrast to this, in most cases permanent col-
The parallel existence of multiple iron acquisition onization of the intestine by EcN cannot be achieved
systems (siderophores) is peculiar to the EcN strain. in healthy adults after oral administration, since the
They are thought to be ‘fitness factors’, which prob- present microbiota will try to prevent integration of
ably are necessary for competition with other micro- EcN into the existing microbial consortium. This is
organisms in the gut (69,84,168). It might also be also true for other probiotic strains (171). The indig-
one of the reasons for its ability to colonize or at least enous intestinal microbiota does not differentiate
to persist for some time in the intestine (79). between microbial intruders that might be harmful
Also in humans, long-term intestinal colonization to the host (pathogens) and other microorganisms
by EcN could be achieved following oral administra- that are useful (probiotics) (19). For the established
tion of the strain, but only under certain specific microbial consortium in the gut all foreign micro-
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conditions (96,97,121,169) (see also Colonization organisms entering the ecosystem are competitors
prophylaxis, below). In clinical trials with newborn for the locally available substrates and ecological
babies who received daily oral doses of 108 cfu of niches.
EcN, starting immediately after birth, for the first 5
days of life, the applied strain could be recovered
Toxicological potential and safety aspects
from stool samples for a period of several months
(biosafety)
(96,97). In adult volunteers, intentional disturbance
of the intestinal microbiota was achieved by treat- Investigations on possible adverse or even toxic
ment with ciprofloxacin and metronidazole, followed effects of probiotics are becoming more and more
by an orthograde gastrointestinal lavage. Thereafter, important in the relevant literature (172–179). This
EcN was administered orally for 14 days, and the holds true for probiotics containing new lactic acid
strain could be detected in stool samples after the bacteria that have no traditional use in humans, but
end of medication for individually different periods even more for probiotics with strains from bacterial
of time (up to 129 days in one volunteer) (169). species that also contain pathogenic variants, such as,
Accordingly, in a more recent study, after administra- for example, E. coli.
tion of the EcN strain for 1 week long-term persis- Since the species E. coli includes enteropatho-
tence of EcN in the gut of healthy adults was observed genic and extraintestinal pathogenic strains
only in about 50% of the volunteers after 2 weeks (54,68,180–184), particular attention has been paid
and in less than 10% after 48 weeks (170). to the toxicological and biosafety aspects of the Nissle
Taken together, the results obtained in gnotobi- strain (Table VI). As a general rule for probiotics
otic and conventional animals, in human newborns, from Enterobacteriaceae, selected probiotic candidate
and in adults treated with antibiotics and intestinal strains should not exhibit pathogenic adhesion fac-
lavage clearly show that in principle EcN is able tors and should not be able to form enterotoxins and
to colonize the gut, even in non-human species. cytotoxins or to invade epithelial cells. Moreover,

Table VI. Biosafety aspects of E. coli strain Nissle 1917 (EcN) important for preventive and therapeutic use in human and veterinary
medicine.

Positive biosafety aspects found/considered Negative biosafety aspects tested

Exact species and strain designation: E. coli strain Nissle 1917 No enterotoxin production
Complete serotype determination: serotype O6:K5:H1 No cytotoxin production
Clonal affiliation: a non-pathogenic subclone of the E. coli O6: No pathogenic adhesion factors
K5-lineage No invasiveness
Genetic stability: genetically stable, bad acceptor of foreign plasmid DNA, no No immunotoxicity
stability of artificially introduced foreign plasmid DNA, no transfer of the No blood serum resistance (no risk of sepsis)
strain-specific cryptic plasmids pMUT1 and pMUT2 to other microorganisms No uropathogenicity
Deposit in Official Strain Collection: deposited in the strain collection of No toxicity in germ-free and conventional animals
industrially used strains at the German Collection of Microorganisms and Cell
Cultures (DSMZ) as E. coli DSM 6601
142 U. Sonnenborn et al.
such strains should not carry virulence factors that (EIEC) or Shigella strains (56). In in vitro experi-
are typical for extraintestinal pathogenic E. coli, such ments with HT-29/B6 cells stressed by treatment
as serum resistance or hemolysin production, and with proinflammatory cytokines, EcN did not show
should not show uropathogenicity or provoke immu- translocation across the colonic monolayers, in con-
notoxic effects (19). trast to a uropathogenic E. coli O4 strain (190).

Toxin production Serum resistance


The EcN strain does not produce any of the toxins A common feature of ExPEC strains producing
associated with pathogenic E. coli strains, such as the sepsis and meningitis is their ability to resist the bac-
heat-labile enterotoxins (H-LT), heat-stable entero- tericidal activity of blood serum (59). This charac-
toxins (H-ST), cytotoxins (e.g. cytotoxic necrotizing teristic is called serum resistance. The EcN strain
factor, CNF 1) or shiga-like toxins (SLT I, SLT II) does not show serum resistance when inoculated into
(Table VI). Toxin production was tested in vitro and human, bovine or pig serum. This finding can be
in vivo, using various assay systems (cell culture explained by the presence of its modified (semi-
methods with Y1 adrenal cells and Vero cells, specific rough) O6 surface antigen (55,191). Serum resis-
GM1-ELISA, baby mouse test). Examinations of tance testing was done by employing the classic in
general cytotoxic activity and of the presence of SLT vitro assay of Hughes et al. (60).
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I and SLT II were done according to the method of


Karch and Meyer (185). Testing for CNF 1 activity
Pathogen-specific adhesion factors
was performed as described by Blum et al. (54,180).
In contrast to some ExPEC strains, EcN does not Mannose-resistant hemagglutination is a feature that
produce any hemolysin (54,109). These findings are is common in pathogenic E. coli strains. EcN shows
due to the fact that the EcN strain does not possess no mannose-resistant hemagglutination and forms
any genetic information for the production of no CFA I/II fimbriae, which are closely associated
the above-mentioned toxins, as demonstrated by with the enteropathogenicity of certain E. coli strains
PCR techniques using specific DNA probes (54, (181,192). In the absence of mannose, EcN is capa-
164,180,185). ble of agglutinating erythrocytes, but not in the pres-
Using standard toxicological test methods ence of mannose. This behavior is typical of E. coli
(according to EC Guideline L 383 A: B1) for the strains possessing the so-called common type I fim-
determination of acute toxicity in rats and mice, EcN briae (F1A-fimbriae) (54,193). The test for the pres-
shows no toxic effects after oral administration of up ence of CFA I or CFA II fimbriae was carried out
to 50 ml/kg body weight of a suspension containing by slide agglutination using specific anti-CFA I and
2  1011 viable bacteria/ml (J. Leuschner, unpub- anti-CFA II antisera (181,194,195). Moreover, EcN
lished results). For that reason, an LD50 value could does not express P, M or S fimbriae, which are asso-
not be determined. Also, in young germ-free and ciated with the virulence of certain ExPEC strains
colostrum-deprived piglets, colonization with EcN (196) and does not possess the respective gene loci
did not lead to any toxic effects (93). This finding is for these adherence factors (56).
particularly remarkable, because these animals rep-
resent an infection model of a completely immuno- Uropathogenicity
deficient host organism (186).
The genome of the Nissle strain exhibits high homol-
ogy to that of the UPEC strain CFT073 (56),
Invasiveness and translocation
although genes for classic uropathogenicity traits are
The EcN strain does not have invasive properties. In lacking. This has led some researchers to speculate
vivo testing was done according to the classic method whether the EcN strain might have some virulence
of Sereny (187) by following the development of in the urogenital system (83). However, EcN shows
keratoconjunctivitis after inoculation of bacteria into none of the uropathogenicity traits typical of UPEC
guinea pig eyes. Contrary to known invasive E. strains (196,197) (Table VII). Compared with uro-
coli strains, EcN did not lead to keratoconjunctivitis pathogenic and other tested E. coli strains, it pos-
(G. Böhme, unpublished results). In vitro testing was sesses only a very limited potential for colonization
performed by using different epithelial cell lines of the urinary tract, as was demonstrated by using
(99–101,188,189). Here, EcN also did not show any the transurethrally infected rat model (197). In this
invasive activity, which can be explained by the fact respect, the colonizing ability of EcN is comparable
that the strain does not carry the genes coding for to that of the avirulent laboratory strain E. coli K-12.
the invasion machinery of enteroinvasive E. coli Following transurethral infection of 20 rats with
E. coli strain Nissle 1917 as probiotic 143
Table VII. Comparison of E. coli strain Nissle 1917 (EcN) with non-pathogenic and uropathogenic E. coli (UPEC) strains in vitro and in
a rat transurethral infection model.

Bacterial Bacterial
counts per counts per Lethality (number
gram urinary gram kidney of dead animals/
Hemolysin UPEC-specific Serum bladder (log (log cfu  number of infected
E. coli strains tested Serotype (Hly) fimbriae resistance cfu  SD) SD) animals)

EcN O6:K5:H1 – – – 3.65  1.5 2.68  1.89 0/20


SK 60 O18:K–:H17 – – + 5.60  1.9 2.72  1.65 2/20
SK 64 O18:K5:H– + P + 5.80  2.0 5.26  2.57 4/20
536 wild-type O6:K15:H31 + S + 5.74  1.61 3.89  1.28 6/20
536/21 (non- O6:K15:H31 – – – 2.52  1.0 1.15  1.62 1/20
pathogenic deletion
mutant of strain 536)

EcN, E. coli Nissle 1917; SK 60, E. coli isolate from a pharmaceutical product; SK 64, uropathogenic E. coli isolate (UPEC); 536,
uropathogenic E. coli isolate (UPEC); 536/21, non-pathogenic deletion mutant of UPEC strain 536. Transurethral infection was done
according to Marre et al. (197). Bacterial counts in the respective tissues were determined 1 week after infection (data from R. Marre,
unpublished results).
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5  107 viable cells of EcN, no lethal effects could for many other pathogenicity factors are localized on
be observed in any case, in contrast to tran- high-molecular mass plasmids (183).
surethral infection with uropathogenic E. coli strains
(Table VII). Immunotoxicology
According to its DNA fragment profile, as
revealed by pulsed-field gel electrophoresis (PFGE) To determine the immunogenic and immunotoxic
following restriction of its genomic DNA, EcN potential of EcN, the original EcN strain as well as
belongs to a non-uropathogenic O6:K5 clone, if
compared with DNA profiles of serologically related Table VIII. Sensitivity and resistance of E. coli Nissle 1917 (EcN)
uropathogenic and non-uropathogenic E. coli strains against different antibiotics∗.
of the O6 lineage (J. Hacker, unpublished results; K.
EcN sensitive to EcN resistant against†
Eiteljörge, unpublished results). Macrorestriction
analyses were done according to the methods of Ott Amikacin Cefsulodin
Amoxicillin/clavulanic acid Clindamycin
et al. (58) and Zingler et al. (166,167).
Ampicillin Erythromycin
Azlocillin Metronidazole
Resistance to antibiotics Cefaclor Penicillin G
Cefazolin Quinupristin/dalfopristin
As an isolate from the pre-antibiotic era, EcN is not Cefoperazone Rifampin
resistant to commonly used antibiotics that are effec- Cefotaxime Teicoplanin
Ceftriaxone Vancomycin
tive against gram-negative enterobacteria (tests Cephalothin
according to NCCLS) (198). Furthermore, the strain Chloramphenicol
does not possess any antibiotic resistance plasmid. Ciprofloxacin
The two small cryptic plasmids characteristic of EcN Doxycycline
(pMUT1 and pMUT2) do not carry any genetic Gentamicin
Imipenem
information for antibiotic resistances (53). Besides Latamoxef
this, EcN – like all other E. coli strains – shows natu- Mezlocillin
ral resistance to antibiotics that are mainly directed Nitrofurantoin
against gram-positive bacteria, such as clindamycin, Norfloxacin
erythromycin, metronidazole, penicillin G, rifampin, Pipemidic acid
Piperacillin
and vancomycin (Table VIII). Rifampicin
Streptomycin
Tetracycline
Presence of large plasmids Ticarcillin
Tobramycin
EcN does not possess plasmids of high-molecular Trimethoprim/sulfamethoxazole
mass that are known to carry genes coding for anti- ∗The antimicrobial substances appear in alphabetical order.
biotic resistance and/or pathogenicity traits (183). †Like E. coli in general, EcN shows natural resistance against the listed
For example, the genes coding for invasiveness of antibiotics. As an isolate from the pre-antibiotic era, EcN has no
enteroinvasive E. coli (EIEC) and the genes coding acquired resistance, and no antibiotic resistance plasmids are present.
144 U. Sonnenborn et al.
a recombinant clone of EcN expressing the HA110- could only be induced in germ-free C3H/HeJZtm
120 peptide of influenza A virus on its cell surface mice, not in conventionally housed mutant mice.
have been used (160). For this purpose, the bacteria In healthy young pigs housed under conventional
have been administered orally to different conven- conditions, oral application of EcN did not induce
tionally kept mouse strains (BALB/c, RAG-1–/–, significant changes of immunological parameters in
TCR-HA-transgenic mice, and double transgenic the gut-associated lymphoid tissue (102,103).
mice expressing the influenza HA peptide under the
control of the rat insulin promoter [INS-HA 
Genetic stability
TCR-HA]), and the immunological effects of EcN
on the respective HA110-120-specific CD4+ T cells EcN is genetically stable, both in vitro following
in the gut and in the peripheral lymphatic tissues 100 sequential cultivation cycles (K. Eiteljörge,
have been analyzed. In addition, the original and the unpublished results), and in vivo following implan-
recombinant EcN strain have been tested for coloni- tation and passage through the intestinal tract of
zation capability and for effects on the gut mucosa newborns, which has been shown with EcN re-
of mice, under normal conditions as well as after isolates obtained from stool samples of 14 children,
genetically induced diabetes and after DSS-induced 24 months after administration of EcN suspension
colitis, respectively. Although both strains, the origi- (K. Eiteljörge, U. Sonnenborn, unpublished results).
nal EcN and its recombinant derivative, exhibited Evidence of genetic stability has been provided by
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good colonizing ability in the gut, there were no means of standard comparative macrorestriction
effects on migration, clonal expansion, or state of analysis using PFGE of chromosomal DNA frag-
activation of specific CD4+ T cells. This was true for ments obtained from the original strain and from
healthy as well as for diseased mice. In the double re-isolates as well as by means of specific REP-PCR
transgenic mouse strain, which is considered to be a (166,167,201,202).
highly susceptible model for autoimmune diseases EcN is a poor recipient for conjugative foreign
(genetically induced diabetes) (160), oral adminis- DNA. Testing of EcN for the acceptance of foreign
tration of recombinant EcN had no effect on induc- plasmid DNA was done by determination of conju-
tion or breakdown of T-cell tolerance. gation frequencies following standard conjugation
Further experiments on gnotobiotic IL-2–/– mice techniques with various plasmid groups (incompat-
mono-colonized with the EcN strain revealed that ibility groups, Inc) (203). Conjugation frequencies of
EcN did not induce colitis, in contrast to coloniza- EcN were compared with those of the E. coli K-12
tion with the colitogenic E. coli strain mpk (199). laboratory strain, which served as a positive (100%)
However, contrary to the well-known but ‘degen- control (Table IX, G. Böhme, unpublished results).
erated’ E. coli K-12 laboratory strain, EcN is an The fact that EcN does not take up plasmids of the
E. coli strain that is thought to have retained some IncFI and IncFII types (conjugation frequency 0%)
wild-type characteristics (56). Therefore, the sensi- is of special importance, because in pathogenic E. coli
tivity to EcN in mice is dependent on the genetic
background of the animals (severity of immune dys- Table IX. Test of the ability of E. coli Nissle 1917 (EcN) to serve
function) as well as on the microbial environment as a recipient for foreign plasmid DNA.
(colonized vs germ-free state). This has been shown DNA DNA
in a recent immunotoxicological study in genetically acceptance acceptance
engineered mouse strains (knockout mice) with dif- Inc group (%) Inc group (%)
ferent immunological handicaps. EcN did not exhibit IncB 1 IncK 0
toxicity after a 1-week course of oral administration IncC 10 IncM 0
to germ-free and conventionally kept animals (200). IncD 5 IncN 0
The only exceptions were the double-mutant C3H/ IncFI 0 IncP 5
IncFII 0 IncQ 1
HeJZtm mice, which have defects in the gene cluster IncFIII 0.5 IncT 5
coding TLR-4. TLR-4, which is present on gut epi- IncOF 1 IncU 0.1
thelial cells as well as on different types of lympho- IncH1 0.05 IncW1 0.1
cytes, is responsible for the recognition of the IncH2 0.01 IncW2 0.2
immunologically highly active LPS of gram-negative IncH3 0.01 IncW4 0.1
IncI1 0.5 IncX 0.1
bacteria, such as E. coli. Germ-free mutant mice IncI2 0 IncY 0
experienced enhanced translocation of EcN from the IncJ 0 IncZ 1
gut into the bloodstream, leading to septicemia and
Results of conjugation experiments with plasmids from different
infection of several organs. It is not known whether incompatibility (Inc) groups (G. Böhme, unpublished results).
EcN might show serum resistance in blood serum of Relative values of DNA acceptance (%) are given in comparison
these mice. Interestingly, this infection of gut origin to E. coli K-12 (100%).
E. coli strain Nissle 1917 as probiotic 145
strains these Inc groups often contain virulence liver cirrhosis (219), and interestingly, also in a
plasmids. dermatological allergic disease (polymorphic light
EcN cannot be transformed by the tox-phages of eruptions, ‘sun allergy’) (220).
enterohemorrhagic E. coli strains (EHEC), which
otherwise are capable of transfering the slt genes for
Inflammatory bowel diseases
the production of shiga-like toxins to other recipient
strains (204). Attempts to introduce the slt genes into Inflammatory bowel diseases (IBD) are severe dis-
EcN by means of electroporation have also not been eases of the gastrointestinal tract connected with
successful (204). Phage-encoded genetic informa- enormous suffering for the respective patients. Ulcer-
tion for the production of EHEC-specific shiga-like ative colitis and Crohn’s disease are the most com-
toxins (205–207) is not taken up by EcN. mon forms of IBD (221–223). While the etiological
factors for the onset of IBD are unknown in most
cases, there is intensive knowledge today with respect
Risk group assessment
to the pathogenesis of these diseases, supporting the
EcN has been classified as a risk group I organism hypotheses that an over-reactive immune system on
(lowest risk group for microorganisms) by the official the basis of a genetical predisposition causes inflam-
German Central Commission for Biological Safety mation of the intestinal mucosa (ulcerative colitis) or
(ZKBS, Zentrale Kommission für Biologische of the entire gut wall (Crohn’s disease) (12,13,23,221).
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Sicherheit), due to the lack of toxicity traits and This process is triggered, sustained, and intensified
its extensive molecular genetic and functional by different environmental factors (224,225). Since
characterization. the recognition of the importance of the environmen-
tal factor ‘intestinal microbiota’ for the pathogenesis
of IBD, the application of preparations containing
Clinical efficacy and safety of E. coli Nissle
viable microorganisms (probiotics) to IBD patients
1917 in human medicine
is gaining more and more interest and acceptance in
The licensed drug Mutaflor® is available either in medical, and especially in gastroenterological prac-
capsule form containing lyophilized powder with tice (12,21,23,44,47,226–240).
viable E. coli Nissle 1917 (2.5–25  109 cfu/capsule) In three randomized controlled clinical trials
or as a bacterial suspension (108 cfu/ml). The cap- (RCTs) EcN has been used for the maintenance of
sules are enteric coated in such a way that viable EcN remission in patients with ulcerative colitis and com-
bacteria are released no earlier than in the terminal pared to the gold standard mesalazine. The first study
ileum. This galenic formulation is necessary to pro- was a controlled clinical pilot trial with double-
tect EcN against gastric acidity and the antimicrobial dummy design (241); 103 patients in remission were
action of bile and digestive enzymes in the small enrolled. Fifty-three patients received 6  250 mg
intestine. The suspension contains viable EcN bacte- mesalazine per day (control group), while 50 patients
ria in an electrolyte solution free of carbohydrates and received 2 capsules with EcN per day. The aim of the
nitrogen sources. This preparation has been devel- study was the maintenance of remission during a
oped especially for babies and toddlers, but may also 3-month period. This was controlled by following the
be used by adults with difficulties in swallowing cap- development of the clinical disease activity index.
sules. With respect to the clinical use of Mutaflor®, Life-table analysis revealed no difference between
about nine decades of experience exist. The spectrum groups with respect to the length of time free of
of indications for which EcN has been successfully relapses (103  4 days for the group taking mesala-
used is relatively broad, which was demonstrated in zine vs 106  5 days for the group taking EcN).
a recently conducted prospective post-marketing sur- These results were confirmed by histological exami-
veillance study (51). Controlled clinical trials that are nations and by the personal judgment of the patients
able to prove efficacy are available for the indications with respect to the intensity of their disease at the
ulcerative colitis, chronic habitual constipation, and end of the trial.
acute and protracted (prolonged) diarrhea in young This first study on the usefulness of a probiotic
children. This will be discussed in more detail below. in ulcerative colitis was followed by another random-
In addition, several pilot studies useful for the gen- ized controlled clinical trial, which was conducted for
eration of hypotheses have been executed so far and a 12-month period, including patients with active
gave hints for possible effectiveness of this probiotic ulcerative colitis (242). This trial was based on the
for other indications. This concerns IBS (208–210), experience that acute relapses in IBD patients could
collagenous colitis (211), pseudomembraneous colitis be initiated and intensified by strongly adherent E.
(212,213), proctitis (214), Crohn’s disease (215), coli bacteria (243). The question was whether EcN
diverticulosis (216), pouchitis (217), halitosis (218), was able to maintain remission as effectively as
146 U. Sonnenborn et al.
mesalazine after initial treatment with antibiotics and thus being suitable for equivalence testing. Patients
corticosteroids. In all, 116 patients with active ulcer- were randomly assigned to two study groups and
ative colitis were enrolled and were randomly assigned received either 3  500 mg mesalazine or two cap-
to 2 groups in a double-blind, double-dummy fash- sules with EcN for a period of 1 year. The results of
ion to receive either 3  800 mg mesalazine or 2  all patients recruited for the study (intention-to-treat
2 capsules with EcN. All patients received predniso- analysis) as well as the results of those patients fulfill-
lone to stop the intestinal inflammation and, in addi- ing all criteria of the study protocol (per-protocol
tion, a 1-week course of gentamicin. Those patients analysis, n  222) revealed that treatment with EcN
who were not in remission after 12 weeks on pred- was equivalent to treatment with mesalazine. In the
nisolone were excluded from the further trial. Patients EcN group 36.4% of the patients experienced a
in remission (44 on mesalazine, 39 on EcN) then relapse during the course of 1 year; in the mesalazine
received per day either 3  400 mg mesalazine or group 33.9% relapsed (Figure 21). The one-sided
1  2 capsules with EcN for the following 12 months. equivalence test showed a p value of 0.003. This
The efficacy of both forms of therapy was calculated study proved that EcN represents an effective alter-
with respect to the numbers of relapses occurring native to mesalazine for maintaining remission in
during the study period of 1 year. The result was that patients with ulcerative colitis.
no differences could be detected between the two On the basis of these study results, EcN has been
treatment groups: 73% of the patients receiving included as an effective therapeutic agent for the
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mesalazine and 76% of the patients receiving EcN maintenance of remission in patients with ulcerative
had a relapse during this time (p  0.0059, one-sided colitis into the treatment guidelines of the German
test for proof of non-inferiority). Also the duration and the Austrian gastroenterological associations as
of the remission phase was not significantly different well as in the guidelines of the European Crohn and
between the two treatment groups (mesalazine, Colitis Organisation (ECCO) (244–247).
206 days; EcN, 221 days), which is also reflected by Recently, similar results were obtained in an open
the Kaplan–Meier plot of patients in remission in the controlled pilot study in children and teenagers with
course of the study (Figure 20). ulcerative colitis. In this study, 24 patients (mean age
To confirm these results, another RCT with 14.6 years) received two capsules with EcN per
double-dummy design was initiated to prove that day for a 1-year period and 10 patients (mean age
EcN is not inferior to mesalazine for maintenance of 14.0 years) received 1.2 g mesalazine per day for the
remission in patients with quiescent ulcerative colitis same time course. After 1 year, 25% of patients in
(155). This large double-blind study was conducted the EcN group and 30% of patients in the mesalazine
in 10 European countries and included 327 patients, group experienced a relapse (248).

100 Duration of remission EcN


Mesalazine
90

80
Patients in remission(%)

70

60

50

40

30

20

10
0
0 50 100 150 200 250 300 350
Days

Figure 20. Comparison of mesalazine (5-aminosalicylic acid, 5-ASA) vs E. coli Nissle 1917 (EcN) for remission maintenance in patients
with ulcerative colitis (242). A total of 116 patients with active ulcerative colitis were randomly assigned to one of two treatment groups
in a double-blind clinical trial with double-dummy design. Patients who had reached remission by standard corticosteroid therapy within
12 weeks were then given either mesalazine or EcN for 1 year. The Kaplan–Meier plot shows the percentage of patients in remission during
the course of the study (straight line, EcN; dotted line, mesalazine). With respect to clinical efficacy and time course of remission, there
were no statistically significant differences between the two study groups.
E. coli strain Nissle 1917 as probiotic 147
100 The EcN and the placebo capsules were taken at
Per-protocol-analysis breakfast each day. The following doses were applied:
Patients with relapses (%)

(n = 222)
80 3  2 capsules per day on days 1 and 2, 1  4 cap-
sules per day from day 3 onwards for a total of 8
60 Equivalence significant with p = 0.003
weeks. Within 4 weeks the number of bowel move-
36.4%
ments in the EcN group increased significantly (4.9/
40 33.9% week, p  0.001) compared with the placebo group
(2.6/week) (Figure 22). While patients in the EcN
20 group experienced a further increase in bowel move-
ments during the next 4 weeks (up to 6.0/week), the
0
EcN Mesalazine
number of bowel movements of patients in the pla-
cebo group decreased to 1.9/week. The study design
Figure 21. Clinical equivalence study between mesalazine included a change-over to the other kind of treatment
(5-aminosalicylic acid, 5-ASA) and E. coli Nissle 1917 (EcN) for for patients who did not experience an effect of either
maintaining remission of ulcerative colitis (155). A total of 327
patients with quiescent ulcerative colitis were recruited and
medication (less than three bowel movements per
randomly assigned to a double-blind clinical multicenter trial with week) during the first 4 weeks. With respect to this,
double-dummy design that was performed in 10 European 13 placebo patients changed to EcN treatment,
countries. Patients received either mesalazine or EcN for 1 year. which resulted in an increase of bowel movements
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At the end of the study, 222 patients could be included in the during the next 4 weeks up to 5.15/week. On the
per-protocol analysis. Of these, 40/110 (36.4%) in the EcN group
and 38/112 (33.9%) in the mesalazine group showed a relapse
other hand, two patients changed from EcN to pla-
during the 1-year period of treatment. The data show significant cebo, but did not experience any increase of bowel
equivalence between both treatment groups with respect to clinical movements (2.5 per week).
efficacy (maintenance of remission) (p  0.003). The therapeutic efficacy of EcN in chronically
constipated patients might rather reflect the probiot-
Jöres-Nguyen-Xuan et al. (170) have shown ics’ ability to produce gut motility-enhancing short-
recently in healthy volunteers that mesalazine and chain fatty acids, such as acetic acid (153) (discussed
EcN do not interfere when administered together, earlier), but not the production of gases (CO2, H2),
opening the way for clinical trials on the usefulness since in a study with healthy volunteers EcN has
of a combination therapy for IBD. been shown to exert no effect on intestinal gas
dynamics and abdominal sensation (251).
Chronic constipation
Diarrhea in young children
The effectiveness of EcN to induce a normalization
of intestinal functions could also be shown in patients Recently, efficacy and safety of EcN for the treatment
with chronic constipation. Using a parallel group of acute diarrhea of babies and toddlers were studied
comparison, constipated patients (after stopping the in a randomized placebo-controlled double-blind clin-
use of laxatives) received either 2  15 ml lactulose ical multicenter trial (252). Acute diarrhea was defined
syrup per day (n  52) or 3  1 capsule with EcN as showing three non-bloody evacuations per day with
per day (n  56) (249). After 12 weeks of treatment, reduced consistency (liquid or almost liquid). Acute
in the EcN group the number of bowel movements diarrhea should not last longer than 3 consecutive
increased to 6.3 per week. In the lactulose group the days. A total number of 113 babies and toddlers, aged
number of bowel movements increased to 5.5 per 2–46 months (median 23 months), were recruited for
week. The ease of evacuation improved significantly the study and randomly assigned to the EcN group
in those patients taking EcN compared with those in (n  55) or to the placebo group (n  58). Patients
the lactulose group (p  0.001). Adverse events received either EcN suspension (108 cfu/ml) or an
(mainly flatulence and abdominal cramps) did occur indistinguishable placebo from day 0 until day 9 in a
sometimes, but were seen more often in the lactulose dosage dependent on age: babies (1 year) 1  1 ml
group. suspension/day, toddlers ( 1 to 3 years) 2  1 ml/
In a further study (250) chronically constipated day, and older children ( 3 to 4 years) 3  1 ml/
patients received in a randomized double-blind study day. Before enrollment, the mean duration of diarrhea
either capsules with EcN or placebo capsules over was 1.4 days for the infants in the EcN group and 1.6
a period of 8 weeks. Initially, in a ‘run-in-phase’ all days for the infants in the placebo group. None of the
patients received placebo for 1 week. Only those children was dehydrated when included in the study.
patients who did not have more than two bowel In both treatment groups diarrhea was partly due to
movements during the run-in-phase were thereafter bacterial or viral infections as well as other unspecific
randomly attributed to verum or placebo group. causes that were not further elucidated. The primary
148 U. Sonnenborn et al.

p < 0.001
9
p < 0.001
Maximum
8

Bowel movements / week


Minimum
7
Placebo
6
EcN
5

1 n = 35 n = 35 n = 30 n = 34 n = 16 n = 31
0
0 4 8 Weeks

Figure 22. Double-blind placebo-controlled randomized clinical trial with E. coli Nissle 1917 (EcN) for the treatment of chronic habitual
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constipation (250). Initially, all constipated patients (n  134, two bowel movements per week or less) in a blinded manner received a
placebo medication for 1 week which, however, was handed over to them by the principal investigator as a ‘new biological drug against
constipation’. After the 1-week course on placebo, only those patients who did not respond to this treatment (n  70) were allowed to
enter the real double-blind study and were randomly assigned to receive either EcN or placebo. The figure shows the development of the
main target criterion (number of bowel movements per week) during the 8-week course of the study. EcN was significantly better than
placebo in increasing the weekly frequency of bowel movements after 4 and after 8 weeks on medication (p  0.001 for EcN vs
placebo).

goal of the trial, to reduce the number of bowel move- mean duration until reaching this goal was 2.5 days
ments (less than or equal to three liquid or almost in the verum and 4.8 days in the placebo group
liquid evacuations per day on at least 2 consecutive (Figure 23A). Treatment with EcN thus reduced the
days), was reached by 52/55 patients (94.5%) in the mean duration of diarrhea by 2.3 days. This differ-
EcN group and 39/58 patients (67.2%) in the placebo ence between verum and placebo is statistically
group. By analysis according to Kaplan–Meier the significant (p  0.007).

A Significant difference
p < 0.0007
Gain in time:
diarrhoea

EcN suspension
2.3 days
Acute

Placebo suspension

0 1 2 3 4 5 Days
2.5 days 4.8 days

B Significant difference
p < 0.0001
Gain in time:
EcN suspension
3.3 days

∅ 5.8 days prolonged diarrhoea Placebo suspension

–5 –4 –3 –2 –1 0 1 2 3 4 5 Days

2.4 days 5.7 days

Figure 23. Two double-blind placebo-controlled randomized clinical trials have been performed with E. coli Nissle 1917 (EcN) suspension
(108 cfu/ml) for the treatment of acute diarrhea (A) or prolonged diarrhea (B) in young children (252,253) (see text for details). In the
study on acute diarrhea (A), EcN treatment shortened the duration of diarrhea by 2.3 days compared with placebo (p  0.0007). In the
study on prolonged diarrhea (B), EcN treatment shortened the duration of diarrhea by 3.3 days (p  0.0001). These results demonstrate
that EcN suspension is an effective treatment option for diarrheal diseases in infants.
E. coli strain Nissle 1917 as probiotic 149
In a second randomized placebo-controlled Interestingly, with respect to the time-sparing effect
double-blind multicenter study the efficacy of EcN of EcN, a comparison of the efficacy of EcN to that
for the treatment of unspecific protracted diarrhea in of other probiotics (19) also suggested superiority of
babies and toddlers was examined (253). Protracted EcN over several LAB preparations, although a true
diarrhea was defined as follows: more than three comparison between the preparations used is diffi-
non-bloody liquid to almost liquid evacuations per cult because of very different study designs.
day. The diarrhea should have been present for at
least 4 days and not longer than 2 weeks. A total of
Colonization prophylaxis
151 babies and toddlers, aged 1–47 months, who
experienced a prolonged diarrhea as defined above Studies concerning colonization prophylaxis in pre-
were recruited for the study. They were randomly term and full-term neonates have shown that the oral
assigned to the EcN group (n  75) or to the placebo application of EcN during the first days of life resulted
group (n  76). At the beginning of the study the in a long-standing colonization of the gut with this
mean duration of diarrhea was 5.8 days for both probiotic, followed by a reduced colonization with
groups. The number of bowel evacuations during the true and potential pathogens (96,97). In addition, this
last 24 h before the onset of the study was four to intentional colonization resulted in a significant
six, mostly of liquid consistency and with mucus increase of specific immunoglobulins IgA and IgM
shedding. Patients received either the verum or the (121) as well as in an early induction of natural
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placebo suspension from day 0 until day 20, applying immune defenses (122). This points to the develop-
the same age-dependent dosage scheme as in the ment of the gut immune system in the direction
study on acute diarrhea described above. To prevent towards an infection-preventing state, which is
dehydration because of loss of liquid and electrolytes, reflected by an anti-allergic Th1-dominated immune
all patients initially received an oral rehydration ther- situation (111). Since children of allergic parents have
apy (ORT) for 4 h according to the WHO Guideline a high risk of developing allergies early in life, inten-
‘Treatment of diarrhoea (2001)’ in an outpatient tional colonization of the gut with EcN might turn out
setting. to be an effective alternative for allergy prevention.
The main goal of this study was to achieve a
decrease of bowel movements (three or less liquid or
Safety aspects in clinical use
almost liquid evacuations per day for at least 4 con-
secutive days). Treatment with EcN should have EcN-treatment is generally well tolerated. No serious
demonstrated superiority compared with placebo on adverse drug reactions have been reported from the
days 7 and 14. The primary aim of the study was explorative and confirmatory clinical trials conducted
reached by 59/75 (78.7%) of patients in the EcN to date to demonstrate efficacy and tolerability of
group and by 45/76 (59.2%) of patients in the EcN for different indications. Flatulence, which is
placebo group on day 7. After 14 days this goal occasionally observed, especially at the beginning of
was reached by 70/75 (93.3%) of the patients on the probiotic therapy, is suspected to be due to the
EcN and by 50/76 (65.8%) of the placebo patients metabolic activity of EcN in the gut. This ailment
(Figure 23B). Therapy was stopped for 13 patients probably represents individual overdosing and does
because of ineffectiveness (2 patients on EcN, 11 not require a cessation of medication nor specific
patients on placebo). These patients were treated treatment, since in general it vanishes after reducing
thereafter with other drugs. Using sequential group the dose. In clinical investigations on the tolerability
analysis it could be shown that already on day 7 there of EcN in healthy volunteers, up to nine capsules per
was a strong tendency for superiority of EcN therapy day (about 1011 cfu/day) were tolerated without any
over placebo (p  0.0758). The difference between problems (E. Zieseniss, unpublished results).
the two groups increased in the course of the study High-risk patients, such as preterm infants and
and was significant on day 14 (p  0.0017). Using also full-term babies, in whom the intestinal mucosal
Kaplan–Meier analysis, the time to reach the main barriers are not yet fully developed, are nevertheless
goal of the study was calculated to be 2.4 days in the tolerant to a daily oral application of 1 ml EcN sus-
EcN and 5.7 days in the placebo group. Treatment pension (108 cfu/day). This has been shown repeat-
with EcN reduced the mean duration of protracted edly during the course of several clinical trials on
diarrhea for about 3.3 days. neonates (96,97,121,122). Although the application
In summary, probiotic therapy with the Nissle of EcN suspension to newborn infants directly after
strain showed statistically significant superiority birth (1 ml once a day for 5 days) resulted in an early
compared with placebo for acute as well as for pro- induction of the development of both the gut and the
longed (protracted) diarrhea in babies and toddlers. systemic immune system (121,122), in a study on
150 U. Sonnenborn et al.
healthy adult male volunteers the application of two (placebo group, n  89; EcN group, n  83), and
capsules with EcN once a day for a total of 3 days the confirmatory controlled study comprised 163
did not reveal significant changes in any of the calves (placebo group, n  81; EcN group, n  82).
immune parameters tested (cytokines, receptor mol- In both studies the number of animals experiencing
ecules) (R. Gruber, unpublished results). Also, diarrhea were counted. Diarrhea was defined as loose
neither induction of autoimmune phenomena nor or watery excrements on at least 2 following days. In
immunotoxic effects have been observed. These find- the first study, 58 animals (65.2%) in the placebo
ings underline the clinical safety of the EcN strain. group experienced diarrhea, but only 22 animals
(26.5%) in the EcN group. In the confirmatory clin-
ical trial, 51 animals (63%) in the placebo group, but
Clinical efficacy of E. coli Nissle 1917 in
only 10 animals (12.2%) in the EcN group showed
veterinary medicine
diarrhea. This difference of 50.8% (p  0.001) rep-
Until the redefinition of the term probiotic by Fuller resents a decrease in diarrhea episodes in the animal
in 1989 (30), viable microorganisms (LAB) were population of more than 80% and proves the high
used as feed additives or medical remedies mainly in efficacy of EcN in the prophylaxis and therapy of
animals, although human experiences have existed for diarrhea in neonatal calves (133).
several thousand years by the traditional consumption The anti-diarrheic efficacy of EcN was also
of fermented foods. In animal breeding the addition assessed in a pig model of intestinal bacterial infection
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of viable bacteria to the feed was thought to stimulate (256). Here, 1010 viable cells of porcine enterotoxi-
the growth of the animals, similar to the prophylactic genic E. coli strain Abbotstown (EcA) were given via
use of low-dose antibiotics, in order to increase the an orogastric tube to weaned piglets (n  7) at day
efficacy of feed exploitation followed by the enhanced 21 postpartum. At 48 h after challenge electrophysi-
production of meat, milk, and eggs and to improve ological parameters of isolated jejunal epithelium
the general health condition of the animals (254). At were assessed in Ussing chambers. In agreement with
that time, there was more speculation than proof as clinical signs of diarrhea, tissue of challenged animals
regards the scientific basis of clinical efficacy and the showed an overshoot of secretory response after stim-
modes of action of probiotics. Since the scientific ulation of the cAMP-mediated second messenger
interest has concentrated in humans on the elucida- pathway by forskolin, indicating higher excitability of
tion of the modes of action of such preparations, the chloride secretory channels under the infection. These
number of studies on the use of probiotics in domes- data were compared with the respective measure-
tic animals has decreased. Nevertheless, infectious ments of jejunal tissues from animals that received a
diarrhea is the leading cause of morbidity and mortal- daily dose of 1010 viable cells of EcN over 10 days
ity in young piglets and calves, induced by shortening before EcA challenge (n  4), with a group on EcN
of the lactation period, industrialized maintenance only (n  4), and with a group of untreated animals
conditions, and inadequate feeding. Often more than (controls, n  6). EcN pretreatment completely abol-
50% of newborn calves are affected by diarrhea ished clinical signs of secretory diarrhea and the over-
between the seventh and the tenth day of life, which shoot of secretory response of intestinal epithelium
reduces the development of the animals and may be upon stimulation with forskolin, seen in mono-infected
the cause of about 8% of all newborn deaths (255). animals. These results demonstrate clinical efficacy of
The efficacy of a suspension with EcN for pre- EcN in preventing gut infection and secretory diar-
venting diarrhea in newborn calves has been tested rhea induced by enterotoxigenic E. coli in pigs.
in a pilot study as well as in a confirmatory placebo-
controlled blinded clinical trial (133). In both studies
Concluding remarks
the animals received 15 ml EcN suspension (108
cfu/ml) on the day of birth, before the first intake of Until recently, the use of living microorganisms (pro-
colostrum, and then continuously once a day until biotics) for therapeutic purposes in general has been
day 10 or 12. The oral application of EcN suspension considered some kind of alternative or comple-
was executed each morning before conventional mentary medicine (257). Fortunately, this view has
feeding was performed. The control group of animals changed considerably in the last couple of years,
received a similar placebo suspension (electrolyte which is reflected by the growing interest of basic
solution) also before feeding in the morning. The biomedical sciences and clinical medicine with
newborn calves were fed colostrum on the first day respect to this group of ‘active substances’. The
of life. Colostrum was then replaced by the addition increasing interest is reflected by the exponentially
of pooled milk or commercially available milk growing numbers of scientific papers and published
replacer. In the pilot study 172 calves were included RCTs dealing with probiotics (see Figure 1).
E. coli strain Nissle 1917 as probiotic 151
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COMMENT

U. Sonnenborn & J. Schulze: The non-pathogenic Escherichia coli


strain Nissle 1917 – features of a versatile probiotic

This article is a comprehensive review considering a by ‘unpublished results’. Usually, I dislike similar
specific strain of Escherichia coli, which has been used statements so much that the authors have to re-write.
as a probiotic for several decades. Over the years Why have I broken my own rule this time? The rea-
MEHD has published similar reviews on leading son is very simple: the results reflect indications on
probiotic products, and they have all been very well exciting ongoing research. E. coli Nissle 1917 is more
received. I am quite sure that will also be the case for vivid than ever.
the present review. It is an amazing story about an
amazing strain and with an amazing future strategy. Tore Midtvedt,
My concern has been some statements documented Editor-in-Chief

ISSN 0891-060X print/ISSN 1651-2235 online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.3109/08910600903462558