Until the late 19
th
century, microbes were the major cause of death in humans. Ironically, the infectious
nature of most diseases was not recognized. The treatment, if any, was focused
on strengthening the general immunity. This changed with the identification
of pathogens by Pasteur and Koch. The knowledge about microbial infections quickly
expanded and reduced dramatically their impact on humanity. However these advances
referred exclusively to mono-infections. The situation remained unchanged for
polymicrobial diseases. A polymicrobial involvement is suspected in caries,
pharyngo-tonsillitis, vaginosis, inflammatory bowel disease (IBD) and colon
cancer. Research data on coronary heart disease, stroke and autoimmune diseases
suggest that pathogens trigger the illness, however positive proof and understanding
of causality are lacking. The current medical strategies for these diseases
are therefore directed toward managing symptoms, conditioning immunity, and
the search for the genetic background.
Most of the polymicrobial infections are probably not recognized. The reason
for this unawareness is a lack of appropriate tools. Since Robert Koch and Louis
Pasteur, we define a pathogen as a microorganism, which is isolated from a diseased
person, absent in a healthy person and causes a disease upon transfection to
a healthy person. The value of Koch’s principles is however limited in case
of polymicrobials. The polymicrobial community can not be grown elsewhere by
transfection of single strains and the investigation of isolated microorganisms
does not explain how the polymicrobial community functions or why it can flourish
under conditions, which are deadly for each of the constituents. Their composite
structure in relation to propagation and response to environmental challenges
need to be monitored and studied in order to understand polymicrobial infections.
Unfortunately, the polymicrobial communities can not be grown in culture. The
environmental microbiologists, however, developed different tools to analyze
microbiota
in situ.
MATERIAL AND METHODS
One of the methods to analyze microbiota
in situ is the ribosomal RNA
fluorescence
in situ hybridization (FISH). Depending on metabolic activity,
each bacterial cell contains 10
4-10
8
ribosomes. Each ribosome includes an RNA molecule. Some areas of the ribosomal
RNA are strain-specific, other are more universal. Based on sequences of the
ribosomal RNA, probes can be synthesized to bind specifically to organisms of
interest. Using probes labelled with different fluorescent dyes, we can simultaneously
visualize different types of microbes within complex communities. Over 100 FISH
probes are currently available and allow explicit analysis of intestinal bacteria.
It is not necessary that the bacteria are alive at the time of the investigation.
The FISH investigations can be carried out any time and repeated, if the material
is properly fixated (1, 2).
We have investigated biopsies from more than 10000 patients and controls using FISH in order to search for microbial roots of IBD. Human bowel is cleaned before the colonoscopy. To investigate the composition of the mucosa adjacent bacteria throughout the intestine without cleansing, we studied sections of whole mice intestine.
We tested the mobility of intestinal bacteria
in vitro with a viscous
gel layer containing different additives enclosed between two cellulose membranes
which were placed on blood agar to attract bacteria (
Fig. 1). The viscosity
of the gel was adjusted by varying the concentration of agarose from 0.2% to
2%. Mixtures of enteric bacteria were overlaid onto the simulated mucus. After
28 hours of anaerobic growth, membranes were fixated, sectioned, and then examined
by FISH (3).
|
Fig. 1. Mucus simulation in
vitro. |
We have also investigated stool probes from patients in form of faecal cylinders. These are punched out of the stool by the use of drinking straws, the stool is fixated, embedded in paraffin, cut to slices and hybridised with FISH probes representing 86 different bacterial groups (8).
Microscopy was performed with a Nikon e 600 fluorescence microscope. The images were photo documented with a Nikon DXM 1200F color camera and software (Nikon, Tokyo, Japan).
RESULTS
Human
The most striking finding in our studies was a lack of contact between intestinal
bacteria and the mucosa in normal subjects. In most healthy controls (84%),
the intestinal wall throughout the ileum and colon was covered with mucus, which
prevented that bacteria contact the mucosal surface (
Fig. 2). In contrast
to healthy controls, we found a dense coating of bacteria on the intestinal
surface in nearly all patients with IBD. Bacteria adhered to epithelial cells,
entered crypts and were sporadically found within cells. The intracellular bacteria
were located mainly at the bottom of the crypts, which were in most cases empty
of bacteria, but not in the columnar epithelium, which directly contacted the
dense masses of bacteria (
Fig. 3). Although adherent bacteria were present
in nearly all (94%) IBD patients who had not been treated with antibiotics,
the highest concentrations of mucosal bacteria were found in less or macroscopically
non-inflamed regions rather than in the inflamed regions of the intestine. In
inflamed regions, the bacterial concentrations were reduced due to leukocytes
that migrated to the outer regions of the mucus, either preventing access to
the mucus layer or exerting antimicrobial effects (
Fig. 4). Despite high
concentrations of leukocytes and reduced numbers of bacteria in the mucus of
inflamed gut segments of IBD patients, some of these bacteria reached the intestinal
wall leading to development of ulcers, fissures, abscesses and deep tissue infiltrates
(
Fig. 5).
|
Fig. 2. Human colonic wall of healthy controls (84%) is covered with mucus that excludes bacteria from contact with the colonic mucosa |
|
Fig. 3. Prolific Bacteroides
fragilis biofilm completely covers the mucosal surface and enters
crypts in a patient with Crohn’s disease (upper part); focusing on the
intraepithelial bacterial inclusions in the same patient indicates, that
bacteria are located within the tissue and not overlaid (blue arrows-lower
part) |
|
Fig. 4. Leukocytes migrate into the mucus, align in the outer regions and prevent access to the mucosa. |
|
Fig. 5. Ulceration of the epithelial surface in a patient with ulcerative colitis. Bacteria attach to the exposed mucosa. (ulcer ground, arrows) |
The bacterial adherence to the mucosa was not IBD specific. Bacterial concentrations
of 10
9 bacteria/ml or higher were found in nearly
all patients with IBD, but also in patients with self-limiting colitis (Sl-colitis),
coeliac disease, HIV enteropathy, 62% of patients with acute diarrhoea, 52%
of patients with diverticulosis, 45% of patients with carcinoma or polyps, and
in 38% of patients with irritable bowel disease (IBS). However, the mean density
of mucosal bacteria was significantly lower in groups without intestinal inflammation
and the composition of the biofilm was different. Bacteria of the
Bacteroides
fragilis group and
Enterobacteriaceaei were responsible for >60%
of the biofilm mass in IBD, but only for 30% in self-limiting colitis. In contrast,
bacteria that positively hybridized with the Erec (
Eubacterium rectale)
and Fprau (
Fecalibacterium prausnitzii) probes accounted for >50% of
the biofilm in IBS patients, but only for <30% of the biofilm in IBD. Bacteria
other than
Bacteroides,
Enterobacteriaceaei,
Fecalibacterium
prausnitzii or
Eubacterium rectale were predominant in self-limiting
colitis (
Fig. 6,
Table 1).
|
Fig. 6. Bacteroides fragilis
(Bfra Probe) Eubacterium rectale group (Erec Probe), other bacteria (Eub338)
in a patient with inflammatory bowel disease, self-limiting-colitis and
irritable bowel disease. |
Table 1. Percent of bacteria within the biofilm in patients with CD, UC, Slc, IBS and in controls. |
|
For better understanding and comparison of the findings, only microphotographs
hybridized with the same set of probes are shown throughout this overview. Thus,
in the figures
Bacteroides is Cy3-stained and appears yellow,
Eubacterium
rectale group (EREC probe) is Cy5-stained and has red fluorescence, and
all other groups are FITC-stained and appear green. The colours are shown as
they appear through the microscope or camera. Micrographs are not manipulated.
Experimental studies
Our tests with agarose (
Fig. 1) showed that only small coccoid rods of
the
Bacteroides group moved at an agarose concentration of 0.2%. The
long rods of the
Eubacterium rectale group were immobilized (
Fig.
7).
Bacteroides was immobilized and only long rods of
Eubacterium
rectale moved at agarose concentration of 0.5% (
Fig. 8). The movement
of all bacterial groups was inhibited at agarose concentrations of 0.7% (
Fig.
9). The segregation of bacteria in the proximal colon in mice into those
bacteria which contact the mucosa and which are separate from the mucosa is
therefore not a result of adherence of “probiotic” bacteria but is due to moderate
viscosity of the mucus layer in this region, which permits bacteria with a long
curly rod shape to move and contact the mucosa but immobilizes the coccoid or
short rod shaped bacteria.
|
Fig. 7. Bacterial velocity
through gels of different viscosity is species specific. Small coccoid
rods of the Bacteroides group have the highest velocity in gels
with low viscosity, here 0.2% agarose |
|
Fig. 8. Long rods of Eubacterium
rectale group (EREC, red) have the highest velocity in gels with high
viscosity, here 0.5% agarose. |
|
Fig. 9. In 0.7% agarose (arrows); bacteria are absent below the membrane. A gap between the bacteria and the membrane indicates a lack of bacterial movement across the gel layer (double headed arrows). |
Mice
Small intestine of healthy wild type mice contain no bacteria which can be definitively
detected by FISH, corresponding to a bacterial concentration of less than 10
6
bacteria/ml. The few microorganisms found were heterogeneously composed, random,
and without signs of adhesion or contact with the intestinal wall. All of them
were separated from the colonic wall by a mucus layer. The large intestine of
healthy wild type mice contain a highly concentrated mass of bacteria. A distinct
mucus gap devoid of bacteria completely separates the colonic wall from the
highly concentrated bacterial biomass in the distal colon. The width of the
mucus layer increases progressively from the middle to the distal colon. No
bacteria contact the colonic wall. The same segment stained with alcian blue
demonstrates that the gap is indeed filled with mucus (
Fig. 10). The
situation in the distal colon of mice is identical to the situation in human.
In the proximal colon of healthy mice, the situation is completely different
to that observed in healthy human. Luminal bacteria directly contact the colonic
wall in the healthy mouse. However, this contact is selective, while
Eubacterium
rectale contacts mucosa and enters crypts to large numbers,
Bacteroides
is separated from the colonic wall. The differences in arrangement of bacterial
groups are especially obvious in multi-colour FISH visualizing different species
in different colours within the same microscopic field.
Eubacterium rectale
are condensed in extremely dense mats adjacent to the mucosa, which are clearly
demarcated from the rest of the faeces and
Bacteroides (
Fig. 11).
The first impression is that
Eubacterium rectale prevents
Bacteroides
from contact with the mucosa. This impression is wrong. Bacteria which were
separated from the colonic wall were represented by
Bacteroides,
Enterobacteriaceaei,
Clostridium difficile,
Veillonella and other groups. Typical for
these groups was not the biochemistry or phylogenetic relationship, but the
bacterial cell morphology of short coccoid rods (
Fig. 12). Bacteria contacting
the proximal colonic wall in mice were also represented by different groups
belonging to
Eubacterium rectale (EREC),
Bifidobacteriaceae (Bif
probe)
Lactobacillius and other groups. Common for these bacteria was
their shape of long often curly rods. The bacterial shape is important for the
bacterial movements. Short rods are equipped with multiple pili. Pili enable
movements in a watery environment but not in slime. Short rods have additionally
flagella, which like propeller move them through slime. Long curly rods use
complex body movements to screw through gels of high viscosity, but are immobile
in water (4-6).
|
Fig. 10. The mucus completely
separates mucosa from faeces in the distal colon of mice similar to the
situation in man (as shown in Fig. 2). |
|
Fig. 11. The separation of
bacteria in the proximal colon of mice is selective, Eubacterium rectale
(EREC) and its subgroup (Physco) enter crypts, Bacteroides has
no contact with the colonic wall. |
|
Fig. 12. Short rods of Bacteroides,
Enterobacteriaceae, Clostridium difficile, and the Veillonella
groups have no contact with the colonic wall in mice. |
The presence of the mucus barrier in the proximal colon of mice can be clearly
demonstrated in germ-free mice mono-associated with
Enterobacter cloacae
– a bacterium with a short coccoid form. The distinct mucus layer and separation
of bacteria from the colonic wall can be observed in both the distal and proximal
colon. Bacteria are perfectly separated in the distal colon, however in the
proximal colon some bacteria can be found inside of isolated vacuoles of the
goblet cells, especially at the bottom of crypts (
Figs. 13,
14).
The undifferentiated epithelial cells at the base of crypts are primarily mucus-secreting
cells, whereas differentiated cells of the columnar epithelium are mainly absorptive
cells, removing water and electrolytes from the mucus. The epithelial stem cells
at the crypt base proliferate and replace surface cells within 4–8 days. The
dissemination of
E. cloacae in crypt bases and goblet cells outline sones
of lower viscosity and confirms independently that during the journey from the
crypt base toward the surface epithelium crypt cells become increasingly differentiated
and absorptive. The adsorptive cells of the crypt neck and of the epithelial
cells of the columnar epithelium dehydrate the mucus layer. Dehydration makes
the mucus layer solid and impenetrable for bacteria and protects sites of mucus
production and the mucosa from encounters with potential pathogens. The lower
viscosity of the mucus at the crypt base promotes emptying of crypts and prevents
obstruction, but as a drawback it may make these types of cells more vulnerable
to invasion by potential pathogens. Indeed, invasion of epithelial cells by
E. cloacae was observed exclusively at the crypt bottom, whereas no
E.
cloacae-containing cells were observed within the cytoplasm of the columnar
epithelial cells in mono-associated mice. Interestingly, crypt abscesses, which
are typical histomorphologic findings in human self-limiting colitis and IBD,
are also more abundant toward crypt bases.
|
Fig. 13. Proximal colon of
mice mono-associated with Enterobacter cloacae. Despite the separation
by the mucus layer, bacteria can be found inside of single vacuoles of
the goblet cells, especially at the bottom of crypts where the viscosity
of the mucus is lower. |
|
Fig. 14. Distal colon of mice
mono-associated with Enterobacter cloacae. Bacteria are perfectly
separated from the intestinal wall. |
Effect of dextrane sodium sulphate on the bowel of mice
What happens if the viscosity of the mucus layer is reduced for example by addition
of detergents? The addition of dextrane sodium sulphate (DSS) in
in vitro
experiments makes the gels penetrable for bacterial movements at viscosity levels,
which normally completely immobilise bacteria (
Fig. 15). In mice, the
addition of DSS to food leads to a colitis. In DSS colitis, leukocytes migrate
into the colonic lumen and line up at the border between mucus and faeces (
Fig.
16). However even this leukocyte response can not stop the migration of
bacteria toward the mucosa. In the distal colon,
Bacteroides circumvents
the leukocytes, passes through mucus, adheres to the mucosa, and causes deep
tissue infiltration (
Fig. 17). The inflammation in the DSS animal model
is restricted to the large bowel, although the substance is provided in the
drinking water and should have theoretically the same effect throughout the
intestine. However bacterial concentrations in the small intestine of mice are
extremely low compared to bacterial concentrations in the colon. Mucus barrier
failure has therefore fewer consequences in the small intestine than in the
large intestine.
|
Fig. 15. DSS supplement to
gel or to the suspension of faecal bacteria enhances bacterial movements.
In DSS supplemented gels, short coccoid bacteria such as Bacteroides
move up to agarose concentrations of 0,6%. The movements of long rods
(EREC) across the mucus can be observed up to concentrations of 0,9%. |
|
Fig. 16. Leukocytes (arrows) migrate into the lumen of the large intestine. |
|
Fig. 17. Bacteroides
crosses the mucus (left). The same microscopic field in DAPI (right) shows
leukocytes (large blue nuclei) migrate into the mucus and hinder Bacteroides
from moving towards the mucosa. Normally only single leukocytes are present
in the mucus. |
Experiments in IL-10 gene-deficient mice treated with carboxymethyl cellulose
Beate Sydora (Alberta University, Canada) has treated IL-10 gene-deficient mice
with 2% carboxymethyl cellulose (CMC) dissolved in water. Normally IL-10 knock-out
mice develop colitis in adult age. The small intestine is not involved. The
pattern of distribution of inflammation is in accordance with murine bacterial
colonization. IL-10 knock-out mice have usually no bacteria in the small intestine
and high bacterial concentrations in the large intestine. In the CMC experiments
of B. Sydora, the controls which consisted of mice treated with water only,
had no inflammation and no bacteria between villi in the small intestine. However
bacteria and leukocytes were found between villi in the proximal parts of the
small intestine in half of the CMC treated mice. (
Fig. 18) The intensity
of changes increased in the distal direction. High bacterial concentrations
were found within crypts of Lieberkuhn in the ileum of all CMC treated IL-10
knock-out mice (7). These findings resembled visually the situation, which can
be observed in the ileum of patients with Crohn’s disease (
Fig. 19).
|
Fig. 18. Proximal jejunum, IL-10 KO mice. The arrows indicate bacteria and leukocytes between villi. |
|
Fig. 19. Ileum of patients with Crohn’s disease shows the similarities to the proximal jejunum in the IL-10 KO mouse, treated with carboxymethyl cellulose, the arrows indicate leukocytes between villi. |
The biostructure of faecal microbiota in stool samples of human
The evaluation of therapies remodelling the mucus barrier affords simple and
effective criteria for efficacy, which are independent of subjective complaints.
FISH investigation of bioptic material is an important method, however biopsy
can not be performed repeatedly just for the study of the effects of therapy.
However the disturbance of the mucus layer leads to changes in the biostructure
of faecal microbiota, which can be investigated. We developed a method to investigate
the biostructure of faecal microbiota. Faeces proved to be highly spatially
organized. Healthy faecal microbiota can be divided into habitual bacterial
groups and occasional bacteria, present only in subgroups of patients, either
diffusely or locally condensed. With regard to the faecal mucus, bacteria could
be divided in faecomucous, mucophob and mucotrop. We found that the stool is
covered with mucus which is free of bacteria in healthy persons (
Fig. 20).
The mucus secretion was increased in patients with diarrhoea. The superficial
mucus layer was thicker and mucus could also be found within faeces enclosed
in form of broad septa or multiple striae (
Fig. 21). In ulcerative colitis,
the mucus was significantly reduced compared to all disease control groups and
to healthy controls. The surface of the faeces was covered with a layer of leukocytes
instead. The occurrence of leukocytes stresses the advantages of using stool
cylinders over faecal homogenates, since no leukocytes are located within the
faecal masses (
Fig. 22).
|
Fig. 20. Stool cylinder, thionin blue staining. |
|
Fig. 21. Distribution of mucus in healthy patients and in patients with diarrhoea. |
|
Fig. 22. Leukocytes covering the surface of the faecal cylinder. |
So far, we have investigated more than 5000 faecal cylinders. The evaluation
of 12 of the most representative bacterial groups in healthy, non-inflammatory
disease controls, UC, and CD revealed many characteristic details, which enable
the discrimination between these conditions. The most prominent features in
IBD were: reduction of the mucus thickness especially in UC, progressive decrease
in the concentrations of the habitual bacteria and disintegration of their web
structure, spheroid precipitation of
Bacteroides to isolated island in
patients with UC, increased concentrations of leukocytes in the mucus and on
the surface of faeces in UC, reduction and loss of
Faecalibacterium prausnitzii
in CD, high concentrations by excellent fluorescence of
Faecalibacterium
prausnitzii in UC, increased concentrations and occurrence of mucotrop
Enterobacteriaceaei
with decreased concentrations of mucotrop. Verucomicrobiaceae (Hel274) in both
CD and UC patients, increased concentrations of faecal cylinders.
Enterobacteriaceaei in CD with low concentrations of faecal
Enterobacteriacae
in patients with UC, reduced occurrence of
Eubacterium hallii and
E.
cylindroids bacteria in CD, and elevated concentrations of
Bifidobacteriaceae
and
Atopobium in patients with UC. The dynamics in concentrations and/or
occurrence of
Faecalibacterium prausnitzii, faecal
Enterobacteriaceaei,
Bifidobacteria, Atopobium,
Eubacterium cylindroides,
E. hallii
and leukocytes were strikingly opposite in UC and CD, allowing differentiation
between both diseases and indicate that these diseases are distinctly different
entities and not just different expressions of the same inflammatory process
(
Table 2). However, the quantitative assessment of 2 parameters: leukocytes
at the faeces/mucus border and
Faecalibacterium prausnitzii concentrations
were sufficient to diagnose active CD and UC with a 79/80% sensitivity and 98/100%
specificity.
Table 2. Results of investigations of stool cylinders. |
|
The lack of sensitivity in the investigation of faecal cylinders in order to diagnose active CD and UC (79/80%) was due to overlap between Crohn’s disease and UC and intermediate colitis, and the lack of specificity (98/100%) was due to overlap between Crohn’s disease and coeliac disease/carcinoid of the small bowel. No overlap occurred between IBD and healthy controls, self-limiting colitis, and non-inflammatory disease subjects. In fact, none of the subjects from the healthy or the non-inflammatory control groups matched criteria for IBD.
DISCUSSION
It has been previously assumed that the enormous masses of bacteria present
in the intestine directly contact the intestinal wall. The non-pathogenic bacteria
are tolerated, while the pathogenic bacteria are responded to. Dysfunction of
the immunologic balance would lead to overreaction to normal non-pathogenic
faecal components, thus initiating and sustaining chronic inflammation. Unfortunately,
the residents of the large bowel can not be clearly divided into good and evil.
However, many of indigenous bacteria are pathogenic.
Escherichia coli
causes sepsis,
Bacteroides causes abscesses,
Enterococci cause
endocarditis,
Clostridium histolyticum causes gas gangrene. We call these
bacterial groups normal inhabitants of the human colon since they can be found
in every healthy person. Let us assume that the host can recognize within the
faecal mass more or less pathogenic bacteria and specifically hinder them on
contact. This response would have to eliminate single bacterial groups from
the polymicrobial mixture without affecting all other bacteria – an implication
which is difficult to believe.
The FISH analysis of the mucosal flora clearly indicates that the host does
not tolerate the indigenous flora or its parts, it ignores it in whole. The
bacterial concentrations within the large intestine can reach extremely high
concentrations of 10
11 bacteria/ml, but the mucus
barrier efficiently separates colonic bacteria from the colonic wall making
any response unnecessary.
Viscous mucus covers the intestinal wall, disables bacterial movements, and protects epithelial cells from contact with bacteria. Leukocytes migrate into and patrol within the mucus layer executing surveillance function without any collateral damage. The sticky outer mucus surface offers the opportunity for probiotic strains to grow and build protective interlaced layers, making it even more difficult for pathogenic strains to reach the mucosa.
The inflammation takes place only after the mucus barrier is broken and the
defence is overwhelmed. Since the beginning of the 20
th
century, there has been a steady increase in reported cases of both Crohn’s
disease and ulcerative colitis and the peak has obviously not been reached.
This increase in IBD is mainly affecting the developed world, especially populations
with high living standard and urban areas. Statistically the frequency of the
disease correlates with the introduction of tap water, soap and improvement
in the living conditions. The hygiene hypothesis argues therefore, that improved
hygiene and a lack of exposure to microorganisms of various types have sensitized
our immune system, leading to inadequate reaction to harmless bacteria in our
environment. Out of this speculation have come recommendations to allow young
children a reasonable amount of contact with dirt, pets, and other potential
sources of infection as well as therapy with helminths for IBD. The statement
that exposure to microbes in the city is lower than in the country population
is basically wrong. The vegetables and fruits on our table are imported from
Greece, Portugal, New Zealand, South Africa, and Australia. They import a vast
variety of microorganisms that were previously unknown to the consumer. The
mobility of the modern society has led to a profound and rapid exchange of bacteria
worldwide which was never encountered in the suburban world.
The
in vivo effects of the dextrane sodium sulphate (DSS) detergent in
mice and in the mucus simulation model however reveal other possible potential
side effects of cleanliness and urbanisation. Traces of the detergents that
make our dishes shine are ingested with our food. The “cleaning” effects of
ingested home soaps on colonic mucus have been never investigated. Detergents
make the objects clean, they do not sterilize them. Emulsifiers that are added
to many foods to achieve a desired consistency may also have effects on the
intestinal mucus. The recent data on Il-10 gene-deficient mice support this
hypothesis. CMC is extensively used in the food industry, because cellulose
is so abundant and cheap and the emulsifying and thickening properties of CMC
are useful. The substance is added to food to stabilize emulsions, for instance
in ice cream, to dissolve ingredients such as cacao in order to make perfect
chocolate and sugar icing, to boost the flavour of the natural aroma and to
keep bread fresh and soft. It can be found in toothpaste, chewing gum, a variety
of baked goods, candies, sausages, ketchup and other sources. It is a filling
and stabilizing component of most pills and it is a main substitute for gluten
in manufactured gluten free products. Actually CMC is everywhere in quantities
which are larger than those given with the drinking water to the mice in Sydora’s
experiments. The annual amount of CMC utilized by the food industry is constantly
increasing. Presently there are no quantitative restrictions on its use, and
its addition to food does not even require to be declared. CMC is, however,
not the only emulsifier broadly used by the food industry. The list of emulsifiers
which are permitted by the EU is too long to fit in a single page. Emulsifiers
are practically everywhere starting with Konjak. Many other factors can influence
the mucus barrier (
Table 3). Bile acids, for example, are natural emulsifiers.
Normally they are completely resorbed in the ileum and do not reach the colon.
In patients with ileum resection, the resorption is disturbed, bile acids reach
the colon and induce diarrhoea. Coeliac disease is regarded as an allergic response
although the exact structure within the gluten molecule which is allergic could
not be defined. We do know that symptomatic coeliac disease is always ongoing
with bacterial overgrowth in the small bowel. The link between bacteria and
glutens is poorly understood. Glutens are however naturally occurring emulsifiers.
It could be that bacteria make glutens harmful.
Table 3. Factors affecting mucus barrier. |
|
Smoking stimulates mucus secretion but does not increase (probably does diminish) the mucus viscosity. The epidemiologic studies indicate that smoking is beneficial for ulcerative colitis but detrimental for patients with Crohn’s disease. A thicker mucus barrier could indeed explain why smoking could be protective in UC patients but have no effect in Crohn’s disease, where bacterial suppression is more important, than bacterial separation.
Stress interferes both with mucus production and regulation of the mucus viscosity. It is a known fact that, in IBD patients, stress leads to acute exacerbations of the disease.
Multiple other factors including defensins, probiotics, enteral pathogens, the inflammation itself, genetic background etc. interfere with the mucus barrier function. As long as the mucus barrier is compromised, a conflict between the organism and the pathogens inhabiting the colon in large numbers and diversity is inevitable.
What can be done to improve the mucus barrier? There are multiple options to
do so (
Table 4). Prednisolone is a very potent drug. As a glucocorticoid
it stimulates the mucus secretion. Its mineral corticoid activity increases
the water resorption, thereby increasing the viscosity gradient within the intestinal
mucus layer. Development of substances which can selectively control the mucus
barrier without the typical side effects of prednisone could be of extreme advantage
for IBD treatment.
Table 4. Possible ways to remodel the mucus barrier. |
|
We have previously mentioned that the columnar epithelial cells are differentiated and mainly resorptive, while crypt cells are immature stem cells and mainly secretory. A balance between both is under tumor necrosis factor (TNF) control. Cell turnover is increased during inflammation. Anti TNF reduces the apoptosis of differentiated epithelial cells and that may explain why, of many known mediators of inflammation, only anti-TNF antibodies have a clinically proven significance. The development of drugs with an effect on apoptosis regulation of the epithelial turnover should be considered in the future.
Antibiotics can effectively reduce the number of pathogens contacting the mucosa.
They have however no direct influence on the mucus barrier and they can not
sterilize the polymicrobial colonic microbiota. As soon as antibiotics are withdrawn
because of increasing microbial resistance, the situation gets reversed. In
the long-term, antibiotics are generally ineffective in IBD. The mucus barrier,
however, can be compromised not only by environmental or genetic factors but
also by specific pathogens such as
Serpulina,
Fusobacteria,
Enterobacteriaceaei,
or
Gardnerella. These bacteria can specifically form adherent biofilms
on the epithelial surface compromising the mucus barrier and allowing migration
of other indigenous bacteria into the mucosa. The specific identification of
such a colonization and eradication of it by specific antibiotic treatment could
be advantageous.
Mesalazine suppresses bacterial biofilms
in vivo by mechanisms, which at present are not clear. Different to antibiotic therapy, the suppression with mesalazine does not seem to induce bacterial resistance. It is possible that the suppressive effects of mesalazine could be further expanded, when the mode of action is clarified.
The reduction of the detergent and emulsifier burden in our food was mentioned. We do not know at present which of the substances may reach the colon and accumulate in the human body. These questions still need to be further investigated.
The stimulation of the immune response is an eligible aim. Previous trials with interferon, GM-CSF were half-heated and inconsequent. PEG interferon was for example not tested at all. The therapeutic potential could be enormous.
After all, probiotics may act as some kind of living vaccines using attenuated strains and stimulating mucosal immunity. Actually we do not know how probiotics work. However, since the influence of antibiotics on polymicrobial microbiota is limited, the use of biologicals as indigenous microbiota is intriguing. At present, all available probiotics use bacterial strains, which work marginally in the human large intestine (less than 0.01%). They were selected mainly for ease of culture, storage, transport and stability within food products. The probiotic potential of anaerobes, which constitute the mass of the indigenous flora of the large intestine, has not been studied.
CONCLUSIONS
The intestinal wall is effectively protected from direct contact with potentially
harmful bacterial groups such as
Bacteroides,
Enterobacteriaceaei,
Enterococci, and
Clostridium histolyticum, which are indigenous
and highly concentrated in the colon. A well-developed mucus barrier and not
the epithelial cell layer is the first line of defence against a variety of
enteric pathogens. Before bacteria can adhere and invade the mucosa, they must
first traverse the mucus barrier. When pathogens penetrate mucus and adhere
to epithelial cells, inflammation clears the mucosa from bacterial contact and
mucus from the bacteria, thus re-establishing the status quo.
The rising incidence of IBD over the last century may result from a disturbance of the mucus barrier function caused by excessive use of detergents and emulsifiers and from changes in the types and numbers of bacteria in our surroundings.
Against this background, IBD can be viewed as a polymicrobial infection that is characterized by a sustained broken mucus barrier with subsequent bacterial migration toward the mucosa and proliferation of complex bacterial biofilms on the epithelial surface.
As long as the mucus barrier function is impaired, the inflammatory process cannot successfully clear bacteria from the mucosal surface and immunosuppressive therapy remains the main therapeutic option. Other therapeutic principals including regulation of the mucus secretion and viscosity, suppression of bacterial biofilms, eradication of occasional pathogens, probiotics and immunostimulation are however also possible and should be increasingly considered and evaluated in the future.
As a consequence of the inflammatory response, the composition and structure of faecal microbiota is changed. The structural changes can be exactly quantified and used to monitor the disease activity. Based on the biostructure of faecal cylinders, Crohn’s disease and ulcerative colitis can be distinguished from each other and other disease controls.
The possibility to monitor the disease activity in faecal samples will allow us to intensify the search for alternative therapeutic strategies aimed at cure of IBD instead at symptom control.
Conflict of interests: None declared.
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