FIELD OF THE INVENTION

The present invention relates generally to methods for selecting probiotic lactic acid bacterial strains capable of increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal, and the use of such strains to reduce the risk for microorganisms, antigens or other agents from entering and coming in contact with the epithelium in the gastrointestinal tract of animals, including humans. The invention also comprises administration of such strains to animals, including humans, which have lost or are at risk of losing the firmly adherent mucus layer or have or are at risk of having an altered or reduced thickness of the firmly adherent mucus layer.

BACKGROUND OF THE INVENTION

Probiotics are defined as microorganisms that provide health benefits when consumed. For example, The Food and Agricultural Organization of the United Nations define probiotics as "live microorganisms which when administered in adequate amounts confer a health benefit on the host".

The mucous membrane covers all body cavities or passages that are externally exposed or are internal organs. Cells in the mucous membrane secrete mucus that lubricates the membrane and acts as a physical barrier protecting the epithelial surfaces and prevents pathogens and other agents from entering the body. Mucosal membranes can, for example, be found in the respiratory, gastrointestinal, urogenital, visual and auditory tract of animals.

The large intestine holds several layers of protection against luminal microbes and pathogens. The pre-epithelial level of defence consists of a continuous mucus layer with accumulated immunoglobulins and antimicrobial peptides that keeps the bacterial load at a distance from the intestinal wall. The mucus can be divided in an adherent layer that is attached firmly to the epithelium (the firmly adherent layer), and a more luminal loosely adherent layer.

At the level of the epithelial cells lining the intestine, secreted antimicrobial peptides and tight junction proteins work together to keep the bacteria from entering the mucosa (i.e. act as the epithelial barrier). Below the epithelial cells, in the lamina propria and the submucosa, a large number of resident intestinal immune cells (leukocytes) ready to engulf invaders and to secrete danger signals (cytokines and chemokines) comprise another level of protection.

Under normal conditions, bacteria colonize the outer (loose) mucus layer where the mucus serves as nutrients for the bacteria and the bacterial microbiota provides the host with vitamins and colonocytes with nutrients as they ferment undigested carbohydrates. The inner firmly adherent mucus layer is devoid of or contains very low numbers of bacteria.

If the firmly adherent mucus layer is lost or thin, bacteria and other agents are found within the firmly adherent mucus layer as well as in the mucosa draining lymph nodes; hence the barrier function has been overridden. This may cause health issues.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that certain strains of lactic acid bacteria, in particular certain strains of Lactobacillus reuteri, could increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract in vivo without increasing the thickness of the loosely adherent mucus layer. This is an advantageous finding for therapy of various diseases that affect the gastrointestinal tract as the ability to increase the thickness of the firmly adherent mucosal layer can result in the improvement, repair or restoration of the pre-epithelial barrier or intestinal lining/mucus lining in diseases where this barrier or lining has been damaged, reduced, lost or otherwise adversely affected. This can in turn result in preventing or reducing potentially damaging microorganisms, antigens or other agents from penetrating through to the underlying epithelial cells and mucosa of the gastrointestinal tract and causing problems such as infection or inflammation. Such lactic acid bacteria can also conveniently be used to prevent or reduce such damage to the pre-epithelial barrier in appropriate subjects, for example those at risk of such damage occurring. The fact that only an increase in mucus thickness in the firmly adherent mucus layer and not the loosely adherent mucus layer was found could further be advantageous when it comes to certain conditions where an increased loosely adherent mucus layer is undesirable, as for example in Crohn's disease.

Such lactic acid bacteria, in particular certain strains of Lactobacillus reuteri, can thus be used in the present invention to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal. Such lactic acid bacteria, in particular certain strains of Lactobacillus reuteri, can also be used to treat or prevent various diseases or conditions associated with the gastrointestinal tract in animals, in particular diseases where the firmly adherent mucus layer is thin or absent.

As the observed ability to increase the thickness of the firmly adherent mucus layer in vivo is to a certain extent dependent on the characteristics of the particular strain of lactic acid bacteria, the present invention also provides methods of selecting appropriate bacterial strains.

Thus, a primary object of the present invention is to provide a method for the selection of lactic acid bacterial strains with the capability to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract of animals, including humans. Another object of the invention is the use of such strains to deliver a health benefit to a host, for example to reduce the risk for microorganisms, antigens and other agents to enter or come in contact with the epithelium in the gastrointestinal tract in animals, including humans.

Another object of the invention is the use of such strains to reduce the risk for microorganisms, antigens and other agents to enter or come in contact with the epithelium in the gastrointestinal tract in animals, including humans, at risk for losing or having a thinner firmly adherent mucus layer thickness.

Another object of the invention includes but is not limited to lactic acid bacterial strains of Lactobacillus reuteri which are suitable for use in such methods, more specifically Lactobacillus reuteri AT CC PTA 4659.

In preferred embodiments of the invention the increase in the thickness of the firmly adherent mucus layer takes place without an increase in the thickness of the loose mucus layer (also referred to herein as the loosely attached or loosely adherent mucus layer). Thus, preferred strains do not have the capability to increase the thickness of the loosely adherent mucus layer in the gastrointestinal tract of said animal. Put another way, preferred strains selectively promote an increase in the thickness of the firmly adherent mucus layer in the gastrointestinal tract as opposed to an increase in the thickness of the loosely adherent mucus layer.

The present invention thus relates to a new method of selecting lactic acid bacterial strains, in particular strains of Lactobacillus reuteri, which are useful as probiotics and in therapy. This new method involves the screening and selection of strains of lactic acid bacteria which have the capability to increase the thickness of the firmly adherent mucus layer in an animal but preferably do not increase in the thickness of the loosely adherent mucus layer. As discussed above, the lactic acid bacterial strains selected by this method are useful as probiotics and in prevention or treatment of diseases of the gastrointestinal tract by way of the ability to increase the thickness of the firmly adherent mucus layer in an animal and preferably not to increase the thickness of the loosely adherent mucus layer. Indeed, to our knowledge, strains of Lactobacillus reuteri have never before been reported to be able to increase the firmly adherent mucus layer thickness in vivo.

Thus, one aspect of the present invention provides a method of selecting a lactic acid bacterial strain or a probiotic lactic acid bacterial strain with the capability to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal, wherein said method comprises screening a lactic acid bacterial strain for the capability to increase the thickness of the firmly adherent mucus layer in an animal and selecting a strain of bacteria which has said capability. Once an appropriate strain has been selected using the method of the present invention it can then be used for increasing the thickness of the firmly adherent mucus layer (and preferably not increasing the thickness of the loosely adherent mucus layer) in the gastrointestinal tract of an animal.

Thus, a further aspect of the present invention provides a lactic acid bacterial strain obtainable by the selection method of the invention, for use in increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal.

Alternatively viewed, the present invention provides a lactic acid bacterial strain for use in increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal.

Preferred features of this strain and its uses are described elsewhere herein.

Methods of increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal are also provided, said methods comprising the

administration of a lactic acid bacterial strain obtainable by the selection method of the invention, or the administration of a lactic acid bacterial strain wherein said lactic acid bacterial strain is capable of increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal, to said animal in an amount effective to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract of said animal.

Preferred features of the strain and its uses are described elsewhere herein. Such methods can also be regarded as methods of treatment of an animal.

Also provided by the present invention is the use of a lactic acid bacterial strain obtainable by the selection method of the invention, wherein said lactic acid bacterial strain is capable of increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal, in the manufacture of a composition or medicament for use in increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of said animal.

Alternative embodiments provide the use of a lactic acid bacterial strain, wherein said lactic acid bacterial strain is capable of increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal, in the manufacture of a composition or medicament for use in increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract of said animal. Preferred features of the strain and its therapeutic uses are described elsewhere herein.

Administering the lactic acid bacterial strains discussed herein, for example those selected according to the present method, to an animal will result in an increase in the thickness of the firmly adherent mucus layer in the gastrointestinal tract that could be beneficial to said animal for several reasons.

The administration of the probiotic strains in said methods of treatment and uses of the invention is carried out in pharmaceutically or physiologically effective amounts, to subjects (animals) in need of treatment. Thus, said methods and uses may involve the additional step of identifying a subject in need of treatment, e.g. by identifying a subject which may benefit from an increase in thickness of the firmly adherent mucus layer in the gastrointestinal tract, for example, by identifying a subject with a reduced thickness of the firmly adherent mucus layer or a subject suffering from diseases or conditions associated with a reduced thickness of the firmly adherent mucus layer as described elsewhere herein, in particular a lost or thin firmly adherent mucus layer. Thus, subjects in need of treatment can be identified in any appropriate way, e.g. by detecting biomarkers or other indicators of reduced thickness of the firmly adherent mucus layer, or of diseases or conditions associated with a reduced thickness of the firmly adherent mucus layer as described elsewhere herein.

As will be outlined elsewhere herein, preferred methods and uses are in the treatment and/or prophylaxis (prevention) of any conditions or diseases of the gastrointestinal tract of an animal which will benefit from increased thickness of the firmly adherent mucus layer in the gastrointestinal tract, for example, diseases or conditions associated with a reduced thickness of the firmly adherent mucus layer, in particular a lost or thin firmly adherent mucus layer.

The present invention also provides products and compositions containing said strains which have the capability to increase the thickness of the firmly adherent mucus layer (and preferably do not have the capability to increase the thickness of the loosely adherent mucus layer). As the methods of the invention provide an appropriate means to identify new strains with such capability, such new strains and products or compositions comprising said new strains form preferred embodiments of the invention. Thus, a strain selected for its ability to increase the thickness of the firmly adherent mucus layer (and preferably not to increase the thickness of the loosely adherent mucus layer) using the selection methods of the invention is also provided.

Other objects and advantages will be more fully apparent from the following disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 . (A) Oral administration of L. reuteri strains 4659 or R2LC significantly increased firmly adherent colonic mucus layer thickness. This increase was present also in mice which received 3% DSS for 7 days. (B) Thickness of the loosely adherent mucus accumulated over 60 min after removal did not differ between the treatment groups. ^* p < 0.05 vs control n = 6 in all groups.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF The relationship between a host and its microbes is complex and has been developing over many years of co-evolution. As such microbes can have beneficial effects on their host, there is therefore also a need to understand specific interactions between microbes and its host which are related to a specific disease or other situations influencing the health of the host so that the most appropriate probiotic strains can be selected and used to counteract such adverse developments.

The mucus layer lining the gastrointestinal tract forms the first line part of the intestinal barrier. The inventors herein have found that certain lactic acid bacterial strains, including certain strains of Lactobacillus reuteri, can increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract, in particular in the colon. Such an increase in thickness can in turn benefit the host by for example preventing or treating diseases of the gastrointestinal tract. The increase of the firmly adherent mucus layer was observed without an increase of the loosely adherent mucus layer. This can be a further advantage in the treatment or prevention of certain conditions in which an increase in the thickness of the loosely adherent mucus layer is undesirable, for example diseases which are associated with increased levels, increased production of, overproduction of, or increased thickness of the loosely adherent mucus layer, for example Crohn's disease.

For example, the increase in thickness of the firmly adherent mucus layer can increase the protection of the epithelial cells of the gastrointestinal tract from damage, injury or inflammation, for example it reduces the risk of microorganisms, antigens or other agents to enter or come into contact with the epithelium in the gastrointestinal tract, thereby enhancing or strengthening the pre-epithelial barrier. Thus, the invention provides the ability to enhance or strengthen or improve the gastrointestinal tract barrier, e.g. the colonic barrier, more specifically the ability to enhance or strengthen or improve the pre-epithelial barrier. The firmly adherent mucus layer not only acts as a physical barrier in the gastrointestinal tract but can also act to sequester beneficial agents such as anti-bacterial peptides which can reduce bacterial translocation across the epithelial barrier to the underlying tissue or mesenteric lymph nodes. Thus, the ability to increase the thickness of the firmly adherent mucus layer as described herein can result in an improved physical barrier and an improved functional barrier at the pre-epithelial/mucus level (e.g. in terms of preventing or reducing bacterial translocation).

Gastrointestinal diseases are complex diseases which often have multiple factors (are multifactorial) or symptoms which result in the manifestation of disease. For example, some gastrointestinal diseases will have an inflammatory component, some will have a mucosal component, for example some kind of damage to or disfunction of the epithelial layer or some kind of damage to or disfunction of the pre-epithelial mucus layer. Some gastrointestinal diseases will have more than one or all of these components, or indeed additional components, and often it will depend on the stage of disease or how severe the disease is.

The effect of the present invention thus occurs at least at the pre-epithelial level, i.e. the mucus level, in particular at the level of the firmly adherent mucus layer. This can be contrasted with for example effects at the epithelial barrier, e.g. by having an effect on the epithelial cells of the gastrointestinal tract, e.g. by strengthening the epithelial barrier in order to for example reduce translocation of bacteria to the underlying mucosa. Although it is possible, and indeed preferable in some embodiments, that the bacterial strains used in the invention have multiple functional effects, the effect on increasing the thickness of the firmly adherent mucus layer is always present.

Further optional functional effects that the bacterial strains used in the present invention might have in addition to the ability to increase the thickness of the firmly adherent mucus layer might include one or more of the ability to improve or strengthen the function of the epithelial barrier, to improve or strengthen the function of the pre-epithelial barrier/mucus barrier in ways other than by increasing the thickness of the firmly adherent mucus layer, or the ability to reduce inflammation in the gastrointestinal tract. In embodiments of the invention it is preferred that the bacterial strains do not have the capability to increase the thickness of the loosely adherent mucus layer.

The present invention thus provides the treatment or prevention of gastrointestinal diseases or conditions characterised by defects, e.g. structural or functional defects, in the pre-epithelial mucus barrier or mucus layer, in particular in the firmly adherent mucus layer. The treatment or prevention of gastrointestinal diseases with reduced barrier function at the mucus layer level, e.g. diseases associated with impaired mucus barrier (mucosal) defense in the gastrointestinal tract, are thus also provided.

The present invention is useful for the treatment and/or prevention of gastrointestinal diseases associated with reduced thickness of the firmly adherent mucus layer, in particular a lost (or absent) firmly adherent mucus layer or a thin firmly adherent mucus layer (e.g. a firmly adherent mucus layer which is thinner or significantly thinner than the firmly adherent mucus layer normally found in a healthy patient or subject (or a population thereof), or thinner than the firmly adherent mucus layer previously found in the same patient (or subject). The present invention can also be used to treat or prevent diseases associated with any damage to the firmly adherent mucus layer as it provides a way of thickening and repairing this mucus layer. In all embodiments described herein the term "disease(s) associated with" can also refer to "disease(s) characterised by".

The term "firmly adherent mucus layer" as used herein is a term of the art which refers to the inner layer of mucus which is closest to the epithelial cell lining of the gastrointestinal tract and which is attached (firmly or physically attached) to the epithelium. The firmly adherent mucus layer is distinct from the more luminal loosely adherent layer which is not attached to the epithelium. In experimental models, the loosely adherent layer (which is also a term of the art) can be removed from the surface of the gastrointestinal tract relatively easily, for example using a pipette or the like. The firmly adherent mucus layer however is physically attached to the epithelium and would for example require physical intervention or chemical intervention to remove it.

The term "reduced thickness of the firmly adherent mucus layer" includes a reduction in thickness which is significant enough to cause physiological problems or symptoms of disease in the gastrointestinal tract of the subject or animal. However, equally the methods and uses of the present invention can be useful before such clinical manifestations arise. Thus, the invention can be used in subjects with any level of firmly adherent mucus layer thinning, for example any measurable or significant thinning (e.g. thinning at a level which is statistically significant, e.g. with a probability value of <0.05) when compared to the thickness of the firmly adherent mucus layer normally found in a healthy patient or subject (or a population thereof), or thinner than the firmly adherent mucus layer previously found in the same patient or subject. In some patients, the firmly adherent mucus layer might be lost or absent. Thus, it can be seen that references to a lost or thin firmly adherent mucus layer is not necessarily a reference to loss, absence or thinning in the whole gastrointestinal tract but also applies to loss, absence or thinning in parts or regions of the gastrointestinal tract.

Equally, as the present invention is particularly suited to the prevention of diseases and conditions which affect the gastrointestinal tract, and in particular for preventing a gastrointestinal disease associated with reduced thickness of the firmly adherent mucus layer, the present invention can also be carried out on healthy subjects. A "healthy subject" or "healthy patient" or "healthy animal" as referred to herein is a subject or animal which is not demonstrating symptoms of or is not suffering from or has not been diagnosed with a disease of the gastrointestinal tract, or otherwise suffering from any gastrointestinal tract problems, for example does not have a reduced thickness of the firmly adherent mucus layer (in other words the thickness of the firmly adherent mucus layer in such a subject is comparable to or not significantly different from the thickness observed in a normal healthy subject or population of subjects with no gastrointestinal disease).

Appropriate subjects with reduced thickness of the firmly adherent mucus layer which are suitable for use in the present invention could be readily recognised or diagnosed by a person skilled in the art, by for example taking appropriate measurements of the thickness of the firmly adherent mucus layer in a subject using appropriate techniques which would be well known and described in the art, for example using colonoscopy, sigmoidoscopy or by analysis of a biopsy, for example by histological analysis of a gastrointestinal tract section. Although the methods and uses of the present invention are suitable for use in treating or preventing any disease associated with reduced thickness or damage to the firmly adherent mucus layer, particular examples of appropriate diseases or conditions are diseases associated with abnormal intestinal permeability, or diseases associated with damage to the intestinal lining, in particular the intestinal mucus lining/layer. For example, said uses involve the repair and rebuilding of the intestinal lining due to the ability of the strains used to increase the thickness of the firmly adherent mucus layer.

Other suitable diseases would be other gastrointestinal diseases including food allergies. Particular examples might be diseases selected from the group consisting of colitis, Crohn's disease, inflammatory bowel disease (IBD), irritable bowel syndrome and

diverticulosis.

Although the methods and uses of the present invention are useful in all phases and severities of gastrointestinal diseases which reduce thickness in the firmly adherent mucus layer (i.e. in subjects with no or mild disease symptoms, mild-to-moderate disease symptoms, moderate-to-severe disease symptoms, or severe disease symptoms, or in subjects with early-stage, mid-stage or late-stage disease), the present invention finds particular use in patients with early stage disease or in patients having no or mild symptoms or mild-to-moderate symptoms. The methods and uses of the present invention also have particular use in patients which are in remission (or in a remission phase, e.g. a symptom- free phase) from a gastrointestinal disease which reduces thickness in the firmly adherent mucus layer, in order to prevent, delay or reduce the severity of the disease if it returns (e.g. to prevent, delay or reduce the severity of disease flares). Indeed this is another example of a preventative use of the invention.

The methods and uses of the present invention are particularly suitable for the treatment or prevention of ulcerative colitis or Crohn's disease.

Ulcerative colitis is a gastrointestinal disease which can often have different phases. Thus, over the course of the disease, the symptoms of ulcerative colitis patients can vary in severity, for example ulcerative colitis patients can be classified as having no or mild disease symptoms, mild-to-moderate disease symptoms, moderate-to-severe disease symptoms or severe disease symptoms.

Active ulcerative colitis is a term used to describe a phase (called a flare-up) of disease where colitis symptoms are present. In between flare-ups or active phases of disease, patients with ulcerative colitis have phases which are generally symptom free, which are called periods of remission. Although the methods and uses of the present invention are useful in phases of active ulcerative colitis with all the above symptoms (i.e. mild disease symptoms, mild-to-moderate disease symptoms, moderate-severe disease symptoms or severe disease symptoms), the present invention finds particular use in patients with early stage ulcerative colitis or ulcerative colitis with patients having no or mild symptoms or mild- to-moderate symptoms (mild-to-moderate colitis). The methods and uses of the present invention also have particular use in patients which are in remission (or in a remission phase, e.g. a symptom-free phase) from ulcerative colitis in order to prevent, delay or reduce the severity of the next flare-up or active phase, e.g. can be used for maintaining remission or preventing, delaying or reducing active ulcerative colitis. Indeed this is another example of a preventative use of the invention.

Other examples of diseases which can affect mucus layer thickness, in particular firmly adherent mucus layer thickness, in the gastrointestinal tract and which can thus be treated or prevented by the methods or uses of the present invention are conditions associated with stress, in other words, stress induced gastrointestinal conditions. For example, some forms or episodes of irritable bowel syndrome can be caused or exacerbated by stress.

The methods and uses of the present invention can also be used in the treatment or prevention of other diseases of the gastrointestinal tract where damage to the firmly adherent mucus layer or thinning of the firmly adherent mucus layer takes place. Exemplary diseases which will cause such damage or thinning are infections caused by pathogenic organisms, e.g. bacteria, viruses or parasites, for example Helicobacter pylori.

The present invention thus finds particular use in the subset of patients with gastrointestinal tract problems that have reduced mucus thickness, in particular a reduced thickness in the firmly adherent mucus layer. For example, the methods and uses of the present invention can be used to treat stages of or patients with gastrointestinal conditions or diseases where the firmly adherent mucus layer has become more thin/has reduced thickness (e.g. from a previous level), or is otherwise thinner than normal, or is otherwise damaged, but where no other signs or symptoms of gastrointestinal disease are being presented, for example no significant, or limited, inflammatory response (or no significant, or limited, inflammation of the gastrointestinal tract) is as yet being manifested or no significant translocation of microorganisms across the epithelial barrier to the underlying mucosa or the mucosal lymph nodes is yet occurring. Thus, the methods and uses of the present invention are particularly useful for early stage diseases or low grade diseases of the gastrointestinal tract, for example early stage inflammatory bowel disease such as colitis (and in particular ulcerative colitis) or Crohn's disease, irritable bowel syndrome and diverticulosis, or early stages of stress-induced conditions or early stages of infection with pathogenic organisms, as described elsewhere herein.

In this way, the methods and uses of the invention can prevent or delay the progress of disease to a more severe form, or result in an end-point which is a less severe form of disease or symptoms of disease than would be expected without treatment. In addition, the finding that increase of the firmly adherent mucus layer can be observed without an increase in the thickness of the loosely adherent mucus layer is particularly advantageous in the treatment or prevention of certain conditions in which an increase in the thickness of the loosely adherent mucus layer is undesirable, for example diseases which are associated with increased levels, increased production of, overproduction of, or increased thickness of the loosely adherent mucus layer, for example Crohn's disease.

The results presented herein demonstrate that certain bacterial strains can significantly increase the thickness of the firmly adherent mucus layer (but not increase the thickness of the loosely adherent mucus layer) in healthy subjects as well as in subjects suffering from reduced thickness of the firmly adherent mucus layer. These results show the ability of these strains to have a direct effect on mucus thickness. A significant protective (preventative) effect is also demonstrated.

Thus, as described elsewhere herein, the methods and uses of the invention can be usefully carried out on healthy patients (subjects/animals) or patients (subjects/animals) at risk of or susceptible to developing diseases of the gastrointestinal tract or suffering from conditions of the gastrointestinal tract. Examples of patients (subjects/animals) at risk or susceptible include those with family history of gastrointestinal disease, genetic

predisposition to gastrointestinal disease, allergies or intolerances to certain food-types, psychological problems, a stressful job or lifestyle, or patients (subjects/animals) which have been prescribed with or are being subjected to pharmaceutical treatments which have gastrointestinal problems as a potential side effect. All these patient types are suitable for the preventative (prophylactic) aspects of the present invention.

The methods or uses of the invention can be carried out on any animal or subject or patient, for example humans, or any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, horses, sheep, rabbits, cows, monkey and birds, for example poultry or other farmed or commercially bred or domestic birds such as chickens, turkeys, geese or ducks, preferably chickens or turkeys. Preferably, however, the animals or subjects are mammals, more preferably are humans.

The therapeutic uses of the strains, products and compositions of the invention as defined herein generally result in the reduction or alleviation of the relevant disease or symptoms of disease (e.g. reduced severity of symptoms or disease), for example can result in an increase in the thickness of the firmly adherent mucus layer in the gastrointestinal tract which can in turn result in improvement and preferably a significant improvement in symptoms or disease in the animal.

For example, increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract may result in prevention or reduction in the passage of bacteria or viruses or other potentially damaging agents through the pre-epithelial mucus layer to the underlying epithelial lining of the gastrointestinal tract. This in turn may result in prevention or reduction in the passage of bacteria or viruses or other potentially damaging agents into the underlying sub-mucosa or draining lymph nodes which can cause local or systemic inflammation in the host. Alternatively or additionally, the increase in thickness of the firmly adherent mucus layer can allow healing (e.g. mucosal healing) to take place and hence a reduction in intestinal injury or damage (e.g. colon injury or damage).

In particular where diseases of the gastrointestinal tract are concerned, for example IBD, e.g. colitis (e.g. ulcerative colitis) or Crohn's disease, the therapeutic uses of the strains, products and compositions of the invention can result in significant reduction in ulceration and intestinal damage (e.g. colon damage) or reduction in severity of disease measured for example by a standard method or score known in the art, which may involve the monitoring of one or more of a significant reduction in weight loss, or a significant reduction in blood content of the stools, or a significant improvement in consistency of stools, or a significant reduction in inflammation of the intestine, e.g. the colon. Blood tests can also be used to assess or monitor disease severity, for example to assess anaemia (which can suggest blood loss from the bowel) or to assess levels of inflammation in the body. Levels of appropriate faecal proteins such as faecal calprotectin (a measure of e.g. neutrophil migration to the intestinal mucosa) can also be measured to assess the severity of IBD (e.g. ulcerative colitis or Crohn's disease), with higher levels generally indicating more severe disease.

The severity of colitis (and other gastrointestinal diseases) can be presented as a Disease Activity Index (DAI) score, an example of which has a maximum score of 4 (Cooper et al., 1993, Laboratory investigation; a journal of technical methods and pathology; 69:238- 249). Such a DAI score can be assessed on the basis of clinical parameters as described elsewhere herein such as weight loss, stool consistency and blood content, levels of inflammation and blood tests. The methods and uses of the present invention are

particularly useful for reducing the DAI score in a colitis patient (or a patient with any other gastrointestinal disease, e.g. Crohn's disease or other forms of IBD). Such a reduction in score can be an active reduction in the score after administration of the strains to a patient which is suffering from colitis (or other disease) or, when administered in a preventative aspect of the invention, can be a reduction in the DAI score compared to a score which would be expected if no treatment is given.

The reduction or prevention of colitis (and other gastrointestinal diseases) can also for example be measured by a histological assessment of a sample, e.g. of a biopsy, taken from the gastrointestinal tract of a subject. For example, the stages and severity of IBD, such as ulcerative colitis or Crohn's disease, can be assessed by analysis of goblet cells and/or T-lymphocytes in a mucosal biopsy. More specifically, empty goblet cells can be indicative of ulcerative colitis and the severity thereof, whereas filled goblet cells can be indicative of Crohn's disease and the severity thereof. Areas and amounts of T-lymphocytes can also be assessed in such a biopsy to give an indication of the level and severity of inflammation, e.g. the presence of chronic inflammation seen for example in ulcerative colitis.

Analysis of the histology of the intestinal crypts can also conveniently be used to assess the severity of colitis. Severe colitis can be classified as colitis in which the entire crypt and epithelium system is lost (for example on a scoring system from 0 to 4 this would be given a histological score of 4, with intact crypts, for example representing mild or asymptomatic colitis, being given a score of 0, see for example the system discussed in Cooper et al., supra).

The methods and uses of the present invention can therefore result in an improved, preferably significantly improved, histological appearance or score (e.g. when compared to an appropriate baseline or control level) which signifies less severe disease. For IBD (e.g. colitis or Crohn's disease) conveniently samples can be taken from the distal colon by methods which would be well known and standard in the art.

Reduction in intestinal damage or severity of disease can also conveniently be assessed by using appropriate techniques to examine the intestinal mucosa such as colonoscopy or sigmoidoscopy. A sigmoidoscopy can be used to examine the rectum and lower part of the colon, whereas a colonoscopy can be used to examine the whole of the colon, and, if necessary, the lower end of the small intestine. As ulcerative colitis generally begins in the rectum, an example of early stage ulcerative colitis might be patients where only the rectum and/or lower part of the colon is affected.

Such reduction or alleviation of disease or symptoms thereof (e.g. clinical symptoms or severity) can thus be measured by any appropriate assay, examples of which would be well known to a person skilled in the art. Preferably the reduction or alleviation of disease or symptoms is significant, e.g. clinically significant or statistically significant, preferably with a probability value of <0.05. Such reduction or alleviation of disease or symptoms are generally determined compared to an appropriate control individual or population, for example a healthy animal or subject (or a population thereof) or an untreated or placebo treated animal or subject (or a population thereof), or, conveniently, the same individual subject before treatment.

More generally, an assessment of whether or not an increase in the thickness of the mucus layer, and in particular the firmly adherent mucus layer, had taken place in a subject after treatment in accordance with the invention could readily be measured using appropriate techniques which would be well known and described in the art, for example, using colonoscopy, sigmoidoscopy or by analysis of a biopsy, for example by histological analysis of a gastrointestinal tract section. The term "mucus thickness" when used herein in connection with the thickness of the firmly adherent mucus layer generally refers to the vertical distance from the epithelial lining of the gastrointestinal tract to the edge of the firmly adherent mucus layer and can be measured in any appropriate way. Put another way it refers to the depth of the firmly adherent mucus layer. Conveniently said thickness is measured in μηη.

The term "mucus thickness" when used herein in connection with the thickness of the loosely adherent mucus layer generally refers to the vertical distance from the edge of the firmly adherent mucus layer to the lumen of the gastrointestinal tract and can be measured in any appropriate way. Put another way it refers to the depth of the loosely adherent mucus layer. Conveniently said thickness is measured in μηη or μΓΤΐ/hour.

Preferably such increases in mucus thickness (and indeed other increases or positive effects as mentioned elsewhere herein) are measurable increases, more preferably they are significant increases, preferably clinically significant or statistically significant, for example with a probability value of <0.05, when compared to an appropriate control level or value (e.g. compared to an untreated or placebo treated sample or a sample where no strain is present, or compared to the thickness in a healthy or normal sample, or the thickness in the same sample before treatment).

Thus, a bacterial strain which can increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract is one which can give rise to a measurable increase, preferably a significant increase, more preferably a clinically significant or statistically significant increase in the thickness of the firmly adherent mucus layer in the gastrointestinal tract when compared to an appropriate control level or value (e.g. compared to an untreated or placebo treated sample or a sample where no strain is present, or compared to the thickness in a healthy or normal sample, or the thickness in the same sample before treatment).

Similarly, a bacterial strain which does not increase the thickness of the loosely adherent mucus layer in the gastrointestinal tract is one which does not give rise to a measurable increase, preferably does not give rise to a significant increase, more preferably does not give rise to a clinically significant or statistically significant increase in the thickness of the loosely adherent mucus layer in the gastrointestinal tract when compared to an appropriate control level or value (e.g. compared to an untreated or placebo treated sample or a sample where no strain is present, or compared to the thickness in a healthy or normal sample, or the thickness in the same sample before treatment).

Any probiotic strain or any lactic acid bacterial strain which has the ability to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract in vivo can be used in the present invention. However, in preferred embodiments said strain is a strain of Lactobacillus. Preferred strains for use in the methods and uses of the present invention are Lactobacillus reuteri, in particular Lactobacillus reuteri 4659 (ATCC PTA 4659). As the selection methods of the invention provide an appropriate means to identify new or alternative strains, for example, new or alternative Lactobacillus reuteri strains which have the capability to increase the thickness of the firmly adherent mucus layer in the

gastrointestinal tract (and preferably not to increase the thickness of the loosely adherent mucus layer), in other embodiments of the invention the strain used is not Lactobacillus reuteri 4659 (ATCC PTA 4659). The Lactobacillus reuteri strain ATCC PTA 4659 was deposited under the Budapest Treaty at the American Type Culture Collection (University Blvd., Manassas, VA 201 10-2209, USA) on 1 1 September 2002.

An appropriate mode of administration and formulation of the strains, etc., is chosen depending on the site where increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract is desired. A preferred mode of administration is oral or rectal, however, equally for some treatments other forms of administration will be appropriate.

Although the Examples herein demonstrate the use of appropriate bacterial strains and appropriate doses thereof to increase the thickness of the firmly adherent mucus layer and to treat colitis, it will be appreciated that this is only one example of the diseases of the gastrointestinal tract which can be treated in accordance with the present invention and that appropriate doses of the strains, products and compositions of the invention as defined herein can be chosen depending on the disease to be treated, the mode of administration and the formulation concerned.

For example, a dosage and administration regime is chosen such that the probiotic bacteria administered to the animal in accordance with the present invention can result in an increase in the thickness of the firmly adherent mucus layer in the gastrointestinal tract of said animal and to give rise to the desired therapeutic effects or health benefits. Thus, preferably said dosage is a therapeutically effective dosage which is appropriate for the type of animal and condition being treated. For example, daily doses of 10 ⁴ to 10 ¹⁰ , for example 10 ⁵ to 10 ⁹ , or 10 ⁶ to 10 ⁸ total CFUs of bacteria may be used. A preferred daily dose is around 10 ⁸ total CFUs, e.g. 10 ⁷ to 10 ⁹ or 10 ⁸ to 10 ⁹ .

The invention herein is made possible by studies of probiotic Lactobacillus reuteri strain 4659 and R2LC which have demonstrated their effect upon significantly increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract (but not to increase the thickness of the loosely adherent mucus layer).

The present invention thus provides certain strains of lactic acid bacteria, a method of selecting such strains and products comprising such strains. The bacteria are selected using a screen to select for increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract. This surprising ability to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract has been shown in the Examples to be beneficial for the prevention of colitis and hence the methods and uses of the invention provide a means to treat or prevent any gastrointestinal disease which will benefit from increasing the thickness of the firmly adherent mucus layer in the gastrointestinal tract (or any disease associated with reduced thickness of the firmly adherent mucus layer), for example other gastrointestinal diseases as described elsewhere herein.

As will be clear from the disclosure elsewhere herein, the methods and uses of the prevent invention are suitable for prevention of diseases as well as treatment of diseases. Thus, prophylactic treatment is also encompassed by the invention. For this reason in the methods and uses of the present invention, treatment also includes prophylaxis or prevention where appropriate. Preferred aspects where prevention is envisaged are discussed herein.

Such preventative (or protective) aspects can conveniently be carried out on healthy or normal subjects and can include both complete prevention and significant prevention, for example include the situation where a reduction in the thickness of the firmly adherent mucus layer is prevented (e.g. the thickness is not measurably or significantly reduced compared to that observed in a normal or healthy subject), or a reduction in thickness compared to that observed in a normal or healthy subject is observed but said reduction is reduced compared to the reduction in thickness which would be expected if no treatment is given. Similarly, significant prevention can include the scenario where severity of disease or symptoms of disease is reduced (e.g. measurably or significantly reduced) compared to the severity or symptoms which would be expected if no treatment is given.

Preferably such reductions (and indeed other reductions or decreases as mentioned elsewhere herein) are measurable reductions, more preferably they are significant reductions, preferably clinically significant or statistically significant, for example with a probability value of <0.05, when compared to an appropriate control level or value.

As described elsewhere herein, the determination that certain strains of lactic acid bacteria and in particular Lactobacillus reuteri can be used to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract opens up new possibilities for therapeutic treatments and prevention. As described elsewhere herein, methods of selection of appropriate strains are also provided.

Thus, one aspect of the present invention provides a method of selecting a lactic acid bacterial strain or a probiotic lactic acid bacterial strain with the capability to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal, wherein said method comprises screening a lactic acid bacterial strain for the capability to increase the thickness of the firmly adherent mucus layer in an animal and selecting a strain of bacteria which has said capability.

In some embodiments it is preferred that multiple (more than one) bacterial strains will be subjected to the methods. In preferred embodiments, the strains will also be screened and selected for the capability not to increase the thickness of the loosely adherent mucus layer.

Although the methods are designed to identify bacteria with the capability to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract of an animal, i.e. for capability to have an effect in vivo, the screening step to assess the ability of the bacteria to increase the thickness of the firmly adherent mucus layer in the gastrointestinal tract (and optionally the ability not to increase the thickness of the loosely adherent mucus layer) can be carried out using any appropriate assay, e.g. any appropriate in vitro, ex vivo, in situ or in vivo assay. Put another way, the assay to measure the thickness of the relevant mucus layers can be carried out using any appropriate assay, e.g. any appropriate in vitro, ex vivo, in situ or in vivo assay.

As in vitro assays, for example assays based on the use of cell lines and even explanted tissue, can sometimes be misleading as to results which might be observed in an in vivo situation, it is preferred that an appropriate in vivo assay be used in said screening step to assess the capability of a strain to increase the thickness of the firmly adherent mucus layer. A preferred assay will use an animal in which it is possible to induce or provide a situation where the firmly adherent mucus layer is thinner than normal, for example mice. A preferred assay is given in the Examples. However, other appropriate in vivo assays for measuring the capability to increase the thickness of the firmly adherent mucus layer in an animal would be well known to a person skilled in the art. As discussed elsewhere herein capability of the strains not to increase the thickness of the loosely adherent mucus layer, i.e. to only effect (increase) the thickness of the firmly adherent mucus layer, may also be assessed in the above assays, as such strains are preferred in some embodiments. Indeed, in vivo assays are also the most appropriate for measuring the thickness of the loosely adherent mucus layer.

In some embodiments, the selection methods will be set up so as to assess the capability of the strains to increase the thickness of the firmly adherent mucus layer in an animal, e.g. a healthy animal or a wild type animal, or on a sample, e.g. a healthy mucus or gastrointestinal sample, in which the thickness of the mucus layers, e.g. the firmly adherent mucus layer, is normal. In other embodiments, the selection methods will be set up so as to assess the capability of the strains to increase the thickness of the firmly adherent mucus layer in an animal or sample where the firmly adherent mucus layer is thinner than normal, for example in a diseased animal, or a mucus or gastrointestinal sample taken from a diseased animal, for example an animal with a gastrointestinal disease associated with reduced thickness of the firmly adherent mucus layer. In some embodiments, bacteria will be assessed using both these possibilities (i.e. tested on normal and thin firmly adherent mucus layers) as strains which can increase the thickness of the firmly adherent mucus layer (and preferably not to increase the thickness of the loosely adherent mucus layer) in both these scenarios are preferred for some embodiments.

Because of the downstream uses of the strains which are selected by the methods of the invention, after appropriate strains are selected or isolated, other embodiments will involve the further steps of culturing or propagating such strains, or possibly storing such strains for future uses, for example by freezing.

Any probiotic strain or any lactic acid bacterial strain can be assessed in such methods. However, in preferred embodiments said strain to be assessed in the selection methods is a strain of Lactobacillus, more preferably Lactobacillus reuteri.

Any appropriate method or assay can be used to determine mucus thickness and the capability to increase the thickness of the firmly adherent mucus layer or the capability not to increase the thickness of the loosely adherent mucus layer, and a preferred method is described in the Examples. As described elsewhere herein, the term "mucus thickness" when used in connection with the thickness of the firmly adherent mucus layer refers to the vertical distance from the epithelial lining of the gastrointestinal tract to the edge of the firmly adherent mucus layer. Such methods to measure the thickness of the firmly adherent mucus layer (and hence the capability of a bacterial strain to increase the thickness of the firmly adherent mucus layer) will conveniently involve microscopy, e.g. in vivo microscopy, and will sometimes involve the preliminary step of removing the loosely adherent mucus layer.

Similarly, the term "mucus thickness" when used herein in connection with the thickness of the loosely adherent mucus layer generally refers to the vertical distance from the edge of the firmly adherent mucus layer to the lumen of the gastrointestinal tract.

Appropriate methods to measure this may involve direct measurement of this distance, e.g. using microscopy, or can for example involve measuring the total thickness of the mucus layer (i.e. the sum of the firmly adherent plus the loosely adherent layers), measuring the thickness of the firmly adherent mucus layer (e.g. involving the preliminary step of removing the loosely adherent mucus layer) and then subtracting the thickness of the firmly adherent mucus layer from the total. Alternatively, a measure of the loosely adherent layer can be made by measuring the amount of regrowth of the loosely adherent mucus layer for a period (e.g. one hour) following its removal. In this way by comparing the amount of regrowth in the presence and absence of a particular bacterial strain will provide a means of assessing the capability of this strain to effect (or not) the thickness of the loosely adherent mucus layer. A suitable method is described in the Examples.

Biopsies or colon sections might also be used to measure mucus thickness.

However, these are less preferred where an assessment of the effect on both the firmly adherent and loosely adherent mucus thickness is required as such methods generally do not allow the thickness of the loosely adherent mucus layer to be assessed. Any appropriate in vivo assay can be used to assess mucus thickness in the selection methods of the present invention. However, a preferred and exemplary method is carried out in mice, for example as provided in the Examples. Such methods will generally involve anesthetizing animals and then carrying out in vivo microscopy of the relevant part of the gastrointestinal tract, e.g. the colon, in order to measure the distance between the epithelial cell surface and the outer edge of the firmly adherent mucus layer (to determine the thickness of the firmly adherent mucus layer) and optionally to measure the distance from the edge of the firmly adherent mucus layer to the lumen of the gastrointestinal tract (to determine the thickness of the loosely adherent mucus layer). Such methods to measure the thickness of the firmly adherent mucus layer thus generally also involve the preliminary step of removing the loosely adherent mucus layer, which can for example be done with gentle suction. Such methods may also involve an assessment carried out on an animal with a firmly adherent mucus layer that is thinner than normal. Thus, steps to induce such mucus thinning in an animal may also take place.

Generally, time is allowed for the animal to stabilize following the administration of the anaesthetic and any surgical procedure required to exteriorize or otherwise make accessible the appropriate part of the gastrointestinal tract for measurement.

A convenient and preferred method of measuring mucus thickness involves the use of a micropipette, which has preferably been treated so that it does not adhere to mucus, for example by using a siliconized micropipette. Micropipettes with a tip diameter of 1-3 μηη are appropriate. The micropipette can be pushed through the mucus layer until it reaches the epithelium. Using the measurement of this distance (i.e. the distance the micropipette tip has penetrated), together with the measurement of the angle of insertion of the micropipette, the thickness of the mucus can be calculated for example using simple trigonometry. Preferably multiple measurements will be carried out and the thickness of the mucus reported as a mean value of these measurements.

Optionally a step of visualizing the surface of the mucus is also carried out, for example by adding graphite particles to the relevant gastrointestinal tissue. This will allow both the luminal mucus surface and the epithelial cell surface to be visible through the microscope.

As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated or where the context makes it clear that this is not the case.

In addition, where the terms "comprise", "comprises", "has" or "having", or other equivalent terms are used herein, then in some more specific embodiments these terms include the term "consists of" or "consists essentially of", or other equivalent terms. Other objects and advantages of the present invention will become obvious to the reader and it is intended that these objects and advantages are within the scope of the present invention.

The invention will be further described with reference to the following non-limiting Examples:

EXAMPLES

EXAMPLE 1 - L. reuteri increased firmly adherent colonic mucus layer thickness Animals and experimental design

Eighty nine male C57BI/6 mice (Taconic M&B, Ry, Denmark) weighing between 25 and 30 g (weight before treatment), were kept under standardized conditions at a temperature of 21- 22°C and with 12 h light and 12 h dark cycle. The animals were allowed to acclimatize for 1 week before the experiments started. Mice were randomly divided into six different groups: control, DSS-treated, Lactobacillus reuteri (two strains)-treated and lastly L. reuteri (two strains)+ DSS-treated. L. ret/ier/ ^' -treated mice were given 10 ⁸ bacteria in 0.1 ml PBS. This bacterial suspension was given daily by gavage for 7 days. For the DSS-treated mice, the mucus layer was destroyed by adding 3% dextran sulphate sodium (DSS) (TdB Consultancy AB, Uppsala, Sweden), molecular weight -40 kDa, to the drinking water for 7 days. The mice treated with both L. reuteri and DSS were given L. reuteri for 14 days and 3% DSS in the drinking water for the last 7 days of their L. reuteri treatment. All experimental procedures in this study were approved by the Swedish Laboratory Animal Ethical Committee in Uppsala and were conducted in accordance with guidelines of the Swedish National Board for Laboratory Animals.

Bacterial suspensions

Two different strains of L. reuteri were used: R2LC isolated from rat and ATCC PTA 4659 isolated from human sources (deposited under the Budapest Treaty at the American Type Culture Collection University Blvd., Manassas, VA 201 10-2209, on Sept. 1 1 , 2002). The bacteria were cultivated separately in 200 ml MRS broth (Oxoid, Basingstoke, UK) at 37°C for 20 h. Bacteria were washed once with phosphate buffered saline (PBS), and suspended in 2 ml freezing solution [0.82 g K ₂ HP0 ₄ , 0.18 g KH ₂ P0 ₄ , 0.59 g sodium citrate, 0.25 g MgS0 ₄ x7 H ₂ 0, and 172 ml glycerol (87%) diluted to 1000 ml with ddH ₂ 0]. The bacterial suspensions were stored at -70°C until used. Statistical analysis.

The results are expressed as means ± SEM. For statistical evaluations, one-way ANOVA for multiple comparisons was performed when comparing data between groups. The differences were regarded as significant at p < 0.05.

Surgical preparation and measurements of mucus thickness in vivo

The mice were anesthetized with isoflurane (Forene, Abbott Scandinavia, Solna, Sweden) administered through a breathing mask in a concentration of -2.2% in a mixture of 40% oxygen and 60% air. Body temperature was maintained at 37°C using a heating pad controlled by a rectal thermistor probe. The colonic preparation for in vivo microscopy has been described in detail previously (Atuma et al., 2001 , Johansson et al., 2008). Briefly, the descending colon was exteriorized through a midline abdominal incision and carefully cauterized and opened along the antimesenteric border. With the animal placed on its left side, a part of the colon was loosely draped over a truncated cone with the mucosal side facing upwards. Without touching the mucosa, a chamber sealed with silicon grease (Dow Corning High Vacuum Grease, Dow Corning, Wiesbaden, Germany) was placed on top of the exposed colon and filled with 37°C saline. Temperature of the saline and mucosal chamber was maintained by circulating warm water through the double bottom of the chamber. The colonic mucosa was visualized through a stereo microscope (Leica MZ12, Leica, Heerbrugg, Switzerland) and a 150 W fiber optic lamp transilluminating the preparation from below. After the operational procedure, the mouse was allowed to rest for 30 minutes before conducting mucus measurements. To measure mucus thickness, micropipettes connected to a micromanipulator (Leitz, Wetzlar, Germany) were used. The pipettes were pulled from glass tubing using a pipette puller (pp-83; Narishige Scientific Instrument Laboratories, Tokyo, Japan), achieving a tip diameter of 1-3 μηη. Pipettes were siliconized to prevent mucus adhesion. To visualize the surface of the mucus, graphite particles were added in the mucosal chamber, allowing both the luminal mucus surface and epithelial cell surface to be visible through the microscope. The micropipette was pushed through the mucus layer until the pipette reached the epithelium. This distance (I) was measured in five different locations using a digimatic indicator (IDC Series 543, Mitutoyo, Tokyo, Japan) connected to the micromanipulator. The angle (a) of the pipette (-30°) was measured and the thickness (T) of the mucus calculated using the formula T = I ^* sin a, using a mean value of the distances travelled by the pipette tip. To measure the thickness of the firmly adherent mucus layer, the loosely adherent mucus first was carefully removed by gentle suction using a plastic catheter connected to a syringe. The thickness of the colonic firmly adherent mucus layer is increased after treatment with L. reuteri 4659 or R2LC

During normal conditions, pretreatment with either of the two strains of L. reuteri resulted in a significantly thicker firmly adherent colonic mucus layer compared to untreated mice (Fig. 1A). Following DSS-treatment, the firmly adherent colonic mucus layer was significantly reduced in thickness as compared to untreated mice (Fig. 1 A). However, pretreatment with either 4659 or R2LC prior to DSS-treatment (Fig. 1A) resulted in a significantly thicker firmly adherent mucus layer compared to DSS-treated mice not receiving bacteria. After allowing re-growth of the mucus layer for 60 minutes, the loosely adherent mucus thickness was measured. No differences in accumulation of this mucus could be observed in response to L. reuteri treatment (Fig. 1 B).