Lacticaseibacillus paracasei DG (registered trademark) CNCM I-1572 DSM 34154 for use in a method for treating dysbiosis in patients with IBS

Abstract

The object of the present invention is to use a bacterial strain named Lactobacillus paracasei DG (registered trademark) CNCM I-1572 DSM 34154 for the treatment of dysbiosis, in particular for use in a method for treating dysbiosis in patients with IBS (irritable bowel syndrome), preferably patients without constipation and with a high level of Collinsella aerofaciens in the feces.

Description

Technical Field

[0001] The object of the present invention is to use a bacterial strain (strain) named Lacticaseibacillus paracasei DG (registered trademark) CNCM I-1572 DSM 34154 for the treatment of dysbiosis, in particular for the treatment of dysbiosis in patients with irritable bowel syndrome (IBS), preferably those without constipation and with a high level of Collinsella aerofaciens in the feces.

Background Art

[0002] Technical Background Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders, affecting approximately 11% of the population in the United States and Europe. The recurrent abdominal pain in IBS is related to changes in bowel habits. IBS has conventionally been regarded as a disorder with a psychosomatic component related to bowel motor abnormalities and visceral hypersensitivity. Despite the absence of obvious physiologic irregularities at the level of the gastrointestinal tract, the use of quantitative morphologic and molecular techniques has made it possible to demonstrate changes in the gastrointestinal mucosa and / or lumen at the tissue, cell, and molecular levels in most patients with IBS.

[0003] There is also some experimental evidence suggesting that the qualitative / quantitative composition of the gut microbiota contributes to the pathophysiology of IBS. First, prospective studies have shown that 3% - 36% of intestinal infections cause a marked disruption of the intestinal microbial ecosystem, resulting in a new diagnosis of IBS called post-infectious IBS. Second, in at least one subgroup of patients with IBS, antibodies against flagellin (a globular protein present in the flagella of commensal bacteria that inhabit the human intestine), toxin B, and vinculin (associated with changes in the gut microbiota), as well as increased levels of human β-defensin-2 (an antimicrobial inducible protein), have been identified. These data suggest the presence of a host immune response to components of the gut microbiota. Furthermore, many studies have reported that the composition and stability of the gut microbiota in subjects with IBS change over time. Although these studies are not completely comprehensive and no final conclusions can be reached, they still show that the gut microbiota in patients with IBS is significantly different from controls.

[0004] Further evidence indicates that the composition of the gut microbiota may have some impact on the pathophysiology of the IBS syndrome and is related to the fact that the modulation of the gut microbiota by probiotics and non-absorbable antibiotics can, in some cases, improve the symptoms of patients, indicating that the interaction between gut bacteria and the host is involved in the progression and development of symptoms in patients with IBS.

[0005] Probiotics are defined as live microorganisms that, when administered in appropriate amounts, confer health benefits on the host. Their functions include, for example, preventing the overgrowth of potentially harmful bacteria in the intestine, the ability to enhance the resistance of this anatomical area to pathogen invasion, inducing the secretion of soluble factors such as cytokines and antimicrobial peptides, and strengthening the epithelial barrier function.

[0006] Reviews and meta-analyses of scientific literature generally indicate that microorganisms belonging to the probiotic class can provide therapeutic benefits with respect to IBS symptoms. However, a general beneficial effect on symptoms was only recognized after integrating data from various studies, regardless of the probiotic used. In contrast, analysis of individual subgroups of patients who respond to specific treatments according to the type of probiotic used did not show a statistically significant benefit from administration. Since the species, strains, and dosages of probiotics used in various studies vary widely and, in some cases, even very limited case reports are conducted, it is difficult to reach a specific conclusion as to what the optimal probiotic strategy for the treatment of IBS might be, if any.

[0007] Also, the mechanisms of action by which probiotics exert beneficial effects in humans are mostly insufficient. In the few available clinical trials, it has been reported that some strains have anti-inflammatory properties and / or can affect the qualitative / quantitative composition of the microbiota. In fact, there are specific strains that show an increase in the level of dysbiosis compared to other strains and are present at high concentrations in IBS patients, such as Ruminococcus bromii and Ruminococcus spp.

[0008] Among clinical trials, O’Mahony L. et al. (Gastroenterology 2005, 128) (Non-Patent Document 1) reported that Bifidobacterium longum subsp. Infantis 35624, rather than Lactobacillus salivarius, was found to normalize the ratio of IL-10 / IL-12 interleukin, an indicator of the pro-inflammatory helper T1-type immune response, in patients with IBS. In a study of healthy volunteers, Ferrario C. et al. (J Nutr 2014, 144) (Non-Patent Document 2) reported that ingestion of L. casei DG (registered trademark) (CNCM I-1572) could significantly regulate the levels of bacteria in the Clostridiales (now reclassified as Eubacteriales) order and butyrate (salt) in feces, with potential benefits to host health. Furthermore, D’Inca et al. (Dig Dis Sci 2011, 56) (Non-Patent Document 3) demonstrated that rectal administration of L. casei DG (registered trademark) (CNCM I-1572) significantly decreased the levels of mRNA encoding TLR-4 and IL-1β and significantly increased IL-10 in the colonic mucosa of patients with mild left-sided ulcerative colitis.

[0009] Khlinov et al. (Experimental & clinical gastroenterology, vol.1 (6), 2021-08-31, pages 57-62) (Non-Patent Document 4) reported a study of patients with IBS-C treated with L. paracasei DG containing mebeverine hydrochloride and fructooligosaccharides versus placebo.

[0010] It should be noted that, following the reclassification of the genus Lactobacillus published by Zheng et al. in the scientific journal Int. J. Syst. Evol. Microbiol., 70(4):2782-2858, 2020 (Non-Patent Document 5), the strain L. casei DG (registered trademark) (CNCM I-1572) or L. paracasei DG (registered trademark) (CNCM I-1572) was re-deposited on February 2, 2022 as Lacticaseibacillus paracasei DG I-1572 DSM 34154. Since the above two names always refer to the same strain (bacterial strain), they are interchangeable.

[0011] The study by Cremon C. et al. (UEG Journal, 2018, 6) (Non-Patent Document 6) reported a randomized pilot clinical trial investigating the effect of Lacticaseibacillus paracasei DG I-1572 DSM 34154 on clinical factors and microbiota composition in IBS patients with diarrheal, constipated, mixed, and unclassifiable bowel (alvus). Specifically, administration of this strain significantly decreased the bacterial genus Luminococcus, increased the short-chain fatty acids acetate and butyrate, and decreased IL-15. However, a statistically significant decrease in IBS symptoms could not be demonstrated.

[0012] Therefore, there is still a need for a specific probiotic therapy that can act on the intestinal microbiota and / or the levels of inflammatory factors in patients with IBS, relieve the symptoms of those patients, be used effectively and safely, and overcome the drawbacks associated with known treatments.

Prior Art Documents

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0014] Summary An object of the present invention is to provide a probiotic for use in a method for treating dysbiosis in patients with IBS, preferably patients with non-constipated IBS, and in particular to reduce or decrease the presence of Collinsella aerofaciens in the intestinal microbiota of said patients. Reducing dysbiosis means, for example, reducing the amount of intestinal pathobiont bacteria such as Bacteroides plebeius, Dorea spp., Lachnococcus bromii, Lachnococcus spp., Blautia spp. and Collinsella aerofaciens.

[0015] Another object of the present invention is to provide a probiotic that effectively and safely reduces intestinal symptoms in patients with IBS, preferably patients with non-constipated IBS.

[0016] These and other objects are achieved by the object of the present invention to provide an effective probiotic that treats the dysbiosis of the gut microbiota of patients with IBS and as a result reduces the symptoms of said disease.

Brief Description of the Drawings

[0017]

Figure 1A

Figure 1B

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0018] Description of the Invention According to one aspect of the present invention, there is provided a bacterial strain belonging to the species Lacticaseibacillus paracasei, deposited as Lacticaseibacillus paracasei DG (Lacticaseibacillus paracasei DG) I-1572 DSM 34154, for use in a method of treating dysbiosis of the gut microbiota of a subject having IBS, wherein the subject is classified as non-constipated.

[0019] Preferably, in the subject having IBS, the levels (abundances) of Collinsella aerofaciens, Bacteroides plebeius, Dorea spp., Ruminococcus bromii, Ruminococcus spp., and Blautia spp. are increased.

[0020] Preferably, Collinsella aerofaciens, Bacteroides plebeius, Dorea spp., Ruminococcus bromii, Ruminococcus spp., and Blautia spp. with increased levels (abundances) are present in the feces of a subject in need of having IBS.

[0021] Preferably, the subject is a sufferer of IBS, and preferably, the subject is a sufferer of non-constipated IBS.

[0022] Preferably, the bacteria are administered orally, preferably in the form of Enterolactis (R) Plus capsules, which are human supplements.

[0023] Preferably, the human supplement is preferably administered twice a day, preferably for 4 to 24 weeks, more preferably for 8 to 12 weeks.

[0024] Preferably, each of the capsules contains 1×10 6 to 1×10 12 CFU / capsule, more preferably 1×10 8 to 1×10 10 CFU / capsule, even more preferably 10×10 9 to 50×10 9 CFU / capsule.

[0025] Preferably, the bacteria are present in each of the capsules in a solid form, preferably in a powder, dried or lyophilized form.

[0026] Preferably, the daily dose may include 1 to 4 capsules / day, preferably 2 to 3 capsules / day.

[0027] Preferably, the bacteria are used in a method for treating abdominal symptoms in IBS patients, preferably non-constipated IBS patients.

[0028] According to one aspect of the present invention, it relates to a probiotic based on Lactobacillus casei paracasei DG I-1572 DSM 34154 for use in a method for treating dysbiosis of the gut microbiota in patients with IBS, particularly when the patient is classified as non-constipated.

[0029] According to one aspect of the present invention, it relates to a probiotic based on Lactiplantibacillus paracasei DG (registered trademark) I-1572 DSM 34154 for use in a method of treating a subject having a high level of Collinsella aerofaciens, Bacteroides prevotii, Dorea genus, Lachnococcus bromii, Lachnococcus genus, and Blautia genus in feces. Preferably, the Lactiplantibacillus paracasei DG (registered trademark) I-1572 DSM 34154 is used in a subject who is a patient with IBS, preferably a patient with non-constipated IBS. Preferably, the Lactiplantibacillus paracasei DG (registered trademark) I-1572 DSM 34154 is administered orally, preferably in the form of Enterolactis (registered trademark) Plus capsules. Preferably, the probiotic Lactiplantibacillus paracasei DG (registered trademark) I-1572 DSM 34154 is preferably administered twice a day for preferably 4 to 24 weeks. Preferably, the Lactiplantibacillus paracasei DG (registered trademark) I-1572 DSM 34154 is used for the treatment of abdominal symptoms in IBS patients, preferably non-constipated IBS patients.

[0030] In particular, Lactiplantibacillus paracasei DG (registered trademark) I-1572 DSM 34154 reduces abdominal pain in NC-IBS patients by 40% to 60%. It also improves the fecal type of the same patients.

[0031] As used herein, dysbiosis of the gut microbiota means identifying a state in which the amount and type of microorganisms present in the gastrointestinal tract have changed compared to a normal physiological state, and this change is causally related to a pathological or dysfunctional state.

[0032] The population of IBS patients considered to be non-constipated consists mainly of IBS-D patients who mainly exhibit diarrhea symptoms and IBS-M patients with mixed bowel habits.

[0033] Lacticaseibacillus paracasei DG I-1572 DSM 34154 is currently included in a dietary supplement registered as Enterolactis® Plus and is also currently known as the registered trademark L. casei DG® (CNCM I-1572) or L. paracasei DG® (CNCM I-1572). It should be noted that, following the reclassification of the genus Lactobacillus published by Zheng et al. in the scientific journal Int. J. Syst. Evol. Microbiol., 70(4):2782-2858, 2020 (Non-Patent Document 5), the strain L. casei DG (L. casei DG)® (CNCM I-1572) or L. paracasei DG® (CNCM I-1572) was re-deposited on February 2, 2022 as Lacticaseibacillus paracasei DG I-1572 DSM 34154. The above two names are interchangeable as they always refer to the same strain (bacterial strain).

[0034] The probiotic for use according to the present invention is Enterolactis® Plus, an oral capsule containing 24 billion or more L. casei DG® (Lactobacillus paracasei CNCM I-1572) (Lacticaseibacillus paracasei DG I-1572 DSM 34154) per capsule, which is administered 1 to 3 capsules per day, preferably 2 capsules per day, for 8 to 24 weeks, for example, 12 weeks.

[0035] Preferably, the capsules are administered on an empty stomach, for example, 1 hour before a main meal or 2 hours after a main meal.

[0036] As will be fully demonstrated in the following experimental part, the probiotic for use according to the present invention can improve the abdominal symptoms of patients with non-constipated IBS. This improvement is related to the ability of the probiotic for use according to the present invention to intervene in the intestinal dysbiosis that plagues a specific subgroup of patients with non-constipated IBS, in particular the ability to reduce the presence of Collinsella aerofaciens in the intestinal microbiota of said patients.

[0037] As previously reported, the probiotic for use according to the present invention is Lactobacillus casei paracasei DG I-1572 (DSM 34154), which is orally administered and has been shown to be effective in the treatment of intestinal dysbiosis in patients with non-constipated IBS, particularly when Collinsella aerofaciens is present at high levels in the intestinal microbiota.

[0038] According to another aspect thereof, the object of the present invention is a method for reducing or decreasing the abdominal symptoms of a subject having IBS, the method comprising administering to said subject in need thereof one or more capsules of Enterolactis® Plus for use according to the present invention, for example 2 capsules per day, preferably for 4 to 24 weeks, even more preferably for 8 to 12 weeks. Here, the subject is understood to mean a human.

[0039] The probiotic for use according to the present invention may be used alone or, if desired or necessary, in combination with other substances, provided that such other substances do not interfere with or limit the effects of the present invention.

[0040] The following experimental section shows the results of a clinical trial on the probiotic for use according to the present invention and compares its use with a placebo.

[0041] Experimental section Objective: One of the aims of the present study was to identify markers to recognize non-constipated (NC) IBS patients who may show significant clinical improvement with treatment by the probiotic strain Lactobacillus paracasei DG (abbreviated as LDG).

[0042] Design: Post hoc analysis of samples collected during a multi-center, randomized, double-blind, parallel-group, placebo-controlled trial. In this trial, NC-IBS patients were randomized to receive either LDG capsules at at least 24 billion CFU or placebo capsules twice daily (b.i.d.) for 12 weeks. The primary outcome was a composite response based on improvement in abdominal pain and stool form. The fecal microbiome and intestinal serum markers such as PV1, liver, and kidney function were investigated.

[0043] Results: Responders (R) in the probiotic group (25%) differed from non-responders (NR) with respect to the abundance of 18 bacterial taxa, including Coriobacteriaceae, the genus Dorea, and Collinsella aerofaciens, which were overrepresented in R patients. These taxa also distinguished R (but not NR) from healthy controls. The probiotic intervention significantly decreased the abundance of these bacteria in R but not in NR. Analysis of data from previous trials of IBS with the same probiotic yielded similar results for C. aerofaciens. Finally, C. aerofaciens was positively correlated with markers of PV-1 and liver function.

[0044] Advantageously, the bacterial strain Lactobacillus casei DG® (Lactobacillus paracasei DG I-1572 DSM 34154) - Enterococcus faecium PLUS is effective in NC-IBS patients with a higher presence of potentially pathogenic commensals. Among these, C. aerofaciens has emerged as a potential predictor of probiotic efficacy.

[0045] Purpose of the study Primary purpose: To evaluate the effect of Lactobacillus casei DG® on abdominal symptoms in non-constipated patients with irritable bowel syndrome (IBS), patients with symptoms meeting the Rome IV criteria in the diagnosis of IBS without constipation (i.e., patients with IBS-D and patients with IBS-M).

[0046] Secondary purposes: To evaluate the following parameters: · Presence or absence of IBS symptoms, · Daily stool consistency, · Patient's overall satisfaction, · Overall quality of life, · Psychological disorders, · Intake of rescue medications, · Composition of the gut microbiota and metabolites, · Intestinal permeability, · Recovery of Lactobacillus casei DG® strains in feces.

[0047] Experimental design Multicenter randomized double-blind placebo-controlled parallel-group comparative study This trial was composed of an initial (trial introduction) phase of two weeks, during which data on pain and discomfort in the abdomen and alveus were collected, and these data were useful for establishing the baseline levels used to evaluate treatment efficacy. Subsequently, a 12-week phase (treatment) followed, during which each patient took two oral capsules of Enterolactis® Plus (a single-strain probiotic formulation (or preparation) containing at least 24 billion CFU of L. paracasei DG) per day, or two placebo capsules that were physically indistinguishable from the Enterolactis® Plus capsules. Finally, an early (follow-up) condition lasted for four weeks, during which no capsules were taken. Thus, according to the test designs of Figures 1A and 1B, the total treatment period for each cycle was 18 weeks.

[0048] For each patient, three fecal samples (at the end of weeks 2, 14, and 18 during hospital visits) and three blood samples (at the first hospital visit (time zero) and at the end of weeks 14 and 18 during hospital visits) were collected. These samples were subjected to the analysis of various markers. In particular, for feces, taxonomic characterization of the bacterial population and quantification of short-chain fatty acids (acetate, butyrate, propionate, valerate, isovalerate, lactate, and succinate) were performed, and in blood samples, the following were quantified: permeability markers such as PV-1, liver [alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (Bil), alkaline phosphatase (ALK)] and kidney [blood urea nitrogen (BUN) and creatinine (Crea)] functions.

[0049] Patient The patients selected for this trial were men or women aged 18 years or older who were diagnosed with IBS without constipation according to the Rome IV criteria.

[0050] The diagnostic criteria for IBS are that symptoms should have onset at least 6 months before diagnosis and the criteria should be met for 3 months prior to the test, with recurrent abdominal pain at least 1 day per week, and related to two or more of the following criteria: ○ Related to defecation, ○ Associated with a change in the frequency of stools, ○ Associated with a change in the shape (appearance) of stools.

[0051] Examples of IBS patients without constipation are as follows: · IBS with diarrhea as the main symptom (IBS-D): More than one-fourth (25%) of defecations are Bristol stool form type 6 or 7, · IBS with mixed bowel habits (IBS-M): More than one-fourth (25%) of defecations are Bristol stool form type 1 or 2, and more than one-fourth (25%) of defecations are Bristol stool form type 6 or 7.

[0052] Inclusion criteria · Aged 18 or older and 65 or younger, · A positive diagnosis of IBS without constipation (IBS-D and IBS-M, both males and females) according to the Rome IV criteria, · When the patient is 50 or younger, or when the patient has any of the following warning signs, the result of a colonoscopy performed within 5 years before the screening visit is negative: · A significant weight loss has been recorded in the past 6 months, or · Symptoms occur at night, or · There is a family history of colon cancer, or · Blood is mixed in the stools (excluding blood from hemorrhoids), · If necessary, the results of further relevant screening or examinations are negative, · The ability to comply with the test protocol.

[0053] Exclusion criteria · Patients with IBS-C or IBS-U according to the Rome IV criteria, · Any related organic, systemic or metabolic diseases (especially a significant medical history of heart, kidney, nervous system, psychiatric, neoplastic, endocrine, metabolic or liver diseases) or abnormal test values detected during the screening period of the clinical trial that are considered clinically significant based on pre-defined values (e.g., kidney or liver function levels are more than twice the upper limit reference value), · The presence of established organic bowel diseases including celiac disease, food allergies or inflammatory bowel diseases (Crohn's disease, ulcerative colitis, diverticular disease, infectious colitis, ischemic colitis, microscopic colitis), · Having had a previous major abdominal surgery, · Having any type of active malignancy or a history of malignancy (patients with a history of surgically resected other malignancies and no evidence of recurrence for at least 5 years before trial enrollment are acceptable), · Having established or suspected untreated food intolerance defined by medical history evaluation or, where appropriate, a lactose breath test, · Having used probiotics or topical / systemic antibiotic treatment within the past month, · Frequently or regularly using contact laxatives, · Being a pregnant woman or a woman of reproductive age without an effective contraceptive method, · Being unable to comply with the protocol, · Having received treatment with the investigational drug within the past 30 days, · Having a recent history of alcohol abuse or drug dependence or suspicion of such a history, · Having a red or white flag according to the criteria of the Psychosocial Alarm Questionnaire for Functional Gastrointestinal Disorders of the Rome IV edition.

[0054] Randomization Enrollable patients entered a 2-week trial introduction period and were then randomly assigned in a 1:1 ratio to Enterogermina® Plus treatment or an equivalent sterile product (placebo) with similar color, texture, and taste, and took it twice a day for 12 weeks.

[0055] Efficacy evaluation Primary evaluation item: The proportion of patients showing a composite response over 12 weeks: patients with more than 50% of days recording a decrease of 30% or more from the baseline average score of the most severe abdominal pain and, at the same time, a stool hardness of 5 or less

[0056] Abdominal pain was evaluated using a standard 11-point numerical rating scale (0 = no pain to 10 = the strongest pain conceivable), and for abnormal defecation, the Bristol Stool Form Scale (BSFS) was used to measure the frequency and shape of stools.

[0057] Secondary evaluation items · Reduction of IBS symptoms. This was evaluated as follows: · Reduction of pain: The score indicating the most severe abdominal pain decreased by 30% or more from the baseline on more than 50% of the days during the period, · Composite response (composite response) at 4-week intervals, · Improvement of the overall symptom score: A score of 0 or 1, or an improvement of 2 or more from the baseline, on more than 50% of the days during the period, · IBS symptoms were sufficiently reduced over more than 50% of the period in the past few weeks (answered "yes" in more than 50% of the weeks to the question "In the past week, have your IBS symptoms been sufficiently reduced?"), · IBS-SSS score evaluated at zero time and at the end of treatment after 12 weeks (a decrease of at least 50 points is considered clinically significant), · Improvement of stool hardness: The stool hardness score is 5 or less, · Overall satisfaction with treatment evaluated by the VAS scale, · Assessment of quality of life on a scale of 0 - 100 by the validated Short-Form 12 Items Health Survey (SF-12), · Intake of rescue medications, · Composition of the gut microbiota and metabolites (SCFAs, free amino acids, and biogenic amines), · Intestinal permeability (using blood samples) by assessment of serum levels of zonulin, citrulline, and PV-1, · Recovery of L. casei DG® strain from feces according to the method described by Arioli et al. (Front. Microbiol 2018. 9:1720).

[0058] Safety evaluation · Vital parameters including blood pressure, heart rate, and respiratory rate. · Physical examination including assessment of the state of hair and skin, lymph nodes, eyes, ENT, chest, respiratory system, cardiovascular system, abdomen, urogenital system, pelvis, and rectum. · Clinical examinations: · Blood chemistry: Glucose, blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, alkaline phosphatase, creatinine, · Hematology: Complete blood count and blood composition, platelets. · Adverse events. This is defined as the occurrence of undesirable medical signs, symptoms, or conditions that are continuously monitored during the trial and occur after obtaining informed consent from the patient.

[0059] Discussion Of the total 300 subjects initially recruited, a total of 72 patients in the probiotic group and 70 patients in the placebo group completed this trial. The 300 recruited patients represent only the PP (per protocol) population, which conforms to the trial implementation plan, and does not represent the ITT (intention to treat) population that is usually considered in the primary analysis. Also, within the PP population itself, not all PP patients were considered (about 235), and only the patients after excluding the deviated patients were considered. This selection was due to the fact that in this method, only the patients who completed the trial while taking the probiotic product and were able to collect samples at various visits were considered.

[0060] As expected beforehand, the primary evaluation item of this trial was the percentage of patients who had a composite response over the 12-week probiotic or placebo administration period, that is, on more than 50% of the days during the period, the strongest abdominal pain decreased by more than 30% from the baseline average score, and on the same number of days, the stool hardness score was less than 5.

[0061] According to the primary evaluation item, it was found that 16 patients (23.5%) in the probiotic group and 19 patients (29.2%) in the placebo group showed a positive response (R, responder) to the probiotic for the use according to the present invention.

[0062] Table 1 shows the abdominal pain and stool type at the second visit (the second time of visit) and the fourth visit (the fourth time of visit) of the treated patients.

[0063]

Table 1

[0064] The explanation of the obtained results is as follows.

[0065] (i) Patients who had no pain or had such a low degree of pain at baseline (IBS-SSS total score of 175 or less and NRS of 3 or less) and did not show significant improvement after treatment were also included in the study.

[0066] (ii) The diary card is stated to require daily indication of the severity of "pain / annoyance" (since the annoyance of symptoms can be reported interchangeably with pain, it becomes unclear which symptom the patient reported, and it becomes unclear whether a comparison is being made between pain / annoyance at baseline and / or pain / annoyance at the follow-up visit).

[0067] (iii) Analysis of the daily diaries filled in by patients during the 2-week study introduction period revealed that although compliance with the eligibility criteria was confirmed at baseline, many patients were found not to meet the Rome IV criteria for the diagnosis of IBS-D or IBS-M.

[0068] (iv) As a result, 183 out of 264 patients in the ITT population were excluded, significantly reducing the effective sample size for the purpose of statistical analysis of the results.

[0069] (v) Furthermore, regarding treatment compliance, microbiome analysis showed that in 25 patients in the Enterococcus group, L. casei DG (registered trademark) (Lactobacillus casei paracasei DG I-1572 DSM 34154) (Enterococcus (registered trademark) Plus) was not detected (this means that it is possible that the patients did not take the product sufficiently or, in any case, did not take it correctly). On the other hand, in 5 patients in the placebo group, L. casei DG (registered trademark) was detected (it is a component present in other products freely available at pharmacies, and it is possible that the patients found and took it freely).

[0070] The significant changes in the quantity and type of bacterial populations in the microbiota of subjects administered with placebo were much smaller than those observed in the group of subjects taking Enterolactis® Plus. This suggests that the improvement observed in the R subjects of the placebo group is not related to changes at the microbiota level as in the case of patients treated with Enterolactis® Plus, but rather may be related to psychological factors, i.e., the known psychosomatic elements of IBS.

[0071] To determine whether patients who benefited from probiotic treatment have characteristics that distinguish them from patients who did not show significant improvement (R vs. NR (non-responders)), the levels of bacteria and other markers were compared between two groups of patients. This analysis showed that R patients had significantly higher abundances of several microbiota that show dysbiosis in IBS patients, such as Bacteroides prevotii, Dorea spp., Lachnococcus bromii, Lachnococcus spp., Blautia spp., and Collinsella aerofaciens, than healthy subjects. Specifically, principal component analysis based on the abundances of 46 bacterial taxa that were significantly different between R and NR patients showed that the most important taxonomic group for distinguishing R and NR patients was the Collinsella aerofaciens species.

[0072] A similar analysis was also performed between R and NR patients in the placebo group. In this case, fewer significantly different taxa were observed. Notably, no significant difference in Collinsella aerofaciens was observed between R and NR patients.

[0073] Subsequent statistical analysis showed that the probiotic contained in the Enterolactis® Plus formulation for use according to the present invention resulted in a decrease in Collinsella aerofaciens in responder (R) patients (significant trend, P = 0.0681). In contrast, the concentration of Collinsella aerofaciens did not change upon administration of the probiotic for use according to the present invention in either NR patients or placebo group patients.

[0074] Upon further investigation, it was confirmed that responder subjects to treatment with Enterolactis® Plus for use according to the present invention, particularly non-constipated IBS patients, had significantly higher initial levels of Collinsella aerofaciens in feces than a group of 100 healthy adults (control).

[0075] Furthermore, it was shown that patients in whom the level of Collinsella aerofaciens decreased due to the use of the probiotic for use according to the present invention simultaneously showed a significant decrease in abdominal pain (P = 0.0497) and a tendency towards a decrease in stool form (P = 0.0709). This suggests that this bacterial strain may be mechanistically related to IBS symptoms.

[0076] In addition to demonstrating that the probiotic for use according to the present invention is effective with respect to the ability to reduce abdominal symptoms in non-constipated IBS patients by decreasing the concentration of Collinsella aerofaciens, the experimental data from the described clinical protocol can emphasize the possibility of measuring the concentration of this microorganism as a predictive test for whether a particular patient will respond to the probiotic L. casei DG® for use according to the present invention.

[0077] Quantification of organic acids As previously described by Gargari G, et al. (Environ Microbiol 2018;20:3201-13), organic acids (acetic acid, butyric acid, propionic acid, valeric acid, isovaleric acid, lactic acid, and succinic acid) in fecal samples were detected and quantified by ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HR-MS) using an Acquity UPLC separation module (Waters, Milford, MA) combined with an Exactive Orbitrap MS using an HESI-II probe for electrospray ionization (Thermo Scientific, San Jose, CA).

[0078] Table 2 shows the quantification of organic acids measured at hospital visit V2 and hospital visit V4.

[0079]

Table 2

[0080] Quantification of PV-1 The endothelial permeability marker, plasmalemma vesicle associated protein (PLVAP) / PV-1, was measured in serum samples using a human PVLAP ELISA Kit (Fine test, China). Samples were processed according to the manufacturer's instructions, and absorbance at 450 nm was measured using an Eon plate reader. The absorbance data were then interpolated by a logarithmic standard curve calculated for each plate analyzed.

[0081]

Table 3

[0082] Analysis of liver and kidney function markers The following liver and kidney function markers in the serum sample were evaluated: alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (Bil), alkaline phosphatase (ALK), blood urea nitrogen (BUN), and creatinine (Crea). AST and ALT were evaluated by enzyme administration without activation by pyridoxal phosphate (Cobas 8000 - Roche Diagnostics). Bil and ALK were administered by an enzymatic colorimetric test (Cobas 8000 - Roche Diagnostics). A kinetic enzyme test was performed to evaluate urea, and creatinine was evaluated by a kinetic staining test (Jaffe method) (Cobas 8000 - Roche Diagnostics).

[0083]

Table 4

Claims

1. A bacterial strain belonging to the species Lacticaseibacillus paracasei, deposited as Lacticaseibacillus paracasei DG I-1572 DSM 34154, for use in a method of treating dysbiosis of the gut microbiota of a subject having IBS, wherein the subject is classified as non-constipated.

2. The bacterium for use according to claim 1, wherein the levels of Collinsella aerofaciens, Bacteroides plebeius, Dorea spp., Ruminococcus bromii, Ruminococcus spp., and Blautia spp. are elevated in the subject having IBS.

3. The bacterium for use according to claim 1 or 2, wherein the elevated levels of Collinsella aerofaciens, Bacteroides plebeius, Dorea spp., Ruminococcus bromii, Ruminococcus spp., and Blautia spp. are present in the feces of the subject having IBS in need thereof.

4. The bacterium for use according to any one of claims 1 to 3, wherein the subject is an IBS sufferer, preferably the subject is a non-constipated IBS sufferer.

5. The bacterium for use according to any one of claims 1 to 4, wherein the bacterium is administered orally, preferably in the form of Enterolactis (R) Plus capsules, which are human supplements.

6. The bacterium for use according to any one of claims 1 to 5, wherein the human supplement is preferably administered twice a day, preferably for 4 to 24 weeks, more preferably for 8 to 12 weeks.

7. each of the capsules contains 1×10 ６ to 1×10 １２ CFU / capsule, more preferably 1×10 ８ to 1×10 １０ CFU / capsule, even more preferably 10×10 ９ to 50×10 ９ CFU / capsule, and the bacterium for use according to any one of claims 1 to 6.

8. The bacterium for use according to any one of claims 1 to 7, wherein the bacterium is present in solid form, preferably powder, dried or lyophilized form, in each of the capsules.

9. The bacterium for use according to any one of claims 1 to 8, wherein the daily dose may include 1 to 4 capsules / day, preferably 2 to 3 capsules / day.

10. The bacterium according to any one of claims 1 to 9 for use in a method for treating abdominal symptoms of IBS patients, preferably non-constipated IBS patients.

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