Microbial consortium for use in a rapid screening method for irritable bowel syndrome

Abstract

A new microbial consortium for use in a rapid screening method for irritable bowel syndrome (IBS) and possibly as a target for the therapeutic treatment of an IBS condition in a subject.

Description

[0001] “MICROBIAL CONSORTIUM FOR USE IN A RAPID SCREENING METHOD FOR IRRITABLE BOWEL SYNDROME”

[0002] FIELD OF THE INVENTION

[0003] Some embodiments described here concern a microbial consortium for use in a rapid screening method for irritable bowel syndrome (IBS).

[0004] Some embodiments also concern therapeutic constructs that can target the components of said microbial consortium in a subject, in order to treat the IBS condition.

[0005] BACKGROUND OF THE INVENTION

[0006] Irritable bowel syndrome (IBS) is known to be a chronic gastrointestinal tract disorder affecting millions of people worldwide. Characterized by symptoms such as abdominal pain, bloating, diarrhea and / or constipation, IBS can have a significant impact on a patient’s quality of life. Despite its high prevalence, the exact causes of IBS are not fully understood, but are believed to involve a complex combination of factors, among which the gut microbiota plays a key role (Chey WD, Kurlander J, Eswaran S. Irritable bowel syndrome: a clinical review. Jama. 2015 Mar 3 ;313(9):949-58_).

[0007] The diagnosis of IBS is mainly based on the symptomatology presented by the patient and the exclusion of other potentially similar medical conditions, generally making the diagnostic process very complex.

[0008] WO 2019 / 014714 Al discloses methods for diagnosing a dysbiosis in a subject, methods for determining an appropriate treatment and methods for treating a dysbiosis, in particular it concerns the diagnosis or determination of a subtype of irritable bowel syndrome (IBS).

[0009] WO 2023 / 278352 Al discloses systems and methods for identifying microbial signatures from samples that determine discriminant features between healthy control populations and sick individuals or populations with specific disorders. The microbial signatures are used to diagnose, prognosticate or determine the risk and / or severity of a disease or disorder in a subject, such as autism spectrum disorder or irritable bowel syndrome.

[0010] WO 2020 / 201457 Al discloses methods for diagnosing irritable bowel syndrome (IBS). In this context, there is the need to make available a new tool that allows to perform a rapid screening of IBS effectively and reliably.

[0011] There is also the need to make available therapeutic constructs that can target the components of the above-mentioned microbial consortium in a subject, for the purpose of treating the IBS condition.

[0012] The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

[0013] DESCRIPTION OF THE INVENTION

[0014] The present invention is set forth and characterized in the independent claims, while the dependent claims describe other characteristics of the present invention or variants to the main inventive idea.

[0015] In accordance with the above purposes, some embodiments described here concern a microbial consortium for use in a rapid screening method for IBS. The microbial consortium comprises Bacteroides vulgatus, Prevotella Copri, Bacteroides fragilis, Saccharomyces cerevisiae and Sarocladium strictum.

[0016] Other embodiments concern a rapid screening method for irritable bowel syndrome (IBS), comprising detecting abundance of components of a microbial consortium which includes Bacteroides vulgatus, Prevotella Copri, Bacteroides fragilis, Saccharomyces cerevisiae and Sarocladium strictum in a sample, in particular a fecal sample, of a subject and deriving, from this abundance, an indication that the subject is suffering from IBS condition or a risk assessment that the subject is suffering from IBS condition.

[0017] In some embodiments, detecting abundance of components of a microbial consortium which includes Bacteroides vulgatus, Prevotella Copri, Bacteroides fragilis, Saccharomyces cerevisiae and Sarocladium strictum in a sample, in particular a fecal sample, of a subject includes metagenomic sequencing, in particular shotgun metagenomic sequencing, and taxonomic characterization to create multi-kingdom microbial abundance profiles.

[0018] Other embodiments concern a composition for oral administration and suitable for human consumption comprising one or more therapeutic constructs able to target components of a microbial consortium comprising Bacteroides vulgatus, Prevotella Copri, Bacteroides fragilis, Saccharomyces cerevisiae and Sarocladium strictum in a subject.

[0019] As a non-limiting example, the therapeutic constructs can be or include therapeutic components, such as for example probiotic and possibly prebiotic compounds, which are able to modulate the abundance, in particular reduce the presence, of components of the above-mentioned microbial consortium in a subject.

[0020] As used here, “probiotic” refers to a substantially pure microbe (i.e., a single isolate) or to a mixture of microbes, and may also include any additional component that can be administered to a subject (for example, a human) in order to restore or alter the microbiota or microbiome in the subject. In some embodiments, a probiotic composition can be administered with an agent in order to allow the microbe(s) to survive in the gastrointestinal tract environment, that is, withstand a low pH and / or grow in the gastrointestinal environment.

[0021] As used here, “prebiotic” refers to an agent that increases the number and / or activity of one or more microbes. Such microbes may include microbes to restore or alter the microbiota or microbiome of a subject. Non-limiting examples of a prebiotic include fibers, fructooligosaccharides (FOS, for example oligofructose, inulin, or an inulin-type fructan), galactooligosaccharides (GOS), an amino acid or an alcohol.

[0022] In some embodiments, the composition as above can be for use in the therapeutic treatment of irritable bowel syndrome (IBS).

[0023] In some embodiments, the composition as above can comprise additional probiotics and / or fibers or prebiotics and / or vitamins and / or adjuvants for the health of the bacterial flora.

[0024] In some embodiments, the composition as above can comprise prebiotics, in particular dietary fibers, wherein the dietary fibers comprise insoluble fibers and / or soluble fibers.

[0025] In some embodiments, the composition as above can comprise fortifying food ingredients and / or micronutrients, in particular including one or more omega 3s, minerals, trace minerals, vitamins or a combination thereof.

[0026] In some embodiments, the composition as above can be in liquid, solid, semisolid, lyophilized or powder form.

[0027] In some embodiments, the composition as above can be in the form of, or prepared in the form of, a tablet, capsule or lozenge, possibly of the gastro-resistant type.

[0028] In some embodiments, the microbial consortium described here can be used to process a predictive-diagnostic model able, for example, to provide a subject with a score correlated to the IBS condition (so-called “IBS-score”), which for example can be an integer value comprised between 0 and 1 , indicating the probability of suffering from IBS with microbiome-based origin. This information can be reported as part of an ad-hoc test developed for the specific condition.

[0029] The microbial consortium described here can advantageously be used for a rapid screening to determine the possible IBS condition.

[0030] Some embodiments also concern methods and uses for the therapeutic treatment of the IBS condition, which include administering therapeutic constructs to a subject, for example therapeutic compounds, such as for example probiotic and possibly even prebiotic compounds, which are able to modulate the abundance, for example reduce the amount, of the components of the microbial consortium described here for the purposes of the therapeutic treatment of the IBS condition.

[0031] This administration to the subject can be done according to an indication that the subject is suffering from an IBS condition, or a risk that the subject is suffering from an IBS condition.

[0032] In some embodiments, the aforementioned microbial consortium can be identified, or used as, a marker of disease or pathology associated with the IBS condition.

[0033] In particular, the aforementioned microbial consortium can be reported as an abundant screening marker in subjects suffering from IBS condition.

[0034] In some embodiments, the aforementioned therapeutic construct can comprise one or more of either antibiotic drugs targeted against disease markers, pre- / pro- / syn- / post-biotics, or fecal microbiome transplantation, to help the subject’s gut microbiome achieve a healthy balance.

[0035] In some embodiments, the aforementioned therapeutic construct comprises one or more natural compounds or synthetically derived compounds that target the components of the microbial consortium, identified as disease markers associated with the IBS condition, wherein the natural or synthetically derived compounds are non- toxic and do not cause any adverse effects. Some embodiments provide uses for administering a therapeutic construct to a subject based on an indication and / or risk of IBS condition. Any known technique can be used to administer the therapeutic construct. The administration can utilize at least one of either a consortium / construct of healthy microbes, antibiotic drugs, or pre- / pro- / syn- / post-biotics or fecal microbiome transplantation that would help the subject’s gut microbiome achieve a healthy balance without any adverse health effects. The therapy can be provided in the form of any (or a combination) of the known routes of administration. The therapeutic pathways that can be suggested consider the microbial consortium described here based on their correlation with the microbiome of the IBS condition that may contribute to the therapeutic treatment of the IBS condition by modulating the subject’s microbiome toward a healthy balance. Possible implementations of suitable therapeutic constructs include for example one or more natural or synthetically derived compounds that target the screening markers reported abundant in the diseased subjects, that is, the disease markers identified by the microbial consortium described here, wherein the natural or synthetically derived compounds are non-toxic and do not cause any adverse effects. Any previously reported organism, or any correlated similar organism (similar in genomic composition or characteristic functions) that inhibits the growth of the screening markers reported abundant in the diseased patients, for example even disease markers previously identified in research as nonpathogenic.

[0036] Some embodiments described here also concern methods and uses for the therapeutic treatment of the IBS condition, which comprise (a) detecting a dysbiosis associated with the IBS condition; and (b) administering probiotics and possibly also prebiotics to the subject, which are able to modulate the abundance of the components of the microbial consortium described here, in particular they are able to reduce the abundance of the components of the microbial consortium. The detection (a) can include investigating the abundance of the components of the microbial consortium described here in the subject, for example by analyzing a sample, in particular a fecal sample.

[0037] Any of the methods and uses described here can also include detecting a dysbiosis associated with an IBS condition in a sample of the subject. In some embodiments, the sample is a fecal sample.

[0038] In some embodiments, detecting the dysbiosis associated with the IBS condition includes determining the potential for bacterial gene expression in the subject’s sample. In some embodiments, detecting the dysbiosis includes determining the bacterial composition in the subject’s sample.

[0039] In some embodiments, detecting the dysbiosis associated with the IBS condition comprises determining the abundance of the microbial consortium described here in the subject’s sample.

[0040] The term “dysbiosis” refers to a state of the microbiota or microbiome of the gut or other area of the body (for example, mucosal or skin surfaces or any other niche populated by microorganisms) of a subject (that is, the host) in which the diversity and / or function of the ecological network is disrupted, for example, relative to the state of the microbiota or microbiome of the gut or other areas of the body in a control population. Any alteration whatsoever of the microbiota or microbiome of a subject (that is, the host) relative to the microbiota or microbiome of a control population may be considered as a dysbiosis, even if such dysbiosis does not result in a detectable decrease in the subject’s health.

[0041] The term “microbiome” refers to the collection of microorganisms and viruses and / or their genes from a given environment. For example, “microbiome” can refer to the collection of microorganisms and viruses and / or their genes from the gastrointestinal tract of humans. “Microbiota” refers to microorganisms in a specific environment.

[0042] In some embodiments, it can be provided to administer compounds, such as for example probiotics and possibly also prebiotics, which are able to modulate the abundance, for example reduce the amount, of the components of the microbial consortium described here, to the subject(s) once, two or three times, or even more, per day.

[0043] Any one of the methods and uses described here can also include administering another treatment to the subject, for example, one or more drugs, for the aforementioned IBS condition.

[0044] In some embodiments, the subject has previously been identified as having an IBS condition.

[0045] The Applicant believes that the components of the microbial consortium described here and / or the metabolic pathways associated with one or more components thereof could be a potential target for the therapeutic treatment of the IBS condition and / or for purposes of prediction, diagnosis or prognosis of such IBS condition. Some examples of how the components of the microbial consortium could be used as a specific therapeutic target for the IBS condition could include:

[0046] - modulation of the microbiome: targeting the components of the microbial consortium described here to modulate the composition of the gut microbiota in patients with IBS condition could help restore microbial balance and potentially alleviate the symptoms of the IBS condition;

[0047] - probiotic interventions: incorporating probiotics containing strains useful for modulating the abundance of the components of the microbial consortium described here in the treatment regimen for patients with IBS condition could offer a targeted approach to the management of the IBS condition;

[0048] - disease-modifying effects: by targeting the components of the microbial consortium described here it may be possible to modify the progression of the IBS condition. Manipulating the gut microbiota to modulate, for example reduce, the presence of components of the microbial consortium which are correlated to the IBS condition could potentially lead to better outcomes for patients;

[0049] - combination therapies: considering the components of the microbial consortium described here as a therapeutic target may open up possibilities for combination therapies involving the modulation of the microbiome alongside traditional treatments. This integrated approach could offer a comprehensive management of the IBS condition that includes the related bowel dysbiosis.

[0050] EXPERIMENTAL DATA

[0051] The Applicant used its own bio bank of actual fecal samples from subjects who consented to their use for research purposes (N ~ 26,000).

[0052] Within this sample collection, a subset of samples (N=384) were resequenced with shotgun metagenomics method. Of these, half (N=192) had an eubiotic gut microbiota configuration (absence of detectable pathogens + biodiversity within the parameters defined by the healthy reference sample), while the other half (N=192) consisted of samples with a diagnosis of IBS reported in the historical questionnaire administered at the time the analysis kit was purchased.

[0053] This set of samples was taxonomically characterized following the sequencing, creating multi-kingdom abundance profiles (bacteria, fungi, any parasites and DNA viruses present in the different samples). The global microbial profile was then processed using a decision tree based algorithm (RandomForest). Following this analysis, it was possible to highlight a multi-kingdom microbial consortium fundamental in the prediction process (overall performance of the model: pROC AUC 0.71 - Accuracy 70.8%). This consortium is found to strongly promote the IBS condition and the additive effect toward the condition of the set of microorganisms involves a ~ 6000 times higher probability of developing the syndrome (OR: 6063.42 [CI 121-302,000], p value: 1.61e'5) by linear association between the phenotype and the set of microorganisms. In addition to the above, the Applicant has carried out further statistical analyses - linear association and Wilcoxon test - which confirm the relevance of the microbial consortium described here with respect to IBS. The results of these analyses are shown in the following table.

[0054] Taxon Linear association (OR - P) Wilcoxon test (P)

[0055] Saccharomyces

[0056] 11.9 [2.6-55.3] - 0.00168 0.4313 cerevisiae*

[0057] Bacteroides

[0058] 3.2 [1.7-6] - 0.000197 1.216c'11fragilis**

[0059] Bacteroides

[0060] 7.3 [1.8 - 29.2] - 0.00497 0.1324 vulgatus*

[0061] Prevotella

[0062] 11.2 [0.3 - 350.6] - 0.169 0.9403 copri

[0063] Sarocladium

[0064] 6.2 [2.1 - 17.9] - 0.000909 0.006027

[0065] **: significant in both the linear association and also the Wilcoxon test

[0066] *: significant in the linear association only

[0067] OR: Odds Ratio

[0068] P: P value

[0069] As can be seen from the data in the table, all the taxa of the microbial consortium identified are also significantly involved in the pathology even if evaluated individually, with the exception of Prevotella copri, which is instead an extremely relevant taxon within the consortium.

[0070] In the attached drawings, fig. 1 shows the boxplots that highlight the distribution and relative abundance of the 5 microbial taxa of interest across the two groups of individuals analyzed, while fig. 2 shows the result of the multivariate analysis on the subjects analyzed (the percentages represent the variability on the Cartesian axes). Referring to fig. 2, it is important to note the following:

[0071] - the separation between the two groups (Healthy and IBS) in the multivariate space of fig. 2 is significant (Permanova P < 0.001 - 999 permutations), indicating that the overall profile of the gut microbiota between the two groups is statistically different;

[0072] - the fitting of the environmental variables identified by the analysis using the aforementioned decision tree based algorithm is also significant with respect to the ordering (P < 0.001 - 999 permutations) and the different microbial taxa are reported in the multivariate space of fig. 2 using the vectors indicated. This confirms the significant relevance of the individual members of the microbial consortium described here with respect to the IBS condition.

[0073] The Applicant has also found that two of the microbial taxa identified show an involvement in the IBS pathology:

[0074] - Bacteroides fragilis presents strains capable of producing the BFT toxin, causing inflammation and diarrhea (Sears CL2009. Enterotoxigenic Bacteroides fragilis: a Rogue among Symbiotes. Clin Microbiol Rev 22: https: / / doi.org / 10.1128 / cmr.00053-08 and Franco AA, Cheng RK, Goodman A, Sears CL. Modulation of bft expression by the Bacteroides fragilis pathogenicity island and its flanking region. Mol Microbiol. 2002 Aug;45(4): 1067-77. doi: 10.1046 / j.1365-2958.2002.03077.x);

[0075] - Saccharomyces cerevisiae is a yeast known for its fermentative abilities, widely used in the food industry and beyond. Its involvement in spontaneous fermentation syndrome (also known as auto-brewery syndrome) is increasingly evident, which may underlie some cases of IBS (Cordell, Barbara and McCarthy, Justin (2013) A Case Study of Gut Fermentation Syndrome (Auto-Brewery) with Saccharomyces cerevisiae as the Causative Organism. International Journal of Clinical Medicine, 04 (07). pp. 309-312. ISSN 2158-284X and Sm^dra A, Trzmielak M, Goralska K, Dzikowiec M, Brzezianska-Lasota E, Berent J. Oral form of auto-brewery syndrome. J Forensic Leg Med. 2022 Apr;87: 102333. doi: 10.1016 / j.jflm.2022.102333). It is clear that modifications and / or additions may be made to the present invention, without thereby departing from the scope of protection as defined by the claims.

[0076] It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art will be able to achieve other equivalent forms, having the characteristics as set forth in the claims and hence all coming within the field of protection defined thereby.

Claims

CLAIMS1. Microbial consortium for use in a rapid screening method for irritable bowel syndrome (IBS), comprising Bacteroides vulgatus, Prevotella Copri, Bacteroides fragilis, Saccharomyces cerevisiae and Sarocladium strictum.

2. Rapid screening method for irritable bowel syndrome (IBS), comprising detecting abundance of components of a microbial consortium which comprises Bacteroides vulgatus, Prevotella Copri, Bacteroides fragilis, Saccharomyces cerevisiae and Sarocladium strictum in a sample, in particular a fecal sample, of a subject and deriving, from the abundance, an indication that the subject is suffering from IBS condition or a risk assessment that the subject is suffering from IBS condition.

3. Method as in claim 2, wherein detecting abundance of components of a microbial consortium which includes Bacteroides vulgatus, Prevotella Copri, Bacteroides fragilis, Saccharomyces cerevisiae and Sarocladium strictum in a sample, in particular a fecal sample, of a subject includes shotgun metagenomic sequencing and taxonomic characterization to create multi-kingdom microbial abundance profiles.

4. Composition for oral administration and suitable for human consumption comprising one or more therapeutic constructs able to target components of a microbial consortium comprising Bacteroides vulgatus, Prevotella Copri, Bacteroides fragilis, Saccharomyces cerevisiae and Sarocladium strictum in a subject.

5. Composition as in claim 4 for use in the therapeutic treatment of irritable bowel syndrome (IBS).

6. Composition for use as in claim 4 or 5, wherein the composition comprises probiotics and / or fibers or prebiotics and / or vitamins and / or adjuvants for the health of the bacterial flora.

7. Composition for use as in claim 4, 5 or 6, wherein the composition comprises prebiotics, in particular dietary fibers, wherein the dietary fibers comprise insoluble fibers and / or soluble fibers.

8. Composition for use as in any claim from 4 to 7, wherein the composition comprises fortifying food ingredients and / or micronutrients, in particular including one or more omega 3 s, minerals, trace minerals, vitamins or a combination thereof.

9. Composition for use as in any claim from 4 to 8, in liquid, solid, semi-solid, lyophilized or powder form.

10. Composition for use as in any claim from 4 to 9, in the form of, or prepared in the form of, a tablet, capsule or lozenge, possibly of the gastro-resistant type.

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