USE OF BACILLUS AMYLOLIQUEFACIENS AND/OR BACILLUS SUBTILIS FOR THE ENHANCEMENT OF ACTIVE PROBIOTIC COMPOSITIONS OR FOR THE ACTIVATION OF INACTIVE PROBIOTIC COMPOSITIONS, COMPOSITIONS THUS OBTAINED, AND RELATED METHOD

Abstract

This invention relates to the use of Bacillus amyloliquefaciens and/or Bacillus subtilis for the enhancement of active probiotic compositions or for the activation of inactive probiotic compositions for use in the prevention and/or treatment of inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis, the compositions thus obtained, and a method for the enhancement of active probiotic compositions or for the activation of inactive probiotic compositions for use in the prevention and/or treatment of inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis.

Claims

1: A method for treating or ameliorating an inflammatory bowel disease comprising administering to an individual in need thereof a probiotic mixture in an amount sufficient to treat or ameliorate the inflammatory bowel disease in the individual in need thereof, wherein the probiotic mixture comprises a Bacillus amyloliquefaciens and/or a Bacillus subtilis in an amount sufficient to enhance an active probiotic composition in the probiotic mixture or activate an inactive probiotic composition in the probiotic mixture, thereby treating or ameliorating the inflammatory bowel disease.

2: The method of claim 1, wherein the amount by weight of Bacillus amyloliquefaciens and/or Bacillus subtilis in the probiotic mixture is between about 2 and about 50%.

3: The method of claim 1, wherein the active probiotic composition comprises a mixture of bacterial strains comprising: Lactobacillus paracasei DSM 24733, Lactobacillus plantarum DSM 24730, Lactobacillus acidophilus DSM 24735, Lactobacillus delbrueckii subspecies bulgaricus DSM 24734, Bifidobacterium longum DSM 24736, Bifidobacterium infantis DSM 24737, Bifidobacterium breve DSM 24732, and Streptococcus thermophilus DSM 24731,

4: The method of claim 1, wherein the active probiotic composition comprises: from between about 20 to 30% by weight of Streptococcus thermophilus, from between about 20 to 30% by weight of Lactobacillus casei, from between about 30 to 20% by weight of Bifidobacterium breve, and from between about 30 to 20% by weight of Bifidobacterium animalis subsp. lactis.

5: The method of claim 1, wherein the inactive composition of probiotics comprises: Streptococcus thermophilus BT01, Bifidobacterium breve BB02, Bifidobacterium longum (recently reclassified as Bifidobacterium animalis subspecies lactis) BL03, Bifidobacterium infantis (recently reclassified as Bifidobacterium animalis subspecies lactis) B104, Lactobacillus acidophilus BA05, Lactobacillus plantarum BP06, Lactobacillus paracasei BP07, and Lactobacillus delbrueckii subspecies bulgaricus (recently re classified as Lactobacillus helveticus) BD08.

6: The method of claim 1, wherein the Bacillus amyloliquefaciens is characterized as having a 16S RNA comprising a sequence as set forth in SEQ ID NO:1.

7: An active probiotic composition or an activated inactive probiotic composition comprising a total of between about 2 to 50% by weight of Bacillus amyloliquefaciens and/or Bacillus subtilis.

8: The active probiotic composition or the activated inactive probiotic composition of claim 7, further comprising from between about 98 to 50% by weight of a mixture of the following 8 bacterial strains: Lactobacillus paracasei DSM 24733, Lactobacillus plantarum DSM 24730, Lactobacillus acidophilus DSM 24735, Lactobacillus delbrueckii subspecies bulgaricus DSM 24734, Bifidobacterium longum DSM 24736, Bifidobacterium infantis DSM 24737, Bifidobacterium breve DSM 24732, and Streptococcus thermophilus DSM 24731.

9: The active probiotic composition or the activated inactive probiotic composition of claim 7, further comprising: from between about 20 to 30% by weight of Streptococcus thermophilus, from between about 10 to 30% by weight of Lactobacillus casei, from between about 10 to 15% by weight of Bifidobacterium breve, from between about 10 to 15% by weight of Bifidobacterium animalis subsp. lactis, and from between about 50 to 10% by weight of Bacillus amyloliquefaciens and/or Bacillus subtilis.

10: The active probiotic composition or the activated inactive probiotic composition of claim 7, comprising from between about 98 to 50% by weight of a mixture of the following bacterial strains: Streptococcus thermophilus BT01, Bifidobacterium breve BB02, Bifidobacterium longum (recently reclassified as Bifidobacterium animalis subspecies lactis) BL03, Bifidobacterium infantis (recently reclassified as Bifidobacterium animalis subspecies lactis) BI04, Lactobacillus acidophilus BA05, Lactobacillus plantarum BP06, Lactobacillus paracasei BP07, and Lactobacillus delbrueckii subspecies bulgaricus (recently re classified as Lactobacillus helveticus) BD08.

10: The active probiotic composition or the activated inactive probiotic composition of claim 7, wherein the of Bacillus amyloliquefaciens is characterized as having a 16S RNA comprising a sequence as set forth in SEQ ID NO:1.

10: The active probiotic composition or the activated inactive probiotic composition of claim 7, wherein the active probiotic composition comprises: from between about 20 to 30% by weight of Streptococcus thermophilus, from between about 20 to 30% by weight of Lactobacillus casei, from between about 30 to 20% by weight of Bifidobacterium breve, and from between about 30 to 20% by weight of Bifidobacterium anima is subsp. lactis.

13: The method of claim 1, wherein the inflammatory bowel disease is selected from the group consisting of: Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis.

14-18. (canceled)

19: The method of claim 13, wherein the inflammatory bowel disease is Crohn's disease.

20: The method of claim 13 wherein the inflammatory bowel disease is ulcerative colitis.

21: The method of claim 13 wherein the inflammatory bowel disease is indeterminate colitis.

22: The method of claim 13, wherein the inflammatory bowel disease is microscopic colitis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0110] FIGS. 1A-1F show the enhancement of the protective effect associated with the administration of the species Bacillus amyloliquefaciens in addition to an already active bacterial composition with respect to this effect. In particular, FIG. 1A shows the trend of body weight, FIG. 1B shows the daily measurement of the Colitis Disease Activity Index (CDAI) which considers body weight loss, stool consistency and the presence of blood in stool, FIG. 1C shows the length of the colon, FIG. 1D shows the ratio of colon weight to colon length, and FIGS. 1E and 1F show the percentage of immune cells present in the Lamina propria of the colon (FIG. 1E: CD3+IL-10+ T lymphocytes within CD3+ T lymphocytes; FIG. 1F: M2 CD11b+Gr1-IL-10+ macrophages within CD11+Gr1-macrophages). The results are expressed as mean±SEM of 7-10 mice per experimental group. *p<0.05.

[0111] FIGS. 2A-2E show the enhancement of the protective effect associated with the administration of the bacterial species Bacillus amyloliquefaciens in addition to a further bacterial composition that is already active with respect to this effect. In particular, FIG. 2A shows the trend of body weight, FIG. 2B shows the daily measurement of CDAI that considers body weight loss, stool consistency and the presence of blood in stool, FIG. 2C shows the ratio of colon weight to colon length, and FIGS. 2D and 2E show the frequency of Treg CD4+FoxP3+ immune cells present in the Lamina propria of the colon (FIG. 2D: within CD4+ T lymphocytes; FIG. 2E: within all cells in the Lamina propria of the colon). The results are expressed as mean±SEM of 6-8 mice per experimental group. *p<0.05.

[0112] FIGS. 3A-3B show the protective effect associated with the administration of the Bacillus amyloliquefaciens species compared to a bacterial composition not presenting this effect. In particular, FIG. 3A shows the trend of body weight and FIG. 3B shows the daily measurement of CDAI which takes into account body weight loss, stool consistency and the presence of blood in stool. The results are expressed as mean±SEM of 6-8 mice per experimental group. *p<0.05.

[0113] FIGS. 4A-4B show the effect of Bacillus amyloliquefaciens alone as well as in combination with some of the compositions of the invention. In particular, FIG. 4A shows the trend of body weight and FIG. 4B shows the daily measurement of CDAI that considers body weight loss, stool consistency and the presence of blood in the stool. The results are expressed as mean±SEM of 6-8 mice per experimental group. *p<0.05.

[0114] FIGS. 5A-5B show the substantial equivalence of the effects of Bacillus amyloliquefaciens and Bacillus subtilis in the tests performed. In particular, FIG. 5A shows the trend of body weight and FIG. 5B shows the daily measurement of CDAI that considers body weight loss, stool consistency and the presence of blood in the stool. The results are expressed as mean±SEM of 6-8 mice per experimental group. *p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

Experimental Part

[0115] Materials and Methods

[0116] The strain of Bacillus amyloliquefaciens used for the experiments is the known strain characterized by SEQ ID NO: 1 coding for 16S RNA.

[0117] The murine model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to test the anti-inflammatory activity of the compositions under examination. In this model, inflammation of the colon is induced by intrarectal administration of TNBS in a 50% ethanol solution. Ethanol is essential to provide access to intestinal epithelial cells, compromise barrier function and allow TNBS to penetrate the wall of the intestine.

[0118] Since this model is associated with the increased presence of highly activated T cells, which have a key pathogenic role in TNBS-induced colitis, the model itself is suitable to investigate the role of T cells in intestinal inflammation (Elson, C. O. et al., J. Immunol. 157:2174-2185, 1996; Dohi, T. et al., J. Exp. Med. 189:1169-1180, 1999). Nevertheless, immune mechanisms are also involved in the development of TNBS colitis (Fiorucci, S. et al., Immunity 17:769-780, 2002).

[0119] Histologically, following the induction of the disease, transmural inflammation with infiltrates of macrophages, neutrophils and lymphocytes, as well as colon hypertrophy (Kiesler, P. et al., Cell. Mol. Gastroenterol. Hepatol. 1:154-170, 2015) is obtained. Since some immunological and histopathological features are similar to those associated with Crohn's disease, TNBS-induced colitis has been widely used to study a variety of aspects potentially relevant to this disease (Kiesler, P. et al., Cell. Mol. Gastroenterol. Hepatol. 1:154-170, 2015; Rieder, F. et al., Gut 62:1072-1084, 2013).

[0120] C57BL6 male mice of 8 weeks of age purchased from The Jackson Laboratory were used. The animals were kept at the preclinical research center of the University of Perugia.

[0121] The mice were kept in controlled temperature of 22° C. with a light/dark period of 12/12 hours and were acclimatized under these conditions for 7 days before their use in experimental activities. The study was conducted in accordance with Italian law and the experimental protocol was approved by the Ethics Committee of the University of Perugia and the Ministry of Health (permit No. 1126/20-PR).

[0122] For the induction of colitis, mice were kept fasting for 12 h (Day −1). The next day (Day 0), the mice were anaesthetized, and a catheter was inserted into the colon up to 4 cm from the anus. To induce colitis, 1 mg of TNBS dissolved in 50% ethanol was administered by catheter with a 1 ml syringe (injection volume: 100 μl).

[0123] Control animals (untreated, NT) received only 50% ethanol solution. The mice were then monitored daily to assess the course of the disease by measuring body weight and the CDAI (Colitis Disease Activity Index) which includes the percentage of weight lost, stool consistency and the presence of blood in stool (each parameter has a value ranging from 0 to 4).

[0124] At the end of the experiment the animals were sacrificed, and the colon was recovered for the analysis of macroscopic characteristics and for the extraction of Lamina propria cells by means of the Lamina propria Dissociation Kit Mouse (Miltenyi Biotec) for analysis by cytofluorimetry.

[0125] The probiotic compositions were administered daily (from Day 0 to Day 4) by oral probe at a concentration of 50×10.sup.9 cfu of probiotics/kg body weight dissolved in saline solution.

Example 1

[0126] In this example a composition active in the prevention and/or treatment of inflammatory diseases which includes the following 8 bacterial strains has been tested:

[0127] Lactobacillus paracasei DSM 24733,

[0128] Lactobacillus plantarum DSM 24730,

[0129] Lactobacillus acidophilus DSM 24735,

[0130] Lactobacillus delbrueckii subspecies bulgaricus DSM 24734,

[0131] Bifidobacterium longum DSM 24736,

[0132] Bifidobacterium infantis DSM 24737,

[0133] Bifidobacterium breve DSM 24732, and

[0134] Streptococcus thermophilus DSM 24731, commercially available under the VIVOMIXX brand, alone and with the addition of Bacillus amyloliquefaciens.

[0135] FIG. 1 shows the results of the tests carried out according to the methods described above. In the figure, NT=untreated and TNBS=treated with 2,4,6-trinitrobenzenesulfonic acid only.

[0136] The results of the experimental data clearly show that, in the TNBS-induced colitis model, the addition of Bacillus amyloliquefaciens improves the efficacy of VIVOMIXX in preventing the reduction of body weight loss, as well as in inducing improvements in inflammation measured as CDAI and in attenuating TNBS-induced colon morphological changes, such as the ratio of colon weight to colon length. The increase in regulatory type intestinal cells (Treg) within the Lamina propria of the colon is also evident.

Example 2

[0137] In this example a new composition has been tested that has been found to be active in the prevention and/or treatment of inflammatory diseases.

[0138] The new composition included:

[0139] 30% by weight of Streptococcus thermophilus,

[0140] 30% by weight of Lactobacillus casei,

[0141] 20% by weight of Bifidobacterium breve, and

[0142] 20% by weight of Bifidobacterium animalis subsp. lactis.

[0143] FIGS. 2A-2E show the results of the tests carried out according to the methods described above. In the figures, NT=untreated and TNBS=treated with 2,4,6-trinitrobenzenesulfonic acid only.

[0144] The results of the experimental data with the above composition clearly show that, in the TNBS-induced colitis model, this composition exhibits good efficacy in preventing the reduction of body weight loss as well as in inducing improvements in inflammation measured as CDAI and in attenuating TNBS-induced colon morphological changes, such as the ratio of colon weight to colon length. The increase in regulatory type intestinal cells (Treg) within the Lamina propria of the colon is also evident.

[0145] To verify the effects of adding Bacillus amyloliquefaciens to this composition, the composition has been modified as follows:

[0146] 30% by weight of Streptococcus thermophilus,

[0147] 30% by weight of Lactobacillus casei,

[0148] 15% by weight of Bifidobacterium breve,

[0149] 15% by weight of Bifidobacterium animalis subsp. lactis, and

[0150] 10% by weight of Bacillus amyloliquefaciens.

[0151] The results of the experimental data summarised in FIGS. 2A-2E clearly show that, in the TNBS-induced colitis model, the addition of Bacillus amyloliquefaciens improves the effectiveness of the new composition in preventing the reduction of body weight loss, as well as in inducing improvements in inflammation measured as CDAI and in attenuating TNBS-induced colon morphological changes, such as the ratio of colon weight to colon length. The increase in regulatory type gut cells (Treg) within the Lamina propria of the colon is also evident.

Example 3

[0152] In this example a commercial composition including the following bacterial strains has been tested:

[0153] Streptococcus thermophilus BT01,

[0154] Bifidobacterium breve BB02,

[0155] Bifidobacterium longum (recently reclassified as Bifidobacterium animalis subspecies lactis) BL03,

[0156] Bifidobacterium infantis (recently reclassified as Bifidobacterium animalis subspecies lactis) BI04,

[0157] Lactobacillus acidophilus BA05,

[0158] Lactobacillus plantarum BP06,

[0159] Lactobacillus paracasei BP07, e

[0160] Lactobacillus delbrueckii subspecies bulgaricus (recently reclassified as Lactobacillus helveticus) BD08.

[0161] As can be seen from the results shown in FIG. 3, this composition was inactive in the tests performed, most likely due to differences in strain cultivation procedures.

[0162] Nevertheless, the addition of Bacillus amyloliquefaciens has made the composition active in preventing the reduction of body weight loss as well as in inducing an improvement in inflammation measured as CDAI.

CONCLUSIONS

[0163] From FIGS. 4A-4B it is evident the enhancement of the protective effect associated with the administration of the bacterial species Bacillus amyloliquefaciens in addition to bacterial compositions already active with respect to this effect, or the establishment of the protective activity associated with the administration of the same species in addition to bacterial compositions originally not presenting this characteristic.

[0164] Furthermore, FIGS. 5A-5B show a substantial equivalence of the effects associated with the administration of the bacterial species Bacillus amyloliquefaciens and Bacillus subtilis. This equivalence would be explained by the high degree of genetic similarity between the two groups of bacteria.

[0165] In fact, Bacillus amyloliquefaciens and Bacillus subtilis constitute two sister species (species originated by differentiation from a shared ancestor) belonging to the larger group of bacterial species characterized by a high genetic correlation, called Bacillus subtilis group (Fritze, D., “Taxonomy of the genus Bacillus and related genera: the aerobic endospore-forming bacteria”, Phytopathology 94:1245-1248, 2004). The high genetic similarity between the species B. amyloliquefaciens and B. subtilis is clearly demonstrated by the difficulty and/or impossibility to efficiently distinguish these two bacterial groups by comparing the sequences encoding for 16S rRNA (Wang et al., “Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens”, Int. J. Syst. Evol. Microbiol. 58:671-675, 2008), which is the marker of choice for taxonomic identification of bacteria (Woese, C. R., “Bacterial evolution”, Microbiol. Rev. 51:221-271, 1987). In recent years, the development of new generation sequencing techniques made it possible to access a large amount of data concerning the sequence of the entire genome of many bacterial species and to develop investigation methods capable of assessing more reliably and accurately the genetic similarity between microbial groups. In this context, genome typing based on multiple loci extended to the entire preserved genome (core-MLST) represents a potent tool to determine the correlation between different taxonomic groups by means of the simultaneous comparison of hundreds or thousands of genetic markers shared between them. The application of this technique to specific bacterial strains belonging to the B. amyloliquefaciens and B. subtilis species further highlights the great genetic similarity between these taxonomic groups. The reconstruction of the phylogenetic relationships between genomes belonging to the two species, based on the alignment of 1664 gene clusters determined by using a sequence similarity threshold of ≥80% (see dendrogram below), shows that B. amyloliquefaciens and B. subtilis strains are phylogenetically closer to each other than to strains included in their own taxonomic group (groups A, B, C, D).

[0166] The high genetic correlation together with the large number of genes shared between the two bacterial species makes it possible to assume that they may also be equivalent from a functional point of view.

[0167] Materials and Methods

[0168] Genome typing based on multiple loci extended to the entire core-genome was performed on genomes related to the B. amyloliquefaciens and B. subtilis species available in the public database (ftp://ftp.ncbi.nlm.nih.gov/genbank). Five complete genomic sequences were selected for each bacterial species, including those related to the species type strains B. amyloliquefaciens DSM7 and B. subtilis ATCC13952. In order to avoid the introduction of errors due to different genome annotation, all sequences were annotated de novo by the Prokka v1.12 pipeline (Seeman T, “Prokka: rapid prokaryotic genome annotation”, Bioinformatics 15 Jul. 2014, 30(14):2068-9) forcing compliance with Genbank/ENA/DDJB standards. The genomes were imported within the BPGA bioinformatics program (Chaudhari et al., “BPGA-an ultra-fast pan-genome analysis pipeline” Sci. Rep. 6:24373, 2016) and homologous gene groups were determined using a threshold of similarity ≥80% between the protein sequences coded by the gene sequences. The homologous protein groups were aligned separately using the MUSCLE v 3.8.31 (Edgar, R. C., “MUSCLE: multiple sequence alignment with high accuracy and high throughput”, Nucleic Acids Res. 32:1792-7, 2004) and subsequently joined to form concatenameres. The phylogenetic reconstructions were performed using the Approximately Maximum-Likelihood method using the Fasttree 2 program (Price et al., “FastTree 2-Approximately maximum-likelihood trees for large alignments”, PLoS One 5:e9490, 2010).

[0169] FIGS. 5A-5B also show the substantial equivalence of the effects of Bacillus amyloliquefaciens and Bacillus subtilis in the tests performed.