Probiotic activity of Bacillus subtilis metabolites in experimentally induced dysbiosis in mice

Scientific relevance. A promising option for dysbiosis correction is the use of metabiotics, products based on metabolites of probiotic microorganisms. During fermentation, Bacillus subtilis bacteria (strains 3H and 1719) produce metabolites that exhibit probiotic properties in vitro. These observations in vitro motivate an in vivo investigation of B. subtilis metabolite effects on colonic mucosal microbiota in mice in experimentally induced dysbiosis and an assessment  of the potential of B. subtilis metabolites as metabiotics.

Aim. The authors aimed to compare the probiotic activity of B. subtilis 3H and B. subtilis 1719 metabolites and a commercial metabiotic in antibiotic-induced dysbiosis in mice.

Materials and methods. The authors induced experimental dysbiosis in BALB/c mice weighing 18–20 g by intraperitoneal injection of gentamicin. For subsequent correction, the test groups received sorbent-bound B. subtilis metabolites, and the comparison group received a commercial metabiotic containing B. subtilis metabolites (VKPM B-2335(3)3) via intragastric injection   for 21 days. The quantitative and qualitative analysis of colonic mucosal microbiota included microbial culturing and colony identification by MALDI-TOF mass spectrometry.

Results. Antibiotic-induced colonic dysbiosis in mice manifested itself as a decrease in the dominant microbiota and an increase in opportunistic pathogens. After 7 days of metabolite administration, the Lactobacillus population returned to normal in all treatment groups. The mice that received B. subtilis 3H metabolites showed the best results: their Lactobacillus spp. composition corresponded to that of intact animals. The content of Lac+ Escherichia coli returned to 100%   in all treatment groups. After 21  days  of  metabolite  administration,  the  authors  observed the elimination of bacteria (Rodentibacter spp., Aerococcus spp.) and fungi (Trichosporon spp., Kazachstania spp.) in the B. subtilis 3H group; Trichosporon spp. (no effect on Kazachstania spp.) in the B. subtilis 1719 group; and Enterococcus spp., Kazachstania spp., and Trichosporon spp. (no effect on Rodentibacter spp. and Aerococcus spp.) in the commercial metabiotic group.

Conclusions. Metabolites of B. subtilis strains 3H and 1719 help to restore the diversity and abundance of  colonic  microbiota  in  antibiotic-induced  dysbiosis.  The differences  observed in microbiota re-establishment in the treatment groups indicate that there is interstrain variability in the probiotic activity of B. subtilis metabolites.

1. Bukharin OV, Perunova NB. The role of microbiota in the regulation of homeostasis in the human body during infection. Journal of Microbiology, Epidemiology and Immunobiology. 2020;97(5):458–67 (In Russ.). https://doi.org/10.36233/0372-9311-2020-97-5-8

2. Shenderov BA, Sinitsa AV, Zakharchenko MM, Lang C. Metabiotics: Present state, challenges and perspectives. Springer International Publishing; 2020. https://doi.org/10.1007/978-3-030-34167-1

3. Mikhailova NA, Voevodin DA, Lazarev SA. A modern view of pro-/eukaryote interactions in the human body as the basis for development of next-generation probiotics. Journal of Microbiology, Epidemiology and Immunobiology. 2020;97(4):346–55 (In Russ.). https://doi.org/10.36233/0372-9311-2020-97-4-7

4. Hou K, Wu ZX, Chen XY, Wang JQ, Zhang D, Xiao C, et al. Microbiota in health and diseases. Signal Transduct Target Ther. 2022;7(1):135. https://doi.org/10.1038/s41392-022-00974-4

5. Kato I, Sun J. Microbiome and diet in colon cancer development and treatment. Cancer J. 2023;29(2): 89–97. https://doi.org/10.1097/PPO.0000000000000649

6. Nesvizhsky YuV. Study of changes in the human intestinal micro-biocenosis in health and in disease. Bulletin of the Russian Academy of Medical Sciences. 2003;(1):49–54 (In Russ.). EDN: OHAQDJ

7. Uspensky YuP, Baryshnikova NV. Intestinal disbiosis and antibiotic-associated diarrhea in a hospital setting: prevention and correction. Vrach. 2019;(12): 81–5 (In Russ.). https://doi.org/10.29296/25877305-2019-12-21

8. Weiss GA, Hennet T. Mechanisms and consequences of intestinal dysbiosis. Cell Mol Life Sci. 2017;74(16):2959–77. https://doi.org/10.1007/s00018-017-2509-x

9. Żołkiewicz J, Marzec A, Ruszczyński M, Feleszko W. Postbiotics — a step beyond pre- and probiotics. Nutrients. 2020;12(8):2189. https://doi.org/10.3390/nu12082189

10. Shenderov BA, Tkachenko EI, Lazebnik LB, Ardatskaya MD, Sinitsa AV, Zakharchenko MM. Metabiotics — novel technology of protective and treatment of diseases associated with microecological imbalance in human being. Experimental and Clinical Gastroenterology. 2018;151(3):83–92 (In Russ.).

11. Oleskin AV, Shenderov BA. Probiotics, psychobiotics, and metabiotics: problems and prospects. Physical and Rehabilitation Medicine, Medical Rehabilitation. 2020;2(3):233–43 (In Russ.). https://doi.org/10.36425/rehab25811

12. Zabokritskiy NA. The biologically active substances produced probiotic microorganisms of the genera Bacillus and Lactobacillus. The Journal of scientific articles “Health and Education Millennium”. 2015;17(3):80–90 (In Russ.).

13. Ilinskaya ON, Ulyanova VV, Yarullina DR, Gataullin IG. Secretome of intestinal bacilli: a natural guard against pathologies. Front Microbiol. 2017;(8):1666. https://doi.org/10.3389/fmicb.2017.01666

14. Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol. 2005;56(4):845–57. https://doi.org/10.1111/j.1365-2958.2005.04587.x

15. Volkov MYu, Vasilev PG, Vorobejchikov EV, Sinitsa AV, Rogozhin AZ, Kotelnikov RV. Preparation “Bactistatin” for treating diseases of gastrointestinal tract. Patent of the Russian Federation No. 2287335;2006 (In Russ.). EDN: OUOWIH

16. Mikhaylova NA, Kuznetsova TN, Kunyagina OV. Strain of bacteria Bacillus subtilis carrying property antibioticoresistance used for preparation “Bactisporin” making. Patent of the Russian Federation No. 2067616;1996 (In Russ.). EDN: ZALBXX

17. Mikhailova NA, Gataullin AG. Bacillus subtilis 1719 bacterium strain as producer of antagonistically active biomass in relates to pathogenic microorganisms, as well as proteolytic, amylolytic, and lipolytic enzymes. Patent of the Russian Federation No. 2298032;2007 (In Russ.). EDN: TPVIMS

18. Lazarev SA, Arzumanyan VG, Mikhailova NA. Influence of nutrient medium composition on biomass growth and antimicrobial metabolites synthesis of Bacillus subtilis probiotic strains. Bacteriology. 2021;6(2):38–42 (In Russ.). EDN: KGFVMJ

19. Avdeeva YA, Kalucki PV, Korolev VA, Medvedeva OA, Verevkina NA, Kalucki AP. Use of mexidol for correction effects of oxidative stress in experimental dysbiosis. Proceedings of Voronezh State University. Series: Chemistry. Biology. Pharmacy. 2017;(4):43–7 (In Russ.). EDN: ZVEWCF

20. Efimov BA, Kafarskaia LI, Korshunov VM. Modern methods for the evaluation of qualitative and quantitative changes in the characteristics of intestinal and vaginal microflora. Journal of Microbiology, Epidemiology and Immunobiology. 2002;(4):72–8 (In Russ.). EDN: MPKMLJ

21. Nesvizhsky YuV, Bogdanova EA, Zverev VV, Vorobyev AA. Microbiocenosis of parietal mucin in gastrointestinal tract of rats with induced dysbiosis. Journal of Microbiology, Epidemiology and Immunobiology. 2007;(3):57–60 (In Russ.). EDN: HFNMCE

22. Qiao H, Duffy LC, Griffiths E, Dryja D, Leavens A, Rossman J, et al. Immune responses in rhesus rotavirus-challenged BALB/c mice treated with bifidobacteria and prebiotic supplements. Pediatr Res. 2002;51(6):750–5. https://doi.org/10.1203/00006450-200206000-00015