The effect of metabolites from Bacillus subtilis BS-Z15 on the treatment of Candida albicans infection in Kunming mice
Fungal diseases are divided into superficial fungi and deep fungi. Currently, the number of cases of deep fungal infections is increasing, and more than 90% of clinically reported fungal related deaths are related to Cryptococcus, Candida, and Aspergillus. It has become one of the important diseases that affect human quality of life and threaten life, health, and safety. Candida albicans is the most pathogenic Gram positive opportunistic pathogen in the Candida genus, often invading human skin, mucous membranes, and causing visceral or systemic infections. The global mortality rate caused by it is between 36% and 63%, which has become an important issue that urgently needs to be addressed in the public health system and healthcare sector.

The use of antagonistic bacteria to inhibit Candida albicans has been widely reported, with Bacillus accounting for the majority of the screened strains, followed by fungi and actinomycetes. Bacillus subtilis can secrete various antibacterial metabolites and has the advantages of multifunctionality, low toxicity, etc. It is widely used in research fields such as medicine, agriculture, and scientific research. At present, research mainly focuses on the antibacterial substances of cyclic lipopeptides, such as surfactin, iturin, fengycin, and chain peptide antibacterial substances. However, there have been more studies on the antifungal effects of these antibacterial substances in vitro, and fewer studies on their in vivo effects. Currently, it has only been found that iturin has a certain therapeutic effect on mice infected with Candida albicans, and there is less research on other antibacterial substances in Bacillus subtilis metabolites.

Bacillus subtilis BS-Z15 was screened from soil in the Heshuo area of Xinjiang in the early stage. The metabolites extracted from the fermentation broth (MEs) have strong antifungal activity and good antagonistic stability. In the evaluation of the antifungal activity of MEs and the safety of administration to mice, it was found that this strain can effectively inhibit various fungi such as Verticillium dahliae and Aspergillus flavus, and has no toxic effect on mice. This article presents the determination of the minimum inhibitory dose (MIC) of crude MEs extracted from the fermentation broth of Bacillus subtilis BS-Z15 against Candida albicans in vitro, and studies their killing effect on yeast like Candida albicans. By establishing an in vivo model of Candida albicans infection in mice, the therapeutic effect of MEs on it is explored. By studying the inhibitory effects of crude MEs extracted from Bacillus subtilis BS-Z15 fermentation broth on Candida albicans in vitro and in vivo, a solid research foundation has been laid for the development of antifungal drugs with strong antifungal activity and low side effects.

With the current overuse of antibiotics, pathogenic fungi are becoming increasingly resistant to existing drugs, making fungal infections easy to occur, difficult to control, and having certain side effects. Amphotericin B has severe nephrotoxicity, while azoles have severe nephrotoxicity and hepatotoxicity. Therefore, the development of new highly efficient and low toxicity antifungal drugs has received increasing attention.

In vitro experiments, when using MEs at a concentration of 0.33mg/mL, yeast like Candida albicans can shrink, dent, and rupture to death. In vivo models, when infected with Candida albicans, the mouse body is subjected to harmful stimuli, which can trigger an internal immune system response and release a large amount of immune inflammatory factors, leading to inflammatory reactions. The spleen and liver are important organs for immune function and detoxification in mice. In the observation of HE stained liver and spleen sections, it was found that the cells in the liver and spleen of the model group mice were significantly swollen and deformed, with some cell membranes ruptured, unclear cytoplasmic differentiation, a large number of inflammatory cell infiltration, and obvious tissue damage. In the MEs gavage group, the degree of these injuries was significantly improved, with a significant reduction in inflammatory cells and a positive correlation with the dosage of MEs. MEs can alleviate inflammation in organs, thereby restoring tissue cell structure.

Meanwhile, after infection with Candida albicans, it can also cause antioxidant imbalance in mice, leading to tissue damage. The antioxidant balance includes both the degree of lipid peroxidation produced in the body, which is the level of free radical production, and the body’s enzymatic and oxidative reactions. The content of MDA often reflects the degree of lipid peroxidation in the body, while the activity of SOD indirectly reflects the body’s ability to eliminate oxygen free radicals. T-AOC reflects the overall antioxidant level of the body’s enzymatic and non enzymatic systems, and is closely related to health status. Compared with the model group mice, 90mg/kg MEs treatment significantly reduced the degree of lipid peroxidation in the body, significantly increased SOD activity, and thus enhanced the total antioxidant capacity of T-AOC in the mice. At the same time, there was a slight improvement in the 30mg/kg and 60mg/kg dose groups. The determination of total antioxidant capacity also indicates that treatment with gastric MEs can help the body restore normal antioxidant levels, which may be one of the reasons for reducing the degree of organ damage.

There have been numerous reports on the use of antagonistic metabolites to treat Candida albicans infections. Antimicrobial peptide P3 is an artificially synthesized peptide derived from bovine hemoglobin, which can cause Candida albicans to develop indentations, protrusions, and cracks. The fermentation supernatant of Bacillus subtilis LAY can antagonize Candida albicans, and PCR screening revealed genes that can produce polyketide compounds, which have high biological activity. Lei et al. found through studying the metabolite of Bacillus subtilis, Bacillus subtilis, that it can reverse the damage to organ structure and function caused by Candida albicans infection by eliminating the fungal burden on the kidneys and slowing down weight changes in mice. The results of this study are similar to the reported effects of common antimicrobial peptides, and the extraction method of MEs is similar to that of lipopeptide substances. However, in the early stage of the whole genome sequencing study of BS-Z15 in the laboratory, it was found that BS-Z15 Bacillus subtilis does not contain the complete operon of mainstream antagonistic fungal genes, such as surfactant, gibberellin, fenapyr, etc. Therefore, we speculate that the antimicrobial active ingredients in MEs may be new lipopeptide substances. However, whether the specific ingredients and active ingredients directly inhibit the growth of Candida albicans in vivo remains to be further studied through technologies such as LC-MS, MS/MS, NMR, and high-throughput sequencing. In summary, the metabolites extracted from the BS-Z15 fermentation broth exhibit significant in vitro and in vivo anti Candida albicans effects. This study provides experimental evidence for the search for novel antifungal lead compounds from metabolites of Bacillus subtilis.