Isolation and Characterization of a Deoxynivalenol-Degrading Bacterium Bacillus licheniformis YB9 with the Capability of Modulating Intestinal Microbial Flora of Mice

Abstract

Deoxynivalenol (DON) is one of the most prevalent food- and feed-associated mycotoxins. It frequently contaminates agricultural commodities and poses serious threats to human and animal health and leads to tremendous economic losses globally. Much attention has been paid to using microorganisms to detoxify DON. In this study, a Bacillus licheniformis strain named YB9 with a strong ability to detoxify DON was isolated and characterized from a moldy soil sample. YB9 could degrade more than 82.67% of 1 mg/L DON within 48 h at 37 °C and showed strong survival and DON degradation rate at simulated gastric fluid. The effects of YB9 on mice with DON intragastrical administration were further investigated by biochemical and histopathological examination and the gut microbiota was analyzed by 16S rRNA Illumina sequencing technology. The results showed that DON increased the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (Cr), decreased those of immunoglobulin G (IgG) and IgM in serum, and resulted in severe pathological damage of the liver, kidney, and spleen. By contrast, YB9 supplementation obviously inhibited or attenuated the damages caused by DON in mice. In addition, YB9 addition repaired the DON-induced dysbiosis of intestinal flora, characterized by recovering the balance of Firmicutes and Bacteroidetes to the normal level and decreasing the abundance of the potentially harmful bacterium Turicibacter and the excessive Lactobacillus caused by DON. Taken together, DON-degrading strain YB9 might be used as potential probiotic additive for improving food and feed safety and modulating the intestinal microbial flora of humans and animals.

Keywords: Bacillus licheniformis YB9; degradation and detoxification; deoxynivalenol; intestinal microbial flora.

Figure 1

Isolation and identification of Bacillus licheniformis YB9. (a) The growth of YB9 on LB plates containing 1 mg/L DON. (b) Morphology and Gram-staining of YB9 observed under a microscope. (c) Phylogenetic tree of B. licheniformis YB9 based on the 16S rRNA gene sequence. (d) Colony morphologies of YB9 on LB.

Figure 2

YB9 growth (empty triangles) and DON concentration (empty circles) in liquid MM containing YB9 and supplemented with DON as the sole carbon. Liquid MM containing DON (solid circles) and liquid MM containing YB9 (solid triangles) are used as controls.

Figure 3

Degradation of DON and the tolerance of YB9 to SGF. (a) Degradation of DON by YB9 in LB and SGF during a 48 h period. (b) Survival of YB9 in SGF at pH 2, 3, 4, and LB (as control).

Figure 4

YB9 protects against DON-induced liver damage. (a) Relative weight of liver. (b) AST activity and (c) ALT activity of mice treated with normal saline (control group), 5 mg/kg BW DON (DON group), 7 × 10⁸ CFU/ml YB9 (YB9 group) or 5 mg/kg BW DON plus 7 × 10⁸ CFU/ml YB9 (DON+YB9 group), which were administered for 2 weeks. The different letters at the top of the columns mean significant difference (P < 0.05). NS, no significance. (d) Histopathological examination of liver tissues by HE staining. Magnification times, 400 ×.

Figure 5

YB9 protects against DON-induced kidney lesion. (a) Relative weight of kidney. (b) Cr activity of mice treated with normal saline (control group), 5 mg/kg BW DON (DON group), 7 × 10⁸ CFU/ml YB9 (YB9 group) or 5 mg/kg BW DON plus 7x10⁸ CFU/ml YB9 (DON+YB9 group), which were administered for 2 weeks. The different letters at the top of the columns mean significant difference (P < 0.05). NS, no significance. (c) Histopathological examination of kidney tissues by HE staining. Magnification times, 400 ×.

Figure 6

Protection of YB9 against the injury of spleen caused by DON. (a) Relative weight of spleen. (b) IgG and (c) IgM activity of mice treated with normal saline (control group), 5 mg/kg BW DON (DON group), 7 × 10⁸ CFU/ml YB9 (YB9 group) or 5 mg/kg BW DON plus 7x10⁸ CFU/ml YB9 (DON+YB9 group), which were administered for 2 weeks. The different letters at the top of the columns mean significant difference (P < 0.05). NS, no significance. (d) Histopathological examination of spleen tissues by HE staining. Magnification times, 40 ×.

Figure 7

Effects of DON and YB9 on the intestinal flora of mice. (a) Shannon analysis based on alpha diversity of intestinal flora. (b) PCoA analysis based on β-diversity of intestinal flora. (c) The relative abundance at the phylum level. (d) The relative abundance at the genus level. (e) The heatmap analysis of the top-20 relative abundance of the genus in all the samples.