Probiotic Potential of Pediococcus pentosaceus M6 Isolated from Equines and Its Alleviating Effect on DSS-Induced Colitis in Mice

Abstract

Colitis in equines has high morbidity and mortality rates, which severely affects the development of the equine-breeding industry. With the issuance of antibiotic bans, there is an urgent need for healthier and more effective alternatives. In recent years, probiotics have been widely used as microbial feed additives in animal husbandry, playing a crucial role in preventing and treating diarrhea and regulating host immune function. In this study, we isolated and screened a strain with rapid and stable acid production using bromocresol purple, litmus milk coloration tests, and acid production performance assessments. Based on morphological characteristics, physiological and biochemical properties, and 16S rDNA identification, the strain was identified as Pediococcus pentosaceus and named M6. The Pediococcus pentosaceus M6 exhibited stable growth and tolerance to high temperatures, acid and bile salt concentrations, and simulated gastrointestinal fluid environments. The M6 strain demonstrated good antibacterial effects against Escherichia coli, Staphylococcus aureus, and Salmonella. The M6 strain did not produce hemolysis zones on Columbia blood agar plates, indicating its high safety, and was found to be insensitive to 12 antibiotics, including cephalexin and neomycin. Additionally, intervention in mice with dextran sulfate sodium (DSS)-induced colitis alleviated weight loss and shortened colon length. To a certain extent, it regulated the expression of inflammatory cytokines and the gut microbiota within the body and reduced inflammatory cell infiltration and intestinal barrier damage. In summary, the isolated Pediococcus pentosaceus M6 strain exhibited excellent probiotic properties and could alleviate DSS-induced colitis in mice, suggesting its potential application value as a probiotic in animal husbandry.

Keywords: 16S rDNA; Pediococcus pentosaceus; equine; inflammatory bowel disease; intestinal contents.

Figure 1

Results of screening and identification of equine-derived strains. (A) Purification of the M6 strain; (B) microscopic examination of the M6 strain; (C) phylogenetic tree of the M6 strain.

Figure 2

Growth curves and heat, acid, and bile salt resistance of Pediococcus pentosaceus M6. (A) Growth curve; (B) heat resistance; (C) acid resistance; (D) bile salt resistance.

Figure 3

Results of hemolytic properties of strains. (A) Staphylococcus aureus; (B) Pediococcus pentosaceus M6.

Figure 4

Analysis of the results of clinical indices. Note: * indicates p < 0.05; ** indicates p < 0.01. (A) Experimental process for M6-intervened DSS-induced ulcerative colitis in mice; (B) changes in the body weight of the mice; (C) DAI scores; (D,E) length of the colon in each group of mice.

Figure 5

Histopathologic results of the colon of mice in each group. Note: ** indicates p < 0.01, yellow arrows indicate inflammatory cell infiltration. (A) H&E results of colonic tissues in each group under 100×; (B) H&E results of colonic tissues in each group under 200×; (C) histopathologic scores of the colon of mice in each group.

Figure 6

Inflammatory cytokine expression in the serum of each group. Note: ^ns indicates p ≥ 0.05; ** indicates p < 0.01. (A) IL−1β; (B) IL−6; (C) IL-10.

Figure 7

Results of tight junction protein gene expression in the mouse colon tissue by group. Note: ^ns indicates p ≥ 0.05; ** indicates p < 0.01. (A) ZO-1; (B) occludin.

Figure 8

Effect of Pediococcus pentosaceus M6 on the diversity of intestinal flora in the mice with DSS-induced colitis. Note: * indicates p < 0.05. (A) Venn diagram; (B) Chao 1 index; (C) Shannon’s index; (D) Simpson’s index.

Figure 9

Effects of Pediococcus pentosaceus M6 on the structure and differences of intestinal flora in the mice with DSS-induced colitis. Note: * indicates p < 0.05; ** indicates p < 0.01. (A) The relative abundance of bacterial phyla at the phylum level across different groups; (B) the abundance of Bacteroidetes; (C) the abundance of Firmicutes; (D) the relative abundance of bacterial genera at the genus level across different groups; (E) the abundance of Muribaculaceae_unclassified; (F) the abundance of Muribaculum.