Exploring Weissella confusa W1 and W2 Strains Isolated from Khao-Mahk as Probiotic Candidates: From Phenotypic Traits to Genomic Insights

Abstract

Growing interest in probiotics has spurred research into their health benefits for hosts. This study aimed to evaluate the probiotic properties, especially antibacterial activities and the safety of two Weissella confusa strains, W1 and W2, isolated from Khao-Mahk by describing their phenotypes and genotypes through phenotypic assays and whole genome sequencing. In vitro experiments demonstrated that both strains exhibited robust survival under gastric and intestinal conditions, such as in the presence of low pH, bile salt, pepsin, and pancreatin, indicating their favorable gut colonization traits. Additionally, both strains showed auto-aggregation and strong adherence to Caco2 cells, with adhesion rates of 86.86 ± 1.94% for W1 and 94.74 ± 2.29% for W2. These high adherence rates may be attributed to the significant exopolysaccharide (EPS) production observed in both strains. Moreover, they exerted remarkable antimicrobial activities against Stenotrophomonas maltophilia, Salmonella enterica serotype Typhi, Vibrio cholerae, and Acinetobacter baumannii, along with an absence of hemolytic activities and antibiotic resistance, underscoring their safety for probiotic application. Genomic analysis corroborated these findings, revealing genes related to probiotic traits, including EPS clusters, stress responses, adaptive immunity, and antimicrobial activity. Importantly, no transferable antibiotic-resistance genes or virulence genes were detected. This comprehensive characterization supports the candidacy of W1 and W2 as probiotics, offering substantial potential for promoting health and combating bacterial infections.

Keywords: EPS; Weissella confusa; antibacterial activities; probiotic bacteria; whole genome sequencing.

Figure 1

Survival rates of W. confusa strains W1 and W2 strains under different gastric and intestinal conditions. Tests were done on three independent experiments. ** indicates the statistically significant difference between W1 and W2 strains (p < 0.01).

Figure 2

Percentage of bacterial cell attachment to Caco2 cell line and auto-aggregation. Tests were done on three independent experiments.

Figure 3

Exopolysaccharide production on 2% sucrose MRS agar and confirmation test in ethanol (A,B) W1 and (C,D) W2.

Figure 4

Hemolysis test on 5% defibrinated blood agar (A) W1 and (B) W2.

Figure 5

Production of antimicrobial substances (A) acid production of W1, (B) acid production of W2 (left: our studied culture, right: control agar plate), (C) H₂O₂ production of W1, and (D) H₂O₂ production of W2.

Figure 6

Circular Genome Maps of (A) W. confusa W1 and (B) W. confusa W2, illustrating CRISPR–Cas cluster and prophage. The figures were generated by CGview tool.

Figure 7

Functional analysis of gene families of W. confusa W1 and W2 based on distribution of Cluster of Orthologous group (COG) categories.

Figure 8

Illustration of exopolysaccharide-production clusters of W. confusa W1 and W2 strains. (Gene functions were predicted by Prokka and protein functions were defined by UniProt.).

Figure 9

Illustrating the gene clusters encoding the secondary metabolic enzymes in the genomes of W. confusa W1 and W2.

Figure 10

Summary of isolation, phenotypic and genetic characteristics of Weissella confusa strains W1 and W2 from Khao-Mahk in Thailand.