Exploring Bacteroidota strains from human stool and animal feces: phenotyping and functional diversity

Abstract

Bacteroidota, a diverse phylum of bacteria, includes classes whose members are increasingly recognized for their significant contributions to host health, particularly through their antimicrobial properties. This study investigates the functional diversity of 42 new Bacteroidia and Sphingobacteriia strains enriched and identified from diverse hosts, including mouse ceca and human stool samples. Using 16S rRNA gene sequencing, we phylogenetically characterized the strains of the genera Bacteroides, Phocaeicola, and Sphingobacterium and assessed their functional properties related to potential beneficial functions. The strains were evaluated concerning their ability to inhibit biofilm formation of the World Health Organization-declared clinically significant pathogens, including Gram-positive Staphylococcus aureus and Staphylococcus epidermidis, Gram-negative Klebsiella oxytoca and Pseudomonas aeruginosa, and the eukaryotic yeast Candida albicans. Additionally, we investigated bile salt hydrolase and quorum-quenching (QQ) activities of the strains, as these functions contribute to microbial community interactions and host-microbe dynamics. Our findings demonstrate that all examined Bacteroidota strains consistently exhibit a capacity to inhibit biofilm formation but to different extents. Furthermore, 14 strains showed QQ activity, and 39 bile salt hydrolase activity, indicating functional diversity among the isolates. High biofilm inhibition as well as QQ activity against both autoinducers, AHL and AI-2, were predominantly observed in Bacteroides caecimuris and Bacteroides muris. These traits suggest that such strains may play important roles in shaping microbial communities and interfering with pathogens and their communication. Overall, this study provides valuable insights into strain-specific functions that could support future microbiome-based strategies for pathogen control and host health modulation.

Keywords: bacteroidota; bile salt hydrolase; biofilm inhibition; quorum quenching.

Figure 1.

Phylogenetic tree of 16S rRNA gene sequences. neighbor-joining phylogenetic tree of 16S rRNA genes from environmental isolates (labels 2411xxE) and reference type strains in the phylum Bacteroidota. The tree was constructed in MEGA11 using Maximum Composite Likelihood distances with a gamma distribution (shape = 0.5) and partial deletion of gaps (95% site coverage). Bootstrap support (%) from 1000 replicates are shown as circle size at each node. The tree is rooted on A. putrputredinis DSM 17216 (outgroup)and tip labels indicate the host origin: human stool, mouse cecum, tortoise feces, and horse feces.

Figure 2.

Inhibition of biofilm formation by Bacteroidota isolates. The heatmap illustrates the percentage of biofilm inhibition calculated relative to the corresponding biofilm control. Supernatant and three subcellular fractions from the cell extract of Bacteroidota isolates were tested to prevent biofilm formation of C. albicans, K. oxytoca, P. aeruginosa, S. aureus, and S. epidermidis. The evaluation was performed with three biological replicates, each consisting of eight technical replicates. The level of inhibition is indicated by the intensity of the tiles.

Figure 3.

Distribution of biofilm reduction activity across tested pathogens. The graph shows the number of hits with ≥20% biofilm reduction for the pathogens C. albicans, K. oxytoca, P. aeruginosa, S. aureus, and S. epidermidis using different molecular weight fractions (<3 kDa, 3–10 kDa, and >10 kDa) and culture supernatant.

Figure 4.

Potential of Bacteroidota isolates to inhibit biofilm formation of K. oxytoca. Klebsiella oxytoca biofilms were cultured in GC minimal media for 24 h at 30°C, allowing bacterial cells 1 h for adhesion. The cell extracts from 241155E or 241174E were mixed with GC minimal media to generate a final concentration of 0.5%. The supplemented media was renewed every 6 h and continuously flown through the flow cell at a flow rate of 15 µl/h for 24 h at 30°C. (A) SYTO9 was used for biofilm staining and visualized using CLSM. Representative CLSM images with scale bars representing 40 µm. (B) Analysis was performed using Zen Black (version 14.0.22.201) software and Imaris (version 9.9.0). The biofilm-preventing effect was calculated as a percentage value compared to the biofilm control (100%). Biofilm characteristics are presented in a bar plot, representing the means of four biological replicates, each consisting of four technical replicates, along with the respective standard error of the mean. Unpaired t-test was performed with GraphPad Prism 6 software with differences *P < .05, **P < .01, ***P < .001, and ****P < .001 considered significant.

Figure 5.

Effective inhibition of S. epidermidis biofilm formation by 241155E and 241170E. Staphylococcus epidermidis biofilms were generated in Caso broth media for 20 h at 37°C, with bacterial cells allowed 1 h for adhesion. Cell extracts from 241155E or 241170E were then mixed with Caso broth to attain a final concentration of 0.5%. This mixture was renewed every 6 h and continuously flown through the flow cell at a rate of 18 µl/h for 20 h at 37°C. (A) Biofilm staining was performed using SYTO9 and visualized using CLSM, yielding representative CLSM images with scale bars set at 40 µm. (B) Biofilm analysis was conducted using Zen Black (version 14.0.22.201) software and Imaris (version 9.9.0). The biofilm-preventing effect was calculated as a percentage value compared to the biofilm control (100%). The biofilm characteristics were presented in a bar plot, indicating the means of four biological replicates, each comprising four technical replicates, along with their respective standard errors of the mean. Statistical analysis was carried out using an unpaired t-test with GraphPad Prism 6 software, where differences with *P < .05, **P < .01, ***P < .001, and ****P < .001 were deemed significant.

Figure 6.

BSH activity of the isolated Bacteroidota strains. (A) Conjugation of bile acids with glycine or taurine results in the formation of bile salts. These bile salts are hydrolyzed by the microbial enzyme BSH, a process known as deconjugation. (B) BSH activity across Bacteroidota isolates is categorized by the diameter of the precipitation zone in millimeters. (C) Illustration of BSH activity levels, with darker shades representing higher activity. The absence of shades indicates that no BSH activity was detected for those specific isolates.

Figure 7.

Overview of the characteristics of the isolated Bacteriodota strains. The probiotic and antimicrobial properties of the Bacteroidota isolates were tested. For the determination of biofilm-inhibiting activity, biomass was assessed using the crystal violet assay. White squares indicate <20% inhibition, while purple squares indicate ≥20% inhibition. To determine the QQ activities, reporter strains AI1-QQ.1 and AI2-QQ.1 were used (Weiland-Bräuer et al. 2015). White squares indicate no QQ activity, and green squares indicate QQ activity. The BSH activities were determined by measuring the precipitation zone diameter. Ocher squares show the presence of BSH activity, and white squares indicate its absence.