Phenotypic and Genomic Insights into Schleiferilactobacillus harbinensis WU01, a Candidate Probiotic with Broad-Spectrum Antimicrobial Activity Against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter) Pathogens

Abstract

The increasing prevalence of multidrug-resistant (MDR) pathogens, particularly ESKAPE bacteria, necessitates alternative antimicrobial strategies. Probiotics, particularly lactic acid bacteria, protect against pathogenic infections. This study aimed to characterize Schleiferilactobacillus harbinensis WU01, isolated from fermented palm sap, and evaluate its probiotic potential and antimicrobial activity. Its probiotic characteristics were assessed based on low-pH and bile tolerance, auto-aggregation, hydrophobicity, and adhesion to Caco-2 cells. Antimicrobial activity against ESKAPE pathogens was evaluated using the agar well diffusion assay. Whole-genome sequencing (WGS) and in silico analysis were performed to identify bacteriocin-related genes, virulence factors, and antibiotic-resistance genes. WU01 exhibited a strong tolerance to gastrointestinal conditions, with high survival rates under acidic and bile-salt environments. S. harbinensis WU01 demonstrated significant auto-aggregation, high hydrophobicity, and strong adhesion to Caco-2 cells. Antimicrobial assays revealed inhibitory activity against MDR ESKAPE pathogens, which correlated with the presence of bacteriocin-related genes, including those homologous to Carnocin_CP52. Molecular dynamics (MDs) simulations confirmed the interaction of Carnocin_CP52 with bacterial membranes, suggesting a mechanism for pathogen disruption. WGS confirmed the absence of virulence and antimicrobial-resistance genes, confirming its safety for probiotic applications. These findings suggest that S. harbinensis WU01 possesses probiotic properties and antimicrobial activity against ESKAPE pathogens. The combined results highlight its potential application in functional foods and therapeutic interventions.

Keywords: ESKAPE pathogens; Schleiferilactobacillus harbinensis; antimicrobial activity; probiotics; whole-genome sequencing.

Figure 1

Probiotic properties of S. harbinensis WU01. (A) Survival rates of Schleiferilactobacillus harbinensis WU01 under various gastric and intestinal conditions (pH tolerance and pepsin, pancreatin, and bile-salt exposure). (B) Percentage of auto-aggregation (2 h, 4 h, and 24 h), hydrophobicity, and cell adhesion of S. harbinensis WU01 to Caco-2 cells. Statistical analysis was conducted for pH tolerance (pH 2.0, pH 3.0, and pH 6.5) and auto-aggregation across different time points using one-way ANOVA, with significant differences indicated by p < 0.05 (*).

Figure 2

Scanning electron microscopy (SEM) of Caco-2 cells where the S. harbinensis WU01 adhered to the surface of Caco-2 cells: (A) untreated Caco-2 cells as control. (B) S. harbinensis WU01; (C) S. harbinensis WU01 adhered to the surface of Caco-2 cells.

Figure 3

Circular genome maps of S. harbinensis WU01.

Figure 4

Molecular dynamics simulations. (A) Snapshots at critical simulation time points. The Carnocin_CP52 peptide is represented in cyan and was initially positioned approximately 15 Å above the upper leaflet of the bilayer. The lipid bilayer consists of 80% DMPC (tan) and 20% DOPG (gray). (B) The last snapshot (500 ns) represents the hydrogen bonds between Carnocin_CP52 (cyan color) and lipids. The key interacting residues are Asn55, Arg59, Tyr60, His64, Arg65, and Tyr75.

Figure 5

Morphology of P. aeruginosa (A,B) and P. aeruginosa after treatment with CFS of S. harbinensis WU01 (C,D) observed by SEM. Magnifications were revealed as (A,C) = 10,000×; (B,D) = 20,000×.