Complete Genome Sequence, Metabolic Profiling and Functional Studies revealLigilactobacillus salivarius LS-ARS2is a Promising Biofilm-forming Probiotic with Significant Antioxidant, Antibacterial, and Antibiofilm Potential
Authors: Patra Sinjini , Pradhan Biswaranjan , Roychowdhury Anasuya 
Published: December 2024

AbstractBACKGROUNDProbiotics restore microbial balance and prevent gut-inflammation. Therefore, finding out novel probiotic strains is a demand. As gut-microbe, benefits ofLigilactobacillus salivarius (LS)are established. However, strain-specific detailed studies are limited. Here, we illustrate probiotic attributes of novelLS-ARS2for its potential application as food-supplement and/or therapeutic to improve gut-health.METHODSWhole genome sequencing (WGS) and phylogenetic analysis confirm the strain asLS. To establish probiotic properties, acid-bile tolerance, auto-aggregation, cell-surface-hydrophobicity, biofilm-formation and adhesion-assays are performed. To ensure safety attributes, antibiotic-susceptibility, haemolytic, DNase, trypan-blue and MTT assays are done. ABTS, DPPH, superoxide, hydroxyl free radical scavenging assays are used to determine anti-oxidant potential. Antibacterial assays including co-culture assay with pathogen and pathogenic biofilm-inhibition assays are performed to explore antibacterial efficacy. To characterize metabolic-profile ofLS-ARS2-derived cell-free-supernatant (CFS), HRMS analysis are carried out. Consequently, WGS-analyses predict potential molecular associations related to functional outcomes.RESULTSWe findLS-ARS2as remarkable fast-growing strain that shows acid and bile tolerance (>60% survival rate) indicating promising gut-sustainability. High auto-aggregation capacity (>80%), robust cell-surface hydrophobicity (>85%), and adhesion efficacy to Caco-2 cells illustrate significant potential ofLS-ARS2for gut colonization. Fascinatingly,LS-ARS2is able to form biofilm within 24 h (p<0.0001), rare amongLSstrains, indicating the potential of the strain for efficient stay in the gut. The strain ensures safety attributes.LS-ARS2-WGS analysis recognizes probiotic-specific determinants, predicts genomic stability, identifies orthologous-clusters for diverse functions, and predicts metabolites and bacteriocins. HRMS-studies withLS-ARS2-CFS further validate the presence of diverse beneficial metabolites with antimicrobial and immunomodulatory potential.LS-ARS2shows significant antioxidant properties in ABTS (>60%), DPPH (>10U/mL), superoxide (>70%), and hydroxyl free radical scavenging assays (>70%). Further,LS-ARS2shows antimicrobial activities against Gram-positive Methicillin-resistantStaphylococcus aureus (MRSA)and Gram-negative multidrug-resistant clinical strains enterotoxigenicEscherichia coli, Vibrio cholerae,andShigella flexneri. Anti-Salmonellaeffect ofLS-ARS2is prominent (p<0.0001). Most interestingly,LS-ARS2-CFS inhibitsMRSA-biofilm (p<0.0001), again rare amongLSstrains.CONCLUSIONSLS-ARS2is a novel fast-growing, biofilm-forming probiotic with significant antioxidant, antibacterial, and anti-biofilm potentials, suggesting the promising applications ofLS-ARS2for combating pathogenic biofilms and improving gut-health. However, furtherin vivostudies would facilitate their potential applications.