A Novel Brevinin2 HYba5 Peptide against Polymicrobial Biofilm of Staphylococcus aureus and Enterococcus faecalis.

BACKGROUND: Brevinin2 HYba5 (Peptide 29) is a novel cationic peptide identified from an endemic frog, Hydrophylax bahuvistara. Staphylococcus aureus and Enterococcus faecalis are troublesome biofilm-forming pathogens associated with nosocomial and community-acquired infections and contribute to the severity of infections associated with implanted devices and chronic wounds. Co-existence of both pathogens in biofilm mode contributes to an increased antibiotic resistance, treatment failure and hence persistent disease burden. Identifying a novel and stable, less toxic compound targeting multispecies biofilm with a lower probability of acquiring resistance in comparison to antibiotics is highly warranted. OBJECTIVE: Evaluate the activity of Brevinin2 HYba5 against S. aureus and E. faecalis mixed biofilm. METHODS: The anti-biofilm activity of peptide 29 was tested by Crystal violet assay, Confocal laser scanning Microscopy (CLSM) and MTT Assay. Cytotoxicity of the peptide was tested in RBC and L929 fibroblast cell line. Biofilm inhibitory activity of the peptide was evaluated at different temperatures, pH, serum and plasma concentrations. The antibiofilm potential of the peptide was tested against polymicrobial biofilm by Fluorescent in situ hybridisation (FISH) and plate counting on HiCromeTM UTI Agar media. RESULTS: The peptide 29 could inhibit biofilm formation of S. aureus and E. faecalis individually as well as in polymicrobial biofilm at 75 µM concentration. The peptide maintained its antibiofilm potential at different temperatures, serum and plasma concentrations. Activity of the peptide was high at acidic and neutral pH but found to get reduced towards alkaline pH. The peptide is nonhemolytic and does not exhibit significant cytotoxicity against the L929 fibroblast cell line (92.80% cell viability). CONCLUSION: The biofilm inhibition property makes peptide 29 a promising candidate for the management of S. aureus and E. faecalis biofilm, especially in catheter-associated devices to prevent the initial colonization and thus can ease the burden of pathogenic biofilm-associated infections.

Decoding the proteomic changes involved in the biofilm formation of Enterococcus faecalis SK460 to elucidate potential biofilm determinants.

BACKGROUND: Enterococcus faecalis is a major clinically relevant nosocomial bacterial pathogen frequently isolated from polymicrobial infections. The biofilm forming ability of E. faecalis attributes a key role in its virulence and drug resistance. Biofilm cells are phenotypically and metabolically different from their planktonic counterparts and many aspects involved in E. faecalis biofilm formation are yet to be elucidated. The strain E. faecalis SK460 used in the present study is esp (Enterococcal surface protein) and fsr (two-component signal transduction system) negative non-gelatinase producing strong biofilm former isolated from a chronic diabetic foot ulcer patient. We executed a label-free quantitative proteomic approach to elucidate the differential protein expression pattern at planktonic and biofilm stages of SK460 to come up with potential determinants associated with Enterococcal biofilm formation. RESULTS: The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of proteomic data revealed that biofilm cells expressed higher levels of proteins which are associated with glycolysis, amino acid biosynthesis, biosynthesis of secondary metabolites, microbial metabolism in diverse environments and stress response factors. Besides these basic survival pathways, LuxS-mediated quorum sensing, arginine metabolism, rhamnose biosynthesis, pheromone and adhesion associated proteins were found to be upregulated during the biofilm transit from planktonic stages. The selected subsets were validated by quantitative real-time PCR. In silico functional interaction analysis revealed that the genes involved in upregulated pathways pose a close molecular interaction thereby coordinating the regulatory network to thrive as a biofilm community. CONCLUSIONS: The present study describes the first report of the quantitative proteome analysis of an esp and fsr negative non gelatinase producing E. faecalis. Proteome analysis evidenced enhanced expression of glycolytic pathways, stress response factors, LuxS quorum signaling system, rhamnopolysaccharide synthesis and pheromone associated proteins in biofilm phenotype. We also pointed out the relevance of LuxS quorum sensing and pheromone associated proteins in the biofilm development of E. faecalis which lacks the Fsr quorum signaling system. These validated biofilm determinants can act as potential inhibiting targets in Enterococcal infections.

Metataxonomic approach to decipher the polymicrobial burden in diabetic foot ulcer and its biofilm mode of infection.

Chronic diabetic foot is a global burden affecting millions of people, and the chronicity of an ulcer is directly linked to the diverse bacterial burden and its biofilm mode of infection. The bacterial diversity of 100 chronic diabetic ulcer samples was profiled via traditional culturing method as well as metagenomic approach by sequencing the 16S rRNA V3 hyper-variable region on Illumina Miseq Platform (Illumina, Inc., San Diego, CA). All the relevant clinical metadata, including duration of diabetes, grade of ulcer, presence of neuropathy, and glycaemic level, were noted and correlated with the microbiota. The occurrence and establishment of bacterial biofilm over chronic wound tissues was revealed by Fluorescent in situ Hybridization and Scanning Electron Microscopy. The biofilm-forming ability of predominant bacterial isolates was studied via crystal violet assay and Confocal Laser Scanning Microscopy. The dominant phyla obtained from bacterial diversity analysis were Firmicutes, Proteobacteria, and Actinobacteria. The dominant aerobic pathogens identified by culture method are Pseudomonas, Proteus, Enterococcus, and Staphylococcus, whereas high-throughput sequencing revealed heightened levels of Streptococcus and Corynebacterium along with 22 different obligate anaerobes. The biofilm occurrence in chronic diabetic ulcer infection is well analysed. Herein, we illustrate the comprehensive pattern of bacterial infection and identify the community composition of chronic wound pathogenic biofilm.