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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.    Electronic supplementary material  The online version of this article (10.1186/s12866-019-1527-2) contains supplementary material, which is available to authorized users.

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