In vitro and in silico assessment of anti-biofilm and anti-quorum sensing properties of 2,4-Di-tert butylphenol against Acinetobacter baumannii

Introduction. Acinetobacter baumannii is a nosocomial pathogen with a high potential to cause food-borne infections. It is designated as a critical pathogen by the World Health Organization due to its multi-drug resistance and mortalities reported. Biofilm governs major virulence factors, which promotes drug resistance in A. baumannii . Thus, a compound with minimum selection pressure on the pathogen can be helpful to breach biofilm-related virulence. Hypothesis/Gap Statement. To identify anti-biofilm and anti-virulent metabolites from extracts of wild Mangifera indica (mango) brine pickle bacteria that diminishes pathogenesis and resistance of A. baumannii . Aim. This study reports anti-biofilm and anti-quorum sensing (QS) efficacy of secondary metabolites from bacterial isolates of fermented food origin. Method. Cell-free supernatants (CFS) of 13 bacterial isolates from fermented mango brine pickles were screened for their efficiency in inhibiting biofilm formation and GC-MS was used to identify its metabolites. Anti-biofilm metabolite was tested on early and mature biofilms, pellicle formation, extra polymeric substances (EPS), cellular adherence, motility and resistance of A. baumannii . Gene expression and in silico studies were also carried out to validate the compounds efficacy. Results. CFS of TMP6b identified as Bacillus vallismortis, inhibited biofilm production (83.02 %). Of these, major compound was identified as 2,4-Di- tert -butyl phenol (2,4-DBP). At sub-lethal concentrations, 2,4-DBP disrupted both early and mature biofilm formation. Treatment with 2,4-DBP destructed in situ biofilm formed on glass and plastic. In addition, key virulence traits like pellicle (77.5 %), surfactant (95.3 %), EPS production (3-fold) and cell adherence (65.55 %) reduced significantly. A. baumannii cells treated with 2,4-DBP showed enhanced sensitivity towards antibiotics, oxide radicals and blood cells. Expression of biofilm-concomitant virulence genes like csuA/B , pgaC , pgaA , bap , bfmR , katE and ompA along with QS genes abaI, abaR significantly decreased. The in silico studies further validated the higher binding affinity of 2,4-DBP to the AbaR protein than the cognate ligand molecule. Conclusion. To our knowledge, this is the first report to demonstrate 2,4- DBP has anti-pathogenic potential alone and with antibiotics by in vitro, and in silico studies against A. baumannii . It also indicates its potential use in therapeutics and bio-preservatives.