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.

Molecular evaluation of quorum quenching potential of vanillic acid against Yersinia enterocolitica through transcriptomic and in silico analysis.

Introduction. Yersinia enterocolitica is one of the leading food-borne entero-pathogens causing various illnesses ranging from gastroenteritis to systemic infections. Quorum sensing (QS) is one of the prime mechanisms that control the virulence in Y. enterocolitica.Hypothesis/Gap Statement. Vanillic acid inhibits the quorum sensing and other virulence factors related to Y. enterocolitica. It has been evaluated by transcriptomic and Insilico analysis. Therefore, it can be a prospective agent to develop a therapeutic combination against Y. enterocolitica.Aim. The present study is focused on screening natural anti-quorum-sensing agents against Y. enterocolitica. The effect of selected active principle on various virulence factors was evaluated.Methodology. In total, 12 phytochemicals were screened by swarming assay. MATH assay, EPS and surfactant production assay, SEM analysis, antibiotic and blood sensitivity assay were performed to demonstrate the anti-virulence activity. Further, RNA sequencing and molecular docking studies were carried out to substantiate the anti-QS activity.Results. Vanillic acid (VA) has exhibited significant motility inhibition, thus indicating the anti-QS activity with MQIC of 400 µg ml-1 without altering the cell viability. It has also inhibited the violacein production in Chromobacterium violaceum ATCC 12472, which further confirms the anti-QS activity. VA has inhibited 16 % of cell-surface hydrophobicity (CSH), 52 % of EPS production and 60 % of surfactant production. Moreover, it has increased the sensitivity of Y. enterocolitica towards antibiotics. It has also made the cells upto 91 % more vulnerable towards human immune cells. The transcriptomic analysis by RNA sequencing revealed the down regulation of genes related to motility, virulence, chemotaxis, siderophores and drug resistance. VA treatment has also positively regulated the expression of several stress response genes. In furtherance, the anti-QS potential of VA has been validated with QS regulatory protein YenR by in silico molecular simulation and docking study.Conclusion. The present study is possibly the first attempt to demonstrate the anti-QS and anti-pathogenic potential of VA against Y. enterocolitica by transcriptomic and in silico analysis. It also deciphers that VA can be a promising lead to develop biopreservative and therapeutic regimens to treat Y. enterocolitica infections.

Anti quorum sensing and anti virulence activity of tannic acid and it's potential to breach resistance in Salmonella enterica Typhi / Paratyphi A clinical isolates.

Salmonella enterica Typhi and Paratyphi A are food borne pathogens causing typhoid, which is one of the most important food borne disease in the developing world. S. Typhi and S. Paratyphi A are of much concern as multi drug resistance has been on the rise. The current study is aimed to screen phytochemicals for anti quorum sensing (QS) activity against S. Typhi and S. Paratyphi A. Upon screening with swarming assay, tannic acid (TA) showed highest anti-QS activity with minimal concentration of 400µg/ml. The anti-QS activity of TA was confirmed with C. violaceum ATCC 12,472. TA showed 38-43% and 35-50% of inhibition in cell surface hydrophobicity and EPS production respectively. Through FTIR analysis, it has been observed that EPS of treated cells has a considerable change in protein and peptide. TA has also exhibited drastic reduction in the surfactant production as high as 85-90%. Blood sensitivity and antibiotic sensitivity assay revealed that TA significantly sensitizes the S. Typhi and S. Paratyphi A cells to immune components in human blood and antibiotics. It has reduced the resistance of S. Typhi and S. Paratyphi A cells against amikacin, ampicillin, ciprofloxacin, azithromycin, chloramphenicol and gentamycin, thus revitalized the usage of these antibiotics against drug resistant S. Typhi and S. Paratyphi A infections. The consistency of anti-QS potential of TA was further evaluated and established with another eight clinical isolates of S. Typhi and S. Paratyphi A. Thus TA has been proved as a promising anti QS agent that can be developed as a therapeutic combination against S. Typhi and S. Paratyphi A.