Screening of natural compounds inhibiting biofilm formation by antibiotic resistant enterobacteria using experimental trials and computational molecular modelling

Abstract

The increasing prevalence of antibiotic resistant Enterobacteria poses a significant challenge in healthcare settings, necessitating the development of alternative strategies, such as inhibition of quorum sensing system to combat bacterial infections. Quorum sensing (QS), a crucial communication mechanism used by bacteria, regulates biofilm formation and virulence, contributing to antibiotic resistance. This study aimed to identify natural compounds capable of inhibiting quorum sensing, biofilm formation and motility of Enterobacterial pathogens using a combined approach of experimental trials and structure-based computational molecular modelling. The following strains, Escherichia coli (ATCC 10536), Klebsiella pneumoniae (ATCC 33495) and Proteus mirabilis (ATCC 33583), were used in this study as test-cultures Eight Bacillus strains were used in this study as sources of natural compounds, which potential quorum-sensing inhibitor activities have been collected, identified and predicted as producers of bioactive compounds in previous studies. Composition of extracellular extracts from these Bacillus strains were analysed using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF). Bioactive compounds were extracted from Bacillus sp. and they were assessed individually for their antibacterial, anti-biofilm, anti-motility and anti-quorum sensing activities against E. coli (ATCC 10536), K. pneumoniae (ATCC 33495) and P. mirabilis (ATCC 33583). From the eight studied Bacillus strains, the extracellular extract from B. subtilis strain 07-2 demonstrated the most effective growth inhibition of all three tested pathogens. The bio-monitor strain Chromobacterium violaceum (ATCC 12472) producing violacein pigment in response to QS signals was used to assess the Bacillus sp. extracts for their anti-quorum sensing (AQS) activities using qualitative and quantitative assay. Qualitative results showed no significant AQS activity except for B. cereus strain MJHN10, which exhibited strong AQS activity and the highest violacein reduction of 45.74%. Furthermore, when the extracts were assessed for anti-motility activities, B. velezensis strain At1 extract showed the highest swimming and swarming motility reduction tested on E. coli (ATCC 10536) and P. mirabilis (ATCC 33583). Particularly, this extract reduced swimming motility of E. coli (ATCC 10536) and P. mirabilis (ATCC 33583) by 43.63% and 33.50%, respectively. Regarding to swarming motility, the reduction of 50.50% and 70.05% were observed for E. coli (ATCC 10536) and P. mirabilis (ATCC 33583), respectively. The results on inhibition of biofilm formation recorded by reduced cell-to-surface attachment reduction showed a good inhibition activity of B. cereus strain LJ4 (84.75%), Bacillus sp strain 7M1 (92.74%) and B. cereus isolates BRL02-31 (86.76%) tested on E. coli (ATCC 10536), K. pneumoniae (ATCC 33495) and P. mirabilis (ATCC 33583) consecutively. Whereas B. cereus strain MJHN10 extract demonstrated a good inhibitory activity against biofilm formation by E. coli (ATCC 10536) and K. vi pneumoniae (ATCC 33495) with reduction of 48.71% to 55.46%. B. cereus strain LJ4 showed a strong anti-biofilm formation activity (46.10% reduction) against P. mirabilis (ATCC 33583). This observation demonstrates that the bioactive compounds produced by different Bacillus strains may act specifically on different test-cultures. The Bacillus sp. extracts were further investigated for identification of bioactive compounds using the Ultra Liquid Performance Chromatography (ULPC). The chemical profiling revealed a range of potentially bioactive compounds such as L-prolyl-L-leucine, 6-Benzamidohexanoate, L-Valyl-L-Leucyl-L-prolylglycine, Oxazinoquinolone, D-Norvaline, Sulfurous acid, Indoline, Sulfamoyl formic acid and Azumamide B. In silico techniques were then used to validate the anti-quorum sensing and anti-motility activities of the identified compounds produced by Bacillus sp. Molecular docking was therefore used to screen the compounds with potential to inhibit quorum sensing and motility protein of K. pneumoniae (SdiA), E. coli (SdiA and MotB) and P. mirabilis (MqsR and Mrp). Molecular docking results showed that 3,5-dihydroxy-4-ethyl-trans-stilbene exhibited the best docking score of -10.01 kcal/mol followed by Bacilysin with a docking score of -9.322 kcal/mol when docked against SdiA protein. Compound 3-amino-3-deoxyglucose showed the highest docking score of -6.722 kcal/mol and -5.606 kcal/mol against of MqsR and MotB, whereas L-Valyl-L-Leucyl-L-prolylglycine showed the best docking score of -10.269 kcal/mol against Mrp. Additionally, molecular dynamics (MD) simulations of the promising protein-ligand complexes were tested to assess the stability of the complexes throughout the simulation process. The simulation results showed good protein stability of the complexes of 6-benzamidohexanoate and 3-amino-3-deoxyglucose bound to Mrp and MqsR proteins, respectively, with an average root-mean-square deviation (RMSD) value of below 3Å. Complexes of SdiA protein with the selected compound proved unstable with RMSD values higher than 3Å. The drug-likeness predictive results indicated that the compounds selected in this study obeyed the Lipinski rule of five. These results implied that Bacillus sp. compounds may be suggested for further studies as promising QS and motility inhibitors which may reduce virulence of drug-resistant enterobacteria.