Molecular insights into sub-inhibitory ceftriaxone-mediated modulation of Pseudomonas aeruginosa biofilm architecture, quorum sensing networks, and antibiotic-target docking interactions.

BACKGROUND

Pseudomonas aeruginosa biofilm polymer matrix formation contributes to antibiotic tolerance. The antibiofilm effects of sub-minimum inhibitory concentrations (MICs) of ceftriaxone (CTX), the molecular mechanisms by which these sub-MICs modulate biofilm polymer production and quorum sensing (QS), and the binding interactions of CTX with key biofilm regulatory proteins (LasR and RhlR QS receptors) have not been previously investigated.

OBJECTIVES

To determine the role of sub-MIC CTX in regulating biofilm polymer matrix formation, bacterial adhesion, QS gene expression (rhlR and lasR), and to perform molecular docking analysis of CTX interactions with LasR and RhlR QS receptor proteins and biofilm EPS polymer-associated targets.

MATERIAL AND

METHODS

MICs and biofilm formation were determined. The effects of CTX sub-MICs on biofilm formation, adhesion to mouse bladder epithelial cells (BECs), and QS gene expression (rhlR and lasR, by qRT-PCR) were assessed. In silico molecular docking of CTX against the ligand-binding domains of LasR (PDB: 2UV0) and RhlR (PDB: 3T5K) was performed using AutoDock Vina. Interaction fingerprinting with biofilm EPS polymer-associated enzymes (AlgD and PelB) was also performed.

RESULTS

CTX sub-MICs regulated biofilm formation in an isolate-dependent manner, reduced P. aeruginosa adhesion to mouse BECs, and downregulated the rhlR and lasR genes in a concentration-dependent manner. Molecular docking revealed that CTX binds favorably within the ligand-binding pockets of LasR (-8.3 kcal/mol) and RhlR (-7.1 kcal/mol) via hydrogen bonding and hydrophobic interactions, suggesting competitive interference with QS autoinducer binding. CTX also exhibited affinity for AlgD (-7.6 kcal/mol), a key enzyme in alginate polymer biosynthesis.

CONCLUSIONS

CTX sub-MICs modulate biofilm EPS polymer matrix formation and epithelial adhesion by downregulating QS regulatory genes. lasR was more responsive to CTX sub-MIC stress than rhlR. Molecular docking supports a direct molecular interaction mechanism through which CTX may interfere with QS receptor signaling and alginate polymer biosynthesis, providing a structural basis for its antibiofilm activity at sub-inhibitory concentrations.