The last decade has witnessed a massive increase in the rate of mortalities caused by multidrug-resistant Pseudomonas aeruginosa. Therefore, developing new strategies to control virulence factors and pathogenicity has received much attention. One of these strategies is quorum sensing inhibition (QSI) which was developed to control Pseudomonas infection. This study aims to validate the effect of one of the most used β-lactam antibiotics; cefoperazone (CFP) and its metallic-derivatives on quorum sensing (QS) and virulence factors of P. aeruginosa. Assessment of quorum sensing inhibitory activity of CFP, cefoperazone Iron complex (CFPF) and cefoperazone Cobalt complex (CFPC) was performed by using reporter strain Chromobacterium violaceum ATCC 12472. Minimal inhibitory concentration (MIC) was carried out by the microbroth dilution method. The influence of sub-MICs (1/4 and 1/2 MICs) of CFP, CFPF and CFPC on virulence factors of P. aeruginosa was evaluated. Data was confirmed on the molecular level by RT-PCR. Also, molecular docking analysis was conducted to figure out the possible mechanisms of QSI. CFP, CFPF, and CFPC inhibited violacein pigment production of C. violaceum ATCC 12472. Sub-MICs of CFP (128- 256 μg/mL), and significantly low concentrations of CFPC (0.5- 16 μg/mL) and CFPF (0.5- 64 μg/mL) reduced the production of QS related virulence factors such as pyocyanin, protease, hemolysin and eliminated biofilm assembly by P. aeruginosa standard strains PAO1 and PA14, and P. aeruginosa clinical isolates Ps1, Ps2, and Ps3, without affecting bacterial viability. In addition, CFP, CFPF, and CFPC significantly reduced the expression of lasI and rhlI genes. The molecular docking analysis elucidated that the QS inhibitory effect was possibly caused by the interaction with QS receptors. Both CFPF and CFPC interacted strongly with LasI, LasR and PqsR receptors with a much high ICM scores compared to CFP that could be the cause of elimination of natural ligand binding. Therefore, CFPC and CFPF are potent inhibitors of quorum sensing signaling and virulence factors of P. aeruginosa.

Multiple drug resistance poses a significant threat to public health worldwide, with a substantial increase in morbidity and mortality rates. Consequently, searching for novel strategies to control microbial pathogenicity is necessary. With the aid of auto-inducers (AIs), quorum sensing (QS) regulates bacterial virulence factors through cell-to-cell signaling networks. AIs are small signaling molecules produced during the stationary phase. When bacterial cultures reach a certain level of growth, these molecules regulate the expression of the bound genes by acting as mirrors that reflect the inoculum density.Gram-positive bacteria use the peptide derivatives of these signaling molecules, whereas Gram-negative bacteria use the fatty acid derivatives, and the majority of bacteria can use both types to modulate the expression of the target gene. Numerous natural and synthetic QS inhibitors (QSIs) have been developed to reduce microbial pathogenesis. Applications of QSI are vital to human health, as well as fisheries and aquaculture, agriculture, and water treatment.

The number of deaths caused by multidrug-resistant Pseudomonas aeruginosa has risen in the recent decade. The development of quorum sensing inhibition (QSI) is a promising approach for controlling Pseudomonas infection. Therefore, this study mainly aimed to investigate how a plant-source material inhibits QSI to produce an antipathogenic effect for fighting microbial infections. The QSI effect of Trigonella stellata was assessed by using Chromobacterium violaceum ATCC 12472 reporter strain. Trigonella stellata exhibited high QSI activity, and an ethanolic extract of T. stellata was prepared for phytochemical isolation of the most active QSI compound. Nine pure compounds were isolated and identified as kaempferitrin (1), soyasaponin I (2), β-sitosterol-3-O-glucoside (3), dihydromelilotoside (4), astrasikokioside I (5), methyl dihydromelilotoside (6), (3R, 4S)-4, 2′, 4′-trihydroxy-7-methoxy-4′-O-β-d-glucopyranosylisoflavan (7), (3S, 4R)-4, 2′, 4′-trihydroxy-7-methoxyisoflavan (8, TMF), and (+)-d-pinitol (9). These compounds were screened against C. violaceum ATCC 12472, and TMF exhibited a potent QSI. The effect of TMF at sub-minimum inhibitory concentrations (MICs) was assessed against P. aeruginosa virulence factors, including biofilm, pyocyanin formation protease and hemolysin activity. TMF induced significant elimination of QS-associated virulence behavior. In addition, TMF at sub-MICs significantly reduced the relative expression of lasI, lasR, rhlI, and rhlR compared with that in untreated cells. Furthermore, molecular docking was performed to predict structural basis of the QSI activity of TMF. The study demonstrated the importance of T. stellata as a signal modulator and inhibitor of P. aeruginosa pathogenesis.

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