Unlocking the Potential of Pseudomonas aeruginosa QS intermediates as Antimicrobial Synergists Against Three Multidrug-Resistant Enteric Bacteria

Saba Kiran¹Alaa Alhegaili²Noura Al-Dayan²Zubera Naseem¹WAQAR SIDDIQUE¹Itra Ayoub¹Shoaib Iqbal¹Sobia Jabeen¹Fazal E Habib¹Saman Taj¹Ashfaq Hussain¹Rizwan Bashir¹Yasra Sarwar¹Aamir Ali¹Waqar Rauf^1*Georg Jander³Mazhar Iqbal^1*

• ¹National Institute for Biotechnology and Genetic Engineering (Pakistan), Faisalabad, Pakistan
• ²Prince Sattam bin Abdulaziz University, Al Kharj, Saudi Arabia
• ³Cornell University, Ithaca, United States

Antimicrobial resistance in the Enterobacteriaceae poses a global health concern by jeopardizing the effectiveness of antibiotics. The scarcity of new antibiotics has prompted increased interest in natural bioactive secondary metabolites derived from microbial sources and their co-action with existing antimicrobials. In this study, we investigated the bioactivity of crude extracts from Pseudomonas aeruginosa MC9 (accession no. MK530186) and evaluated the in-vitro antimicrobial-augmenting efficacy of its quorum sensing (QS) effectors against multidrug-resistant strains of Salmonella Typhi (S. Typhi-29C), Salmonella Typhimurium (S. Typhimurium-W20), and Escherichia coli (E. coli SS1). The MC9 extract exhibited inhibition zones of 26±1.5, 24±1, and 19±1.5 mm, with minimum inhibitory concentrations of 16, 32, and 256 mg/mL against S. Typhimurium-W20, S. Typhi-29C, and E. coli-E92, respectively. Mass spectrometric analysis revealed the presence of 5-methyl-1(5H)-phenazinone (pyocyanin), rhamnolipids, 4-hydroxy-2heptylquinoline (PQS), and 2-heptyl-3-hydroxy-4(1H)-quinolone (HHQ). Notably, pyocyanin and rhamnolipids exhibited significant antimicrobial activities across a concentration range from 0.04 mg/mL to 50 mg/mL, whereas HHQ and PQS showed no anti-Enterobacteriaceae activity up to 5 mg/mL. Combination assays demonstrated that all four QS effectors potentiate the activity of conventional antibiotics. Pyocyanin showed the highest synergistic effect, with a 300% increase in the inhibition zone when combined with sulfamethoxazole/trimethoprim (23.75/1.25 µg/mL) against S. Typhimurium-W20. Rhamnolipids exhibited a 106% increase in synergy with ceftriaxone (30 µg/mL) against E. coli-SS1, whereas HHQ (10 µg/mL) showed a 257% increase with ampicillin (10 µg/mL) against E. coli-SS1. PQS displayed the highest synergistic effect of 109% with amoxicillin clavulanic acid (30 µg/mL) against E. coli-SS1. Moreover, growth curve analysis revealed a dose-dependent reduction in bacterial growth with sub-inhibitory concentrations of antimicrobials, particularly for the combinations exhibiting the highest synergy across the QS effectors. These findings demonstrate the potential of the QS effectors in reducing the required dosage of antibiotics against resistant Enterobacteriaceae strains and highlight the need to develop a comprehensive understanding of the underlying mechanisms for the co-action of antimicrobials and QS mediators.