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Efflux Pump Inhibition-Based Screening and Anti-Infective Evaluation of Punica granatum Against Bacterial Pathogens
Samreen1, Iqbal Ahmad2, Shirjeel Ahmad Siddiqui1
1Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, 202002, Uttar Pradesh, India.
Abstract:
Drug efflux pumps contribute to bacterial multidrug resistance (MDR), reducing antibiotic effectiveness and causing treatment failures. Besides their role in MDR, efflux pumps also assist in the transportation of quorum sensing (QS) signal molecules and increased the tolerance of biofilms. Recently, the search for efflux pump inhibitors from natural sources, including anti-infective plants, has gained attention as a potential therapy against drug-resistant bacteria. In this study, 19 traditional Indian medicinal plants were screened for their efflux pump inhibitory activity against Escherichia coli TGI. The promising extract, i.e., Punica granatum was subsequently fractioned in the solvents of increasing polarity. Among them, at sub-MIC active EPI fraction was PGEF (P. granatum ethyl acetate fraction), further investigated for anti-infective potential against Chromobacterium violaceum 12,472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MTCC 97. PGEF was also evaluated for in vivo efficacy in Caenorhabditis elegans model. Major phytocompounds were analyzed by mass spectroscopic techniques. At respective Sub-MIC, PGEF reduced violacein production by 71.14% in C. violaceum 12,472. Moreover, PGEF inhibited pyocyanin (64.72%), pyoverdine (48.17%), protease (51.35%), and swarming motility (44.82%) of P. aeruginosa PAO1. Furthermore, PGEF reduced the production of prodigiosin and exoprotease by 64.73% and 61.80%, respectively. Similarly, at sub-MIC, PGEF inhibited (≥ 50%) biofilm development in all test pathogens. The key phytocompounds detected in active fraction include 5-hydroxymethylfurfural, trans-p-coumaric acid 4- glucoside, (-)-Epicatechin 3'-O-glucuronide, and ellagic acid. Interestingly, PGEF also demonstrated anti-infective efficacy against the PAO1-infected C. elegans test model and highlighting its therapeutic potential as an anti-infective agent to combat drug-resistant problems.
Insights
Pomegranate ethyl acetate fraction (PGEF) effectively inhibits bacterial efflux pumps and virulence factors in multiple pathogens. This natural compound shows promise as an anti-infective agent against drug-resistant bacteria and in vivo models.
Area of Science:
- Microbiology
- Pharmacology
- Natural Products Chemistry
Background:
- Bacterial multidrug resistance (MDR) is a growing threat, driven by efflux pumps that reduce antibiotic efficacy.
- Efflux pumps also play roles in bacterial communication (quorum sensing) and biofilm formation, contributing to persistent infections.
- Natural products, particularly from medicinal plants, are being explored as novel efflux pump inhibitors (EPIs).
Purpose of the Study:
- To screen traditional Indian medicinal plants for efflux pump inhibitory activity.
- To investigate the anti-infective and anti-virulence properties of a promising plant extract fraction.
- To evaluate the in vivo efficacy of the active fraction against bacterial infections.
Main Methods:
- Screening of 19 Indian medicinal plants against *Escherichia coli* TGI for efflux pump inhibition.
- Fractionation of *Punica granatum* extract and identification of the active ethyl acetate fraction (PGEF).
- Assessing PGEF's effect on virulence factors (violacein, pyocyanin, pyoverdine, protease, prodigiosin, exoprotease) and biofilm formation in *Chromobacterium violaceum*, *Pseudomonas aeruginosa*, and *Serratia marcescens*.
- Evaluating PGEF's efficacy in a *Caenorhabditis elegans* infection model.
Main Results:
- PGEF significantly reduced violacein production in *C. violaceum* (71.14%) and prodigiosin in *S. marcescens* (64.73%).
- PGEF inhibited key virulence factors of *P. aeruginosa* including pyocyanin (64.72%), pyoverdine (48.17%), and protease (51.35%), as well as swarming motility (44.82%).
- PGEF demonstrated broad-spectrum inhibition of biofilm development (≥50%) across tested pathogens and showed anti-infective efficacy in a *C. elegans* model.
Conclusions:
- The *Punica granatum* ethyl acetate fraction (PGEF) exhibits potent efflux pump inhibitory and anti-virulence activities.
- PGEF contains bioactive phytocompounds like ellagic acid and (-)-Epicatechin 3'-O-glucuronide, contributing to its efficacy.
- PGEF represents a promising natural therapeutic agent for combating bacterial multidrug resistance and infections.
