Antimicrobial efficacy of Punica granatum Lythraceae peel extract against pathogens belonging to the ESKAPE group

Abstract

The improper use and abuse of antibiotics have led to an increase in multidrug-resistant (MDR) bacteria resulting in a failure of standard antibiotic therapies. To date, this phenomenon represents a leading public health threat of the 21st century which requires alternative strategies to fight infections such as the identification of new molecules active against MDR strains. In the last 20 years, natural extracts with biological activities attracted scientific interest. Following the One Health Approach, natural by-products represent a sustainable and promising alternative solution. Consistently, the aim of the present study was to evaluate the antimicrobial activity of hydro-alcoholic pomegranate peel extract (PPE) against MDR microorganisms belonging to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. "ESKAPE" group pathogens. Through semiquantitative and quantitative methods, the PPE showed effective antimicrobial activity against Gram-positive and Gram-negative MDR bacteria. The kinetics of bactericidal action of PPE highlighted that microbial death was achieved in a time- and dose-dependent manner. High concentrations of PPE exhibited antioxidant activity, providing a protective effect on cellular systems and red blood cell membranes. Finally, we report, for the first time, a significant intracellular antibacterial property of PPE as highlighted by its bactericidal action against the staphylococcal reference strain and its bacteriostatic effect against clinical resistant strain in the HeLa cell line. In conclusion, due to its characterized content of polyphenolic compounds and antioxidant activity strength, the PPE could be considered as a therapeutic agent alone or in conjunction with standard antibiotics against challenging infections caused by ESKAPE pathogens.

Keywords: ESKAPE pathogens; antimicrobial activity; in vitro infection model; intracellular antimicrobial action; multidrug-resistant bacteria; natural by-product; pomegranate peel extract.

Figure 1

In vitro antibacterial activity of PPE by the agar well diffusion method. Results of agar diffusion method of PPE against S. aureus ATCC 6538 and MRSA strains (A), P. aeruginosa ATCC 27853 and MDR strains (B), and A. baumannii MDR strains (C). Graphical representation of the results; the MDIZ in mm is reported as the mean of values obtained from assays in triplicate ± SD. Statistical significance was examined using Student’s t-test. Asterisks indicate the statistical significance with respect to the positive control (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05).

Figure 2

Time-killing assay of PPE on ESKAPE group pathogens. Time-killing curve of PPE at different concentrations against MRSA strain 2 and S. aureus ATCC 6538 (A,B), P. aeruginosa MDR strain 1 and P. aeruginosa ATCC 27853 (C,D), and A. baumannii MDR strain 1 (E). NT indicates untreated control. Statistical significance was examined using Student’s t-test. Data represent mean values of three independent experiments, and asterisks denote values statistically significant (*** p < 0.001; ** p < 0.01; * p < 0.05).

Figure 3

Evaluation of cytotoxicity activity of PPE on HeLa and HEK-293 cells. Effects of 50, 100, and 150 μg/μL of extract for an exposure time of 24 and 30 h on HeLa (A) and HEK-293 (B) cell lines. The data are expressed as the percentage of absorbance at 570 nm relative to the control. The data are expressed as the mean ± SD of triplicate experiments. NC, negative control. Statistical significance was examined using Student’s t-test, and asterisks denote values statistically significant (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05).

Figure 4

Effect of PPE on intracellular ROS production in the HeLa cell line. Intracellular ROS production induced by 50, 100, and 150 μg/μL of PPE after 24 h of exposure in HeLa cells. The data are expressed as a percent relative to negative control (NC) ± SD of triplicate experiments. H₂O₂ was used as a positive control. Statistical significance was examined using Student’s t-test, and asterisks denote values statistically significant (*p < 0.05).

Figure 5

Evaluation of anti-hemolytic activity of PPE. The PPE showed a protective effect against the hemolytic activity of H₂O₂ with a percentage of inhibition hemolysis of 104.4% ± 2.5 at 50 μg/μL, 103.2% ± 2.3 at 100 μg/μL, and 85% ± 5.1 with 150 μg/μL. The data are compared to H₂O₂ used as a positive control and to the PBS(1X) used as a negative control. Absorbance was read at 450 nm. The data are expressed as the mean ± SD of triplicate experiments, and statistical significance was examined using Student’s t-test. Asterisks indicate statistical significance (**** p < 0.0001).

Figure 6

Antibacterial activity of PPE in HeLa cells. Survival assay of intracellular S. aureus ATCC 6538 and MRSA strain 2 in HeLa human cells after PPE treatment. HeLa cells (10⁵ cells/well) were infected with staphylococci: (A) S. aureus ATCC and (B) MRSA strain 2 at a multiplicity of infection of 100 treated with GEN and then with PPE at different concentrations or not treated (NT) and re-incubated in Dulbecco’s modified Eagle’s minimal essential medium for the indicated times. After saponin lysis, the numbers of intracellular CFU were recorded. Values are means from at least three independent experiments. The results are shown as the relative number of CFU per well ±SD, and statistical significance was examined using Student’s t-test. Asterisks indicate statistical significance (*** p < 0.001; ** p < 0.01; * p < 0.05).