Figure 2. Effect of PGPE on various traits of P. aeruginosa, in vitro.

‘Control’ in this figure is the vehicle control (0.5%v/v DMSO), which did not exert any effect on growth and pigment production of P. aeruginosa. ( A) Effect of PGPE on growth and QS-regulated pigment production in P. aeruginosa: Bacterial growth was measured as OD ₇₆₄; OD of pyoverdine was measured at 405 nm, and Pyoverdine Unit was calculated as the ratio OD ₄₀₅/OD ₇₆₄ (an indication of pyoverdine production per unit of growth), Pyocyanin Unit was calculated as the ratio OD ₅₂₀/OD ₇₆₄ (an indication of pyocyanin production per unit of growth); Catechin (50 µg/ml) inhibited pyoverdine and pyocyanin production by 17.13±0.06% and 23.65±0.04% respectively, without affecting the bacterial growth. ( B) PGPE acts as a signal-response inhibitor against P. aeruginosa. ( C) P. aeruginosa challenged with antibiotics following pre-treatment with PGPE. ( D) PGPE modulates susceptibility of P. aeruginosa to lysis by human serum: ‘Control’ in serum-dependent lysis assay was PGPE-unexposed cells of P. aeruginosa incubated with human serum. ( E) Effect of PGPE on P. aeruginosa biofilm formation, eradication, and viability. Crystal violet assay was performed to measure biofilm formation, and biofilm eradication, followed by the measurement of OD at 580 nm; Cell viability in biofilm was estimated through MTT assay, wherein OD was measured at 560 nm. ( F) PGPE-treatment reduces the virulence of P. aeruginosa towards C. elegans: Catechin (50 μg/ml) and gentamicin (0.1 μg/ml) employed as a positive controls conferred 100% and 80% protection on worm population respectively. Pre-treatment of bacteria with PGPE at concentrations (2.5, 5, 10, 25, and 100 μg/ml) other than shown in figure allowed 73.75, 75, 77.5, 77.5, and 75% worm survival respectively; DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and PGPE at tested concentrations showed no toxicity towards the worm. ( G) Effect of PGPE on P. aeruginosa growth, Pyoverdine Unit, and Pyocyanin Unit remained unaltered after repeated exposure to PGPE (500 μg/ml). *p<0.05 , **p<0.01 , ***p<0.001. AS, antibiotic susceptibility; QS, quorum sensing; PGPE, Punica granatum peel extract.