Fig. 3
In vitro study of antibacterial activity of the hydrogels. a–c Serial dilution method: Inhibitory effect of different concentrations of Gel-H.P.Cur, Gel-H.P and Gel-H.P.DMSO on P. aeruginosa growth in MH broth during 18 h at 37 °C (mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001). (d–g) Disc diffusion method: The zone formation appeared from the growth inhibition around Gel-H.P.Cur (d and f) and imipenem disc (g) but not Gel-H.P (e) on MH agar plate. The zone of growth inhibition was measured (cm.2) and recorded using a ruler and a digital camera, respectively. h Assessment of P. aeruginosa biofilm formation in the presence of the hydrogels, using crystal violet staining standard method. Inhibitory effect of different concentrations (2.5, 5 and 10 mg/mL) of Gel-H.P or Gel-H.P.Cur on biofilm formation of P. aeruginosa was assayed after 18 h incubation in MH broth at 37 °C. *: statistically significant differences between treated and control groups. Ø: sstatistically significant differences between Gel-H.P and Gel-H.P.Cur with the same concentration (2.5, 5 or 10 mg/mL). Ɛ: statistically significant differences between Gel-H.P (2.5 mg/mL) and Gel-H.P (5 mg/mL), Gel-H.P (2.5 mg/mL) and Gel-H.P (10 mg/mL), Gel-H.P (5 mg/mL) and Gel-H.P (10 mg/mL). Ψ: statistically significant differences between Gel-H.P.Cur (2.5 mg/mL) and Gel-H.P.Cur (10 mg/mL), Gel-H.P.Cur (5 mg/mL) and Gel-H.P.Cur (10 mg/mL). (Mean ± SD, **, ƐƐ, ΨΨ P < 0.01, ***, ØØØ, ƐƐƐ, ΨΨΨ P < 0.001). (i) Inhibitory effect of different concentrations of Gel-H.P (10 mg/mL) or Gel-H.P.Cur (2.5, 5 and 10 mg/mL) on pyocyanin production of P. aeruginosa during 24 h incubation in LB broth at 37 °C. *: statistically significant differences between treated and control groups. Ø: statistically significant differences between Gel-H.P (10 mg/mL) and Gel-H.P.Cur (with different concentrations: 2.5, 5 or 10 mg/mL). Ɛ: statistically significant differences between Gel-H.P.Cur (2.5 mg/mL) and Gel-H.P.Cur (5 mg/mL), Gel-H.P.Cur (5 mg/mL) and Gel-H.P.Cur (10 mg/mL). Ψ: statistically significant differences between Gel-H.P.Cur (2.5 mg/mL) and Gel-H.P.Cur (10 mg/mL). (Mean ± SD, Ɛ P < 0.05, ***, ØØØ, ΨΨΨ P < 0.001). (j and k) Effect of hydrogels (Gel-H.P and Gel-H.P.Cur) at a concentration of 10 mg/mL on the expression level of quorum sensing (QS) regulatory genes (f: lasI/lasR, g: rhlI/rhlR) in P. aeruginosa during 12 h incubation. Both hydrogels significantly reduced the expression of QS genes compared to the control (untreated) group (mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001)