Killing curves of each of antibiotic alone and in presence of α-MSH against clinical MRSA isolate.

<p>(a) Gentamicin, (b) Tetracycline, (c) Ciprofloxacin, (d) Oxacillin, and (e) Rifampicin. Symbols; antibiotic alone (diamond) and antibiotic+α-MSH (8 µg/ml) (square). Experiments were repeated on three independent days. p value ≤0.05 (when multiple comparisons were done among % survival data-sets of different concentrations of same antibiotic with and without α-MSH).</p

Determining bactericidal concentrations of each tested antibacterial agent.

<p>(a) Killing curves showing bactericidal activity of five different tested antibiotics against clinical MRSA isolate. Concentrations of each antibiotic were selected from 0 to 2048 µg/ml. Symbols; Oxacillin (red diamond), Gentamicin (green square), Rifampicin (yellow triangle), Ciprofloxacin (pink square) and Tetracycline (orange circle). (b) Bactericidal activity of α-MSH (2–160 µg/ml) against clinical MRSA isolate. The killing assay was done in triplicate and repeated on three different occasions. *p value ≤0.001, **p value ≤0.01, ***p value ≤0.05.</p

Impact of sub-lethal doses of α-MSH on DNA, RNA and protein synthesis of <i>S. aureus</i> ATCC 29213.

<p>(a) % of radioactivity of thymidine in untreated control (red), treated with 2 µg/ml α-MSH (green), 10 µg/ml α-MSH (purple) and 2 µg/ml of ciprofloxacin (blue); (b) % of radioactivity of uridine in control (red), treated with 2 µg/ml α-MSH (green), 10 µg/ml α-MSH (purple) and 2 µg/ml of rifampicin (blue); (c) % of radioactivity of leucine in control (red), treated with 2 µg/ml α-MSH (green), 10 µg/ml α-MSH (purple) and 2 µg/ml of tetracycline (blue); (d) killing kinetics of 2 µg/ml of α-MSH (diamond), and 10 µg/ml of α-MSH (square) against ∼10⁸ CFU/ml of <i>S. aureus</i> ATCC 29213. Experiments were done in duplicate and repeated on three independent days. *p value ≤0.001, **p value ≤0.01, ***p value ≤0.05.</p

Minimum bactericidal concentration (MBC₅₀ and MBC₉₀) values of selected antimicrobial agents when used alone and in the presence of 8 µg/ml of α-MSH against clinical MRSA strain.

<p>Note: Fold reduction = (MBC₅₀ of antibiotic alone/MBC₅₀ in presence of α-MSH).</p><p>NA*means MBC₉₀ not achieved.</p

Minimum Inhibitory concentration (MIC) values of selected antibiotics against <i>S. aureus</i> strains as determined by broth microdilution assay following CLSI guidelines [25].

<p>Note: (S) Strain susceptible to tested antibiotic.</p><p>(R) Strain resistant to tested antibiotic.</p

Membrane permeabilization of <i>S</i>. <i>aureus</i> by curcumin I, using propidium iodide (PI) through flow cytometry and spectrofluorimetry technique.

<p>A total of 10,000 cells were acquired for each flow cytometry analysis. (a) Histograms of logarithmic <i>S</i>. <i>aureus</i> ATCC 29213 cells labeled with PI and treated with different concentrations of curcumin I (0 μM (control), 25 μM, 50 μM & 100 μM), and positive controls (HNP-1 and nisin) for 2h, (b) percentage of PI uptake by <i>S</i>. <i>aureus</i> as quantified from flow cytometry data and (c) fluorescence intensity of PI in <i>S</i>. <i>aureus</i> cells exposed to curcumin I using spectrofluorimetry. These data represent mean (±SD) of three independent experiments (*** p ≤ 0.05, ** p ≤ 0.01,* p < 0.001).</p

Scanning electron microscopic images of <i>S</i>. <i>aureus</i> cells after 2h exposure to curcumin I.

<p>(a) 0 μM, (b) 25 μM, (c) 50 μM, & (d) 100 μM curcumin I. <i>S</i>. <i>aureus</i> cells exposed to lower concentration of curcumin I show morphological changes including surface roughness, depression, and dent formation, whereas incubation at the higher dose caused leakage of cell material and bursting of cells which were absent in untreated control cells. Similar appearances were found in separate experiments on different days.</p

Fluorescence microscopy assay for viability of <i>S</i>. <i>aureus</i> against curcumin I.

<p>Bacteria were exposed to 25 μM, 50 μM & 100 μM curcumin I and saline (negative control) for 2h at 37°C. Subsequently, bacterial sets were washed in saline and subjected to LIVE/DEAD <i>Bac</i>Light Bacterial Viability Assay. (a) Microscopic images of representative curcumin I treated and untreated bacterial cells, bacteria in red are indicative of dead or membrane damaged cells, whereas, green indicates live/healthy bacteria, (b) relative percentage of live and dead cells in sample exposed to 25 μM, 50 μM & 100 μM curcumin I and saline (control) as quantified from different microscopic images (*** p ≤ 0.05).</p

Time kill kinetics of curcumin I against 10⁴ CFU/ml of four different bacterial cells.

<p>Killing bars for (a) <i>S</i>. <i>aureus</i> ATCC 29213, (b) <i>E</i>. <i>faecalis</i> ATCC 29212, (c) <i>E</i>. <i>coli</i> ATCC 25922, and (d) <i>P</i>. <i>aeruginosa</i> ATCC 25619 by 25 μM, 50 μM & 100 μM curcumin I after 30, 60 & 120 min of incubation. Symbols: Gray, vertical striped and white represent 30, 60 & 120 min exposure of curcumin I, respectively. These data represent mean (±SD) of three independent experiments (*p ≤ 0.001, **p ≤ 0.01, ***p ≤ 0.05).</p

Membrane permeabilization of <i>S</i>. <i>aureus</i> by curcumin I using calcein-AM through flow cytometry.

<p>Logarithmic <i>S</i>. <i>aureus</i> ATCC 29213 was labeled with calcein and analyzed for membrane permeabilization after incubation with different concentrations of curcumin I. A total of 10,000 cells were acquired for each flow cytometry analysis. (a) Histograms showing calcein leakage, on treatment with different concentration of curcumin I (0 μM (control), 25 μM, 50 μM & 100 μM), (b) percentage of calcein leakage from <i>S</i>. <i>aureus</i> cells on treatment with different concentration of curcumin I for 2h as quantified from different days of flow cytometry data. These data represents mean (±SD) of three independent experiments (*p ≤ 0.001).</p