The levels of production of pyocyanin and elastase were evaluated following a previously reported procedure (43 (link)). Briefly, P. aeruginosa strains were cultured in LP medium or HP medium overnight with shaking at 250 rpm at 37°C. To test the production of pyocyanin, 5 ml of culture supernatants was mixed with 3 ml chloroform and shaken vigorously for 30 min at room temperature. The solvent phase was mixed with 1 ml of 0.2 N HCl and shaken for an additional 30 min. The absorbance was measured at 520 nm and normalized against cell density (optical density at 600 nm [OD600]). To test the production of elastase, 500 μl bacterial-cell-free supernatants was mixed with an equal volume of elastin-Congo red buffer (100 mM Tris, 1 mM CaCl2, pH 7) containing 5μ · ml−1 elastin-Congo red (Sigma) and incubated for 2 h at 37°C with shaking at 200 rpm. The reaction mixture was centrifuged to remove insoluble elastin-Congo red, and the absorbance was measured at 520 nm and normalized against cell density at OD600.
Elastin congo red
Manufactured by Merck Group
Sourced in United States, India
Elastin Congo red is a reagent used for the detection and visualization of elastin fibers in histological samples. It is a staining solution that binds specifically to elastin, allowing for the identification and analysis of the elastic components within tissues.
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50 protocols using elastin congo red
1
Quantifying Pyocyanin and Elastase Production in P. aeruginosa
2
Elastolytic Activity Assay of P. aeruginosa
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LasB elastase activity was measured as per the method described by Adonizio et al. (2008) [70 (link)] to determine the elastolytic activity. A culture of P. aeruginosa was incubated with synthesized AgNPs (sub-MIC—1/2, 1/4 and 1/8 MIC). The treated and non-treated cultures were incubated in 900 µL elastin Congo Red buffer (100 mM Tris, 1mM CaCl2, pH 7.5) holding 20 mg of elastin Congo Red (Sigma, Bengaluru, India). A centrifuge was used to remove the insoluble Congo Red elastin after 3 h at 37 °C in a shaker incubator. Afterwards, the collected supernatant absorbance was recorded at 495 nm. The LB medium without synthesized AgNPs was used as a negative control.
3
Elastolytic Activity Measurement in P. aeruginosa
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A method described by Adonizio et al. (2008) [30 (link)] was carried out for the measurement of elastolytic activity. According to the method, a grown P. aeruginosa culture was treated with crude biosurfactants of L. plantarum (sub-MIC-1.5, 3.5 and 4.5 mg/mL). Later on, the entire treated or control culture supernatant was transferred to 900 µL of elastin Congo red buffer (100 mM Tris, 1 mM CaCl2, pH 7.5) comprising 20 mg of elastin Congo red (Sigma®, Bengaluru, India). After 3 h of incubation at 37 °C in a shaker incubator, the insoluble components (elastin Congo red) were removed by the centrifugation process. An absorption measurement was performed by taking supernatant absorbance reading at 495 nm, and an LB medium with or without the crude biosurfactant of L. plantarum served as a negative control.
4
Enzymatic Activity Assay Protocol
5
Elastase Activity Assay for P. aeruginosa
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P. aeruginosa exoproducts and purified LasB were tested for elastase activity using the elastin-Congo red (Sigma, US) method.13 (link) For exoproducts inhibition assay, protease inhibitors cocktail (PIR) composed of 17 mM AEBSF, 2.5 μM Aprotinin, 1 mM Bestatin, 0.1 mM E64, 0.1 mM Leupeptin in DMSO with 8.5 mM EDTA added (Beyotime Biotechnology, Hangzhou) was used to preincubated with 1mg/mL exoproducts at room temperature for 30 min.
6
Quantification of Pseudomonas Virulence Factors
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Exotoxin A was measured according to the method of Shigematsu et al. [22 (link)] and was determined using a commercially available Human Pseudomonas Exotoxin A (PEA) ELISA Kit (Cusabio Biotech Co., Ltd., Hubei, PR China, product code: CSB-E11252 h), according to the manufacturer's instructions. The data were recorded as ng/mL.
Rhamnolipid was quantified by orcinol method, as previously described with a few modifications [23 (link)]. Briefly, 400 μL supernatant from the bacterial culture was extracted twice using 600 μL diethyl ether. The ether layer was transferred to a fresh tube for evaporation. Residues were dissolved in 150 μL H2O, 100 μL 1.6% orcinol (Sigma), and 750 μL 60% sulphuric acid (H2SO4). After heating for 30 min at 80°C, all the tubes were cooled at room temperature for 30 min and absorbance was recorded at 421 nm. The concentrations of rhamnolipid were calculated by multiplying rhamnose values by a coefficient of 2.5, as previously described [24 (link)].
The elastase activity was measured by the elastin-Congo red assay, as previously described [23 (link)]. Briefly, 100 μL supernatant from 24 h LB cultures was added to tubes containing 10 mg of elastin-Congo red (Sigma) and 900 μL Na2HPO4 (pH 7.0). Tubes were incubated for 4 h at 37°C under shaking conditions and the absorbance was recorded at 495 nm after removing the precipitate by centrifugation.
Rhamnolipid was quantified by orcinol method, as previously described with a few modifications [23 (link)]. Briefly, 400 μL supernatant from the bacterial culture was extracted twice using 600 μL diethyl ether. The ether layer was transferred to a fresh tube for evaporation. Residues were dissolved in 150 μL H2O, 100 μL 1.6% orcinol (Sigma), and 750 μL 60% sulphuric acid (H2SO4). After heating for 30 min at 80°C, all the tubes were cooled at room temperature for 30 min and absorbance was recorded at 421 nm. The concentrations of rhamnolipid were calculated by multiplying rhamnose values by a coefficient of 2.5, as previously described [24 (link)].
The elastase activity was measured by the elastin-Congo red assay, as previously described [23 (link)]. Briefly, 100 μL supernatant from 24 h LB cultures was added to tubes containing 10 mg of elastin-Congo red (Sigma) and 900 μL Na2HPO4 (pH 7.0). Tubes were incubated for 4 h at 37°C under shaking conditions and the absorbance was recorded at 495 nm after removing the precipitate by centrifugation.
7
Characterization of Bacterial Enzyme Activity
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AHLs, p-nitrophenyl acetate, azocasein, elastin-Congo red, propidium iodide (PI), and fluorescein isothiocyanate (FITC)-concanavalin A (ConA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The DNA ligation kit, restriction endonuclease, DNA polymerase PrimeScript RT reagent kit, and TB green Premix ExTaq II were purchased from TaKaRa (Dalian, China) and used according to the manufacturer’s recommendations. The E.Z.N.A. plasmid minikit and the E.Z.N.A. gel extraction kit were purchased from Omega (Norcross, GA, USA). The RNeasy MinElute cleanup kit was purchased from Qiagen (Hilden, Germany). All other chemicals and reagents were of analytical grade and purchased from commercial sources unless otherwise stated.
8
Elastase Activity Quantification Assay
9
Elastase Activity Quantification Protocol
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The isolates were grown for 24 h in LB at 37°C with shaking, and the optical densities at 600 nm were measured (Spectronic Biomate 3; Thermo Electron). The cultures were centrifuged, and the elastase activities in the culture supernatants were determined using a previously published protocol (Rust et al., 1994 (link)). In brief, 10 mg of elastin-Congo red (Sigma–Aldrich) was added to 1 ml of concentrated culture supernatant diluted with 2 ml of the following buffer: 30 mM Tris, pH 7.2, 1 mM CaCl2. The mixtures were incubated for 5 h at 37°C with shaking. The results are expressed as the 495 nm/600 nm ratio.
10
Elastase Activity Assay for P. aeruginosa
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