Siderophores form complexes with iron and transport it into the cell during iron-limiting conditions. Siderophores are potent virulence factors which help in establishing severe infection in the host and are known to play a role in regulating quorum sensing in Pseudomonas aeruginosa. Our research aims to quantitatively estimate Siderophore production in Pseudomonas.
Siderophores are extracellular molecules, so total siderophores, along with pyoverdine and pyochelin, are quantified using cell-free supernatant. As pyochelin is not detected in small volume, extraction is performed from a large volume of cell-free supernatant. Recently, LCMS and biosensors have been developed for quantification of pyochelin.
LCMS is a costly setup for small laboratories, whereas biosensors are capable of quantification of pyochelin only. This simple and illustrated protocol uses minimum laboratory reagents and instrumentation and allows siderphores estimation from cell-free supernatant using less time. To begin, take Pseudomonas aeruginosa cultures grown for 24 hours, then set its optical density at 600 nanometers to 0.2 and make suitable dilutions using 0.8%saline.
With a sterile wire loop, streak the bacterial culture on the CAS agar plate. Incubate the CAS agar plate at 30 degrees Celsius for 24 hours. Transfer the culture to a 96-well microtiter plate and measure the optical density at 600 nanometers.
Next, transfer the cultures into centrifuge tubes. Centrifuge the bacterial culture at 4, 650 G for 10 minutes at room temperature. Pipette out 100 microliters of the cell-free supernatant and add it to a well of a 96-well plate.
Then, add 100 microliters of CAS dye to the well. Cover the plate with aluminum foil before incubation. After incubation, take spectrophotometric readings at 630 nanometers and calculate the total quantity of siderophores as percent siderophore unit.
Pipette 100 microliters of the cell-free supernatant into a 96-well microtiter plate, then add 100 microliters of tris-hydrochloric acid to it. Measure the spectrophotometric reading of the solution at 405 nanometers. For pyochelin extraction, first, pipette 100 milliliters of 48 hour grown cultures of Psuedomonas aeruginosa into centrifuge tubes.
Centrifuge the cultures at 4, 650 G for 10 minutes at room temperature. Next, add five milliliters of one molar citric acid to the supernatant. Add 50 milliliters of ethyl acetate to the solution to extract pyochelin.
Filter the organic phase with magnesium sulfate through a syringe filter, then store the organic phase at minus 20 degrees Celsius. Lastly, take spectrophotometric readings at 320 nanometers. The three clinical isolates and the reference strain, PAO1, developed a clear orange halo when grown on CAS agar, indicative of siderophore production.
Significant differences were observed between the total siderophore production of the J3 isolate and PAO1. Spectrophotometric analyses of the pyoverdine extract presented a confirmatory peak at 380 nanometers. While all isolates were positive for pyoverdine production, MR1 and J3 showed lower production relative to the reference strain.
The presence of pyochelin was confirmed by a peak at 320 nanometers. All three clinical isolates produced significantly higher volumes compared to PAO1.
