Quantification of Violacein in Chromobacterium violaceum and Its Inhibition by Bioactive Compounds

Our research aims to identify compounds that inhibit quorum sensing, which regulates key phenotypes. This protocol screens molecules that reduce violacein production without affecting bacterial growth, suggesting quorum sensing interference. Recent studies have identified new bioactive compounds that inhibit quorum sensing without affecting bacterial growth, reinforcing their potential as anti-virulence agents against antibiotic resistant bacteria.

Main challenges include the specificity of quorum sensing inhibition, since compounds may affect the growth or the traits. In this protocol called deference and solubility impact balancing quantification a require careful handling. We established a protocol used in bioactive compounds at sub-inhibitory concentrations that do not affect the bacterial growth.

This is a key premise for starting quorum sensing signaling different from antimicrobial research. We will focus on identifying new quorum state inhibitors and elucidated inhibition mechanisms of communication, especially in bacteria relevant to food and health. To begin, grow a five milliliter culture of chromobacterium violaceum ATCC 12472 in Luria Bertani or LB broth.

Incubate the culture at 30 degrees Celsius in a shaking incubator set at 220 revolutions per minute for 24 hours. Measure the optical density of the overnight culture and adjust it to approximately one times 10 to the power of eight colony forming units per milliliter. Using the LB broth.

For compound preparation, weigh the bioactive compound and dilute it in either pure DMSO or a one to one mixture of DMSO and water depending on the solubility. Take a flat bottom 96 well plate and add LB broth along with the bioactive compound according to a serial dilution scheme. To calculate the required volume of stock solution for the desired concentration in each well apply the formula.

In the first column of each cereal dilution set. Add 196 microliters of Luria Bertani broth to columns three, five, and seven. Then in the subsequent columns used for the dilution, add 100 microliters of Luria Bertani broth to columns four, six, and eight.

To initiate the serial dilution, add four microliters of the bioactive compound to the first well of each set, which corresponds to columns three, five, and seven. After mixing, transfer 100 microliters from each well to the adjacent column. To continue the serial dilution reaching columns four, six, and eight.

Repeat the entire procedure in triplicate for each concentration of every compound using fresh tips to avoid cross-contamination. Add 80 microliters of Luria Bertani broth to rows B through D in all wells. Bringing the total to 180 microliters per well.

Then add 20 microliters of the standardized chromobacterium violaceum culture from the previous preparation to each of those wells, reaching a final volume of 200 microliters. Based on the spectrophotometer compatibility. Cover the microplate with a lid or sealing film to prevent evaporation.

Incubate the sealed plate at 30 degrees Celsius with agitation at 130 revolutions per minute for 24 hours. After incubation, transfer the plate to a drying incubator set at 60 degrees Celsius. Remove the lid and allow it to dry completely for approximately eight hours.

Once dry, remove the plate from the incubator and add 200 microliters of pure DMSO to each well. Cover the plate and incubate it at 25 degrees Celsius with shaking at 130 revolutions per minute for 30 minutes to dissolve the violacein pigment. After incubation, carefully transfer 100 microliters of the dissolved violacein solution to a new microplate.

Avoiding contact between the pipette tip and the well walls. Measure the absorbance of violacein at 595 nanometers in a microplate spectrophotometer with the lid on to avoid contamination. Shake for five seconds before each reading and perform the reading with the lid on to avoid contamination.

To calculate violacein production, first subtract the absorbance of the blank from each sample to exclude background interference, and then calculate the percentage of violacein using the untreated control as the reference with the equation. Three bioactive compounds, resveratrol, farnesol, and linalool were tested for their ability to inhibit violacein production in chromobacterium violaceum ATCC 12472. Farnesol significantly reduced violacein production to 9%at 200 micrograms per milliliter and to 19%at 100 micrograms per milliliter, compared to 100%in the untreated control.

Linalool treatment at both 200 and 100 micrograms per milliliter resulted in violacein production of 32%showing a 68%inhibition relative to control. Resveratrol at 25 micrograms per milliliter, reduced violacein production to 15%while at 12 micrograms per milliliter, it reached 30%compared to 100%in the untreated control. Microplate images visually confirmed reduced violacein pigmentation for all treatments compared to the untreated control.