Studying Copper Nanoparticle-Induced Programmed Cell Death in Bacteria

This research investigate copper nanopartical bactericidal efficacy against three common clinically significant bacteria. It explores cases including ROS generation and potential programmed cell death involvement.

One current challenge is achieving consistent colony counts. I believe this stems from intervention. Cell modernization during dilution stems fake counting with location compared to more experienced counting.

Our group was the first one to use a cell modulator to study copper nanoparticle bactericidal mechanisms, revealing bacterial death involves cellular processes like ROS generation and autophagy.

The protocol addresses the need for a deeper understanding of the mechanism by which copper nanoparticles kill bacteria, particularly focusing on the underexplored area of bacterial programmed cell death pathways.

Using different cell modulators, we confirmed copper nanoparticle-induced bacterial death involves autophagy and ROS overload and identified key pathways supported by reliable triplicate based experiments with a simple desig

[Narrator] To begin obtain commercial copper nanopowders with diameters of 25 nanometers and 60 to 80 nanometers from a commercial supplier. Use one milliliter of one millimolar SDS as a dispersant for each nanoparticle size. Then disperse the nanoparticles using an ultrasonic bath for at least 30 minutes at room temperature. Next, obtain Escherichia coli, Acinetobacter baumannii, and Staphylococcus aureus strains from respective biological resource centers. Culture each bacteria in 10 milliliters of LB broth under aerobic conditions at 37 degrees Celsius. Dilute bacterial cultures in LB medium to reach an optical density of approximately 0.5 at 600 nanometers. Now use stock copper nanoparticle solutions to prepare a range of concentrations between zero to 100 micrograms per milliliter of both sizes. Pipette 500 microliters of the bacterial cultures into microcentrifuge tubes and centrifuge at 3,300 G for 10 minutes at room temperature. After pipetting out the supernatant, add different concentrations of both sizes of the nanoparticles to each tube. Treat control groups with 500 microliters of PBS as a negative control and 500 microliters of 70% alcohol as a positive control. Then incubate all samples with shaking at 200 revolutions per minute at 37 degrees Celsius for 24 hours. After incubation, wash the bacteria with PBS and spread them onto LB agar plates. Incubate the plates at 37 degrees Celsius for 24 hours. The next day, count colonies on each plate for all treatment groups. Treat bacteria with five micromolar SBI for two hours, 0.5 micromolar necrosulfonamide for one hour, 100 nanomolar wortmannin for 30 minutes, or 100 nanomolar Z-VAD for 30 minutes. After all pre-treatment is complete, centrifuge all tubes at 3,300 G for 10 minutes. Then resuspend the pellet in 800 microliters of PBS to wash the pre-treatment modulator. Distribute the resuspended pellets. Centrifuge each tube, then discard the supernatant. Resuspend the pellets in nanoparticle modulator mixtures before incubating with agitation. Now add 70% ethanol and PBS as positive and negative controls. Include blank control groups treated with inhibitors, but no nanoparticles. After incubation, pipette the cell viability reagent at a one to ten volume ratio and incubate. After centrifuging the cultures again, transfer the supernatants to 96-well plates. Measure fluorescence at 560 nanometers excitation and 590 nanometers emission using a microplate reader. Dilute remaining supernatants to 10 to the power of negative five and 10 to the power of negative four and spread on LB agar plates to culture. Count the single colonies the following day. Pipette bacterial cultures into microcentrifuge tubes. Then expose the cultures to 405 nanometer ultraviolet light for three hours. Next, incubate the cultures at 45 degrees Celsius for two hours. Then incubate the bacteria at four degrees Celsius for two hours. After cold treatment, incubate the bacteria in 3% hydrogen peroxide for 30 minutes. Maintain a control group at 37 degrees Celsius in LB broth. Now treat bacteria with 20 or 60 nanometer particles at concentrations between one to 100 micrograms per milliliter for 24 hours. Then wash the treated bacteria twice with PBS. After centrifugation, resuspend the bacterial pellets in a five micromolar solution of 2',7' dichlorodihydrofluorescein diacetate dye. Analyze the fluorescence intensity at 520 or 530 nanometer emission. The colony counts of Escherichia coli were significantly reduced by 20 nanometer copper nanoparticles at one microgram per milliliter and by 60 nanometer copper nanoparticles at five micrograms per milliliter. Staphylococcus aureus showed significant reductions in colony counts at all concentrations of both nanoparticle sizes. In Acinetobacter baumannii, reductions in colony numbers required five micrograms per milliliter of 20 nanometer copper nanoparticles and 10 micrograms per milliliter of 60 nanometer copper nanoparticles. Copper nanoparticle treatments induced reactive oxygen species production in all bacteria, with 20 nanometer particles showing the highest fractions of positive cells at lower concentrations. In contrast, 60 nanometer copper nanoparticle treatments led to consistent reactive oxygen species generation across all concentrations. Programed cell death modulator Z-VAD increased survival of Escherichia coli treated with both nanoparticle sizes at low to moderate concentrations. NSA treatment improved survival of Staphylococcus aureus across all 20 nanometer copper nanoparticle concentrations. Acinetobacter baumannii exhibited improved viability with Z-VAD under one, five, and 10 micrograms per milliliter of both 20 and 60 nanometer copper particle exposure, while NSA increased viabilities at only 10 micrograms per milliliter of 20 nanometer copper nanoparticle exposure.