Antibacterial Activity of a New Silver-Based Antimicrobial Spray
Dr. Misty Thomas
Background : Escherichia coli has previously been shown to evolve and develop resistance to a variety of antibacterial agents including silver. Silver (Ag) has been used as an antimicrobial agent since 1000 BCE; and silver nanoparticles are widely used today in food packaging and surgical garments. In presence of silver, bacteria have the ability to maintain homeostasis through expression of the cusCFBA efflux system, although upon exposure to high concentrations silver is extremely lethal. In our previous work, we evolved silver resistant strains of Escherichia coli K-12 MG1655 and will use these here in this study to evaluate new silver based antimicrobial cleaning sprays. Objective : Analyze the minimum inhibitor concentration (MIC) of four silver-based compounds using both a WT strain of E. coli K-12 MG1655 and a silver resistant strain. Methods : The experiment follows a MIC assay protocol. Make a stock concentration of silver: 10g/L 1:100 dilution of the stock (100uL of silver + 9.9mL of LB media). This will be used for all of the assays (LBAg) = 0.1g/L = 100 mg/L = 100 ug/mL. Take the OD reading of bacterial culture and dilute it to 0.05 to use for MIC (OD) x (how much of your bacteria to add) = (0.05) (2000uL) Add 100uL of LB media into column 1 of a 96 well plate (rows A-C and F-H) (control). Add 95uL of LB media into column 2 of the plate (control). Add 95uL of LB media to columns 3 through 11. Add 95uL of LB and 95 uL of compound to column 12. Take 95uL out of column 12 and add it to column 11, mix up and down by pipetting (100 ug/mL final Ag). Repeat this serial dilution method up to column 3. Take 95uL out of column 3 and discard it. Designate rows A-C to k-12 strain and rows F-H Ag resistant bacteria. Add 5uL to every well that has media EXCEPT for column 1. Put in the Tecan-HP plate reader, select plate ID: BD Falcon 96 Flat Bottom Transparent Polyethylene Terephthalate, and take the OD at 600nm. Put in the incubator to shake for 24 hours. After shaking for 24 hours, take an OD reading at 600nm.
Conclusions : Results show that compound C, inhibits the growth of the WT strain at a compound concentration as low as 1.5625% and Ag resistant strain at 3.125%. Compounds A, B, and D are controls and display growth inhibition significantly higher than that for compound C. Inhibition of bacteria growth required elevated concentrations of 12.5-50% from those particular compounds. The MIC for WT strain is 50% concentration and 3.125% concentration for the resistant of Compound A. The MIC for both WT strain and Ag resistant strain is 25% concentration of Compound B. Compound D inhibits growth of WT strain at 25% concentration and Ag resistant at 12.5% concentration. Future Directions: Test MIC levels of the four compounds on a variety of other bacterial species to gain a broader view of their effectiveness across gram-negative and gram-positive bacteria.
Parsons, Paris, "Antibacterial Activity of a New Silver-Based Antimicrobial Spray" (2019). Undergraduate Research and Creative Inquiry Symposia. 180.