J. Masuda, K. Vig
University of Hawaii at Manoa,
Keywords: bacteria inhibition, peptides, 3D scaffolds
Summary:Escherichia coli (E. coli) are gram-negative bacteria that cause severe foodborne disease outbreaks every year, most recently in 2018 by contaminated romaine lettuce. Likewise, Staphylocccus aureus (S. aureus), a gram-positive bacteria is highly contagious and causes skin infections. Many bacteria develop resistance to current antibiotics, therefore there is a need to develop new bacterial inhibitors. In the present study, two proprietary peptides (p359 and p753) were investigated for their antibacterial activity. Minimum Inhibitory Concentration (MIC) of bacteria for the peptides was determined; p753 and p359 showed MIC of 25μg/mL and 50μg/mL respectively. Likewise, live/dead assay and plate counts of bacteria were done, showing a decrease in bacteria at these concentrations. Toxicity of the p753 and p359 to HEp-2 cells were determined with MTT assay. HEp-2 cells were viable up to 100 μg/mL of both peptides, showing no toxicity to cells. HEp-2 cells were plated in 24-well tissue culture plate and infected with each bacteria at Multiplicity of Infection (MOI) of 1:0.1 for 2D studies. p753 and p359 were added at 25μg/mL and 50μg/mL respectively based on MIC and MTT studies. MTT assay of the 2D cell culture showed that cells infected with bacteria along with peptide showed 80% more cell viability compared to bacteria alone. Antibacterial activity was investigated by live/dead assay, plate count, gene expression, and scanning electron microscope. Live/ dead assay and plate count showed a 95% decrease in bacteria treated with peptide in 2D cell culture. Further studies were done in a 3D system on 3D-printed PLA scaffolds. In 3D system, the live/ dead assay showed a decrease in live bacteria with p753 or p359 application. Plate counting showed up to 70% decrease in bacteria with p753 or p359 application compared to the control. RNA was extracted and qPCR was performed on cells infected with bacteria and bacteria along with peptide. There was a down regulation in gene expression of 16s, KatA, RecA, and grpE in E. coli and down regulated gene expression of SPY1258 in S. aureus with the peptide compared to the control. Further studies will include using different 3D models and testing for cell viability and bacterial inhibition over different time periods. This work was supported by NSF-REU (DBI-1659166) to Dr. Komal Vig (PI) and by NSFCREST (HRD-1241701) to Dr. Shree S. Singh (PI).