Synergistic effect of ultrasound and antimicrobial solutions of cecropin P1 in the deactivation of Escherichia coli O157:H7 using a cylindrical ultrasonic system.

This study investigates the synergistic effect of ultrasonication and antimicrobial action of antimicrobial peptide cecropin P1 on the inactivation of Escherichia coli O157:H7 in a cylindrical ultrasonication system. The inactivation of E. coli at pH 7.4 was performed using: ultrasonication (14, 22, and 47 kHz), cecropin P1 (20 µg/mL), and a combination of both. We found the treatment at 22 kHz, 8W for 15 min of exposure and a combination of ultrasound at higher frequency (47 kHz, 8 W) and cecropin P1 for one minute of exposure were more efficient, reducing the cell density by six orders of magnitude, compared to individual treatments (ultrasound or cecropin P1 only). Dye leakage studies and transmission electron microscopy further validated these results. A continuous flow system was designed to demonstrate synergism of ultrasonication with antimicrobial peptide Cecropin P1 in the inactivation of E. coli; synergism was shown to be more at higher ultrasonication frequencies and power levels. Acoustic cavitation by ultrasonic treatment could drastically improve microbial deactivation by antimicrobial peptides cecropin P1 by increasing their ability for pore formation in cell membranes. A continuous ultrasonication and antimicrobial peptides system can lead to an energy-efficient and economical sterilization system for food safety applications.

Nucleation and growth of pores in 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) / cholesterol bilayer by antimicrobial peptides melittin, its mutants and cecropin P1.

Antimicrobial peptides are one of the most promising alternatives to antibiotics for targeting pathogens without developing resistance. In this study, pore formation in 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) / cholesterol liposome induced by native melittin, its two mutant variants (G1I and I17 K), and cecropin P1 was investigated by monitoring the dynamics of fluorescence dye leakage. A critical peptide concentration was required for dye leakage with the rate of leakage being dependent on peptide concentration above a critical value. A lag time was required for dye leakage for low peptide concentrations that are above the critical value, which decreased at higher peptide concentrations eventually approaching zero. Lag time was found to be in the order I17 K mutant with lower hydrophobicity and higher net charge > G1I with higher hydrophobicity > melittin > cecropin P1. Cecropin P1 exhibited the highest rate of dye leakage followed by melittin, G1I, and I17 K. Size distribution and transmission electron microscopy (TEM) of liposomes exposed to peptides of different concentrations indicated pore formation with accompanied stretching of liposomes at low peptide concentrations for both melittin and cecropin P1. At much higher concentrations, however, size distribution indicated three peaks for both peptides. In both cases, TEM images show that the middle and small peaks are shown to be due to stretched liposome and broken stretched liposome respectively. For melittin, the large peak is due to peptide aggregates as well as aggregates of liposome. For cecropin P1, however, the large peak indicates cecropin P1 aggregates with solubilized lipids thus suggesting carpet mechanism.

Understanding the antimicrobial activity of water soluble γ-cyclodextrin/alamethicin complex.

In our previous investigation (Zhang et. al., J. Functional Foods 40 (2018) 700-706), we have proposed a method of complexation of alamethicin (ALM) with γ-cyclodextrin (γ-CD) to increase the solubility and demonstrated an enhancement in its antimicrobial activity against Listeria monocytogenes. In this study, transmission electron microscopy of L. monocytogenes treated with γ-CD/ALM complex indicated cell lysis due to pore formation. This was further corroborated by fluorescent dye leakage from DMPC/cholesterol liposomes when exposed to γ-CD/ALM complex for molar ratios of 1, 5 and 10. The extent of dye leakage increased with ALM-lipid ratio in the range of 0.00015 to 0.16. Dye leakage of γ-CD/ALM complex was found to be the highest for molar ratio of 5, consistent with our earlier results of antimicrobial activity of the complex against L. monocytogenes. All atom molecular dynamics (MD) simulation showed that γ-CD/ALM complex can effectively bind to the 3:1 POPE/POPG bilayer, a mimic of bacterial cell membrane. In addition, circular dichroism spectrum indicated that ALM in the complex has a helical conformation in solution as well as in the presence of liposome. Transmembrane MD simulation of six γ-CD/ALM complex aggregates in α helical conformation showed water channel with barrel stave like pore structure.