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

Abstract

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.

Figure 1

(A) The sectional view of a cylindrical ultrasonic transducer with the indicated dimensions, (B) The schematic view of continuous liquid treatment using the ultrasonic processing method.

Figure 2

Effect of ultrasound power levels on synergistic effect against E. coli at a fixed frequency (22 kHz) using a batch system. PBS: E. coli without any treatment, PBS + CP1: E. coli treated with cecropin P1 only, US: E. coli treated with ultrasound only, US + CP1: E. coli treated with a combination of ultrasound and cecropin P1. Power density: 1 W (0.5 W/mL); 3 W (1.5 W/mL); 5 W (2,5 W/mL); 8 W (4 W/mL). Tukey’s test: PBS vs PBS + CP1: not significant; PBS vs US: significant difference (** p < 0.01); PBS vs US + CP1: significant difference (** p < 0.01); PBS + CP1 vs US: significant difference (* p < 0.05); PBS + CP1 vs US + CP1: significant difference (** p < 0.01); US vs US + CP1: not significant.

Figure 3

Effect of ultrasound frequencies on synergistic effect at a fixed power level of 8W (4W/mL) using a batch system. Blue: E. coli without any treatment, orange: E. coli treated with cecropin P1 only, grey: E. coli treated with ultrasound only, yellow: E. coli treated with a combination of ultrasound and cecropin P1. Tukey’s test: PBS vs PBS + CP1: not significant; PBS vs US: significant difference (** p < 0.01); PBS vs US + CP1: significant difference (** p < 0.01); PBS + CP1 vs US: not significant; PBS + CP1 vs US + CP1: significant difference (* p < 0.05); US vs US + CP1: not significant.

Figure 4

Effect of residence time on synergistic effect at a fixed frequency (22 kHz) and fixed power level (8W) using a continuous ultrasonic processing system. PBS: untreated E. coli, PBS + CP1: E. coli treated with CP1 only, US: E. coli treated with ultrasonication only, US + CP1: E. coli treated with ultrasonication and CP1. Tukey’s test: PBS vs PBS + CP1: not significant; PBS vs US: significant difference (** p < 0.01); PBS vs US + CP1: significant difference (** p < 0.01); PBS + CP1 vs US: significant difference (* p < 0.05); PBS + CP1 vs US + CP1: significant difference (** p < 0.01); US vs US + CP1: not significant.

Figure 5

Maximum calcein leakage after treatment with cecropin P1 (20 µg/mL) and ultrasonication (3, 5, and 8W). The DMPC/cholesterol liposome was loaded with calcein dye and treated with cecropin P1, ultrasonication, or a combination of both for 5, 10, and 15 min, and then measured the dye leakage intensity. CP1: E. coli treated with CP1 only, US: E. coli treated with ultrasonication only, US + CP1: E. coli treated with ultrasonication and CP1. Tukey’s test: CP1 vs US: significant difference (** p < 0.01); CP1 vs US + CP1: significant difference (** p < 0.01); US vs US + CP1: significant difference (* p < 0.05).

Figure 6

TEM show single or multiple pore formations (red arrow) on E. coli due to ultrasound and cecropin P1 treatment. (A) control, (B) 14 kHz, (C) 22 kHz, (D) 47 kHz, E, and F: a combination of ultrasound (22 kHz) and cecropin P1.