Study of the Membrane Activity of the Synthetic Peptide ∆M3 Against Extended-Spectrum β-lactamase Escherichia coli Isolates

Abstract

Escherichia coli is the most common microorganism causing nosocomial or community-acquired bacteremia, and extended-spectrum β-lactamase-producing Escherichia coli isolates are identified worldwide with increasing frequency. For this reason, it is necessary to evaluate potential new molecules like antimicrobial peptides. They are recognized for their biological potential which makes them promising candidates in the fight against infections. The goal of this research was to evaluate the potential of the synthetic peptide ΔM3 on several extended-spectrum β-lactamase producing E. coli isolates. The antimicrobial and cytotoxic activity of the peptide was spectrophotometrically determined. Additionally, the capacity of the peptide to interact with the bacterial membrane was monitored by fluorescence microscopy and infrared spectroscopy. The results demonstrated that the synthetic peptide is active against Escherichia coli isolates at concentrations similar to Meropenem. On the other hand, no cytotoxic effect was observed in HaCaT keratinocyte cells even at 10 times the minimal inhibitory concentration. Microscopy results showed a permeabilizing effect of the peptide on the bacteria. The infrared results showed that ΔM3 showed affinity for the lipids of the microorganism's membrane. The results suggest that the ∆M3 interacts with the negatively charged lipids from the E. coli by a disturbing effect on membrane. Finally, the secondary structure experiments of the peptide showed a random structure in solution that did not change during the interaction with the membranes.

Keywords: Antimicrobial peptides; Antimicrobial resistance; Extended-spectrum β-lactamase-producing in Escherichia coli; Fluorescence microscopy; Infrared spectroscopy.

Fig. 1

Antimicrobial activities of ΔM3 against A ESBL-producing E. coli isolates: HD7, HD8, HD11 BK43028, EB101, EB107, and EB127, and B E. coli ATCC 25922 strain. The differences with respect to non-treated isolates were obtained by one-way ANOVA where *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001

Fig. 2

In vitro time-kill assessment of the bactericidal activity of ∆M3 on the exponential growth of ESBL-producing E. coli isolates within 12 h of exposure to different peptide concentrations. Each concentration was tested in triplicate for each hour and the data obtained are expressed as the mean ± standard deviation of three independent experiments. The differences with respect to non-treated isolates were obtained by one-way ANOVA where *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001

Fig. 3

Cytotoxic effect of ΔM3 in HaCaT cells treated with different concentrations of peptide for 24 h. Values are expressed as the mean ± standard error of the mean (SEM) of three independent experiments. The differences with respect to non-treated cells were obtained by one-way ANOVA

Fig. 4

Cell permeability assay of ESBL-producing E. coli H8 isolate with the membrane-impermeant dye SYTOX® Green. Cells were treated with different concentrations of ΔM3: A Control, B 1.25 µM, C 2.5 µM, and D 5 µM

Fig. 5

Peak positions of the A symmetric stretching vibration bands of the methylene groups and B stretching carbonyl vibration measured by FT-IR as a function of temperature. Effect of different concentrations of ΔM3 on the E. coli model membrane POPE:POPG (70:30)