DNA Dye Sytox Green in Detection of Bacteriolytic Activity: High Speed, Precision and Sensitivity Demonstrated With Endolysins

Abstract

Introduction: Increasing number of deaths from multi-drug resistant bacterial infections has caused both the World Health Organization and the Centers for Disease Control and Prevention to repeatedly call for development of new, non-traditional antibacterial treatments. Antimicrobial enzymes, including those derived from bacteriophages, known as endolysins or enzybiotics, are considered promising solutions among the emerging therapies. These naturally occurring proteins specifically destroy bacterial cell walls (peptidoglycan) and as such, are capable of killing several logs of bacteria within minutes. Some endolysins cause lysis of a wide range of susceptible bacteria, including both Gram-positive and Gram-negative organisms, whereas other endolysins are species- or even strain-specific. To make wide use of endolysins as antibacterial agents, some basic research issues remain to be clarified or addressed. Currently available methods for testing endolysin kinetics are indirect, require large numbers of bacteria, long incubation times and are affected by technical problems or limited reproducibility. Also, available methods are focused more on enzymatic activity rather than killing efficiency which is more relevant from a medical perspective.Results: We show a novel application of a DNA dye, SYTOX Green. It can be applied in comprehensive, real-time and rapid measurement of killing efficiency, lytic activity, and susceptibility of a bacterial population to lytic enzymes. Use of DNA dyes shows improved reaction times, higher sensitivity in low concentrations of bacteria, and independence of bacterial growth. Our data show high precision in lytic activity and enzyme efficiency measurements. This solution opens the way to the development of new, high throughput, precise measurements and tests in variety of conditions, thus unlocking new possibilities in development of novel antimicrobials and analysis of bacterial samples.

Monitoring phage-induced lysis of Gram-negatives in real time using a fluorescent DNA dye

“…Nonetheless, our results indicate that Sytox green can also have a valuable application for detecting phage-induced damage. A comparable nding was reported by Harhala et al, where Sytox green was used to effectively evaluate bacteriolytic activity of phage endolysins [32], but this…”

Membrane-Active Nonivamide Derivatives as Effective Broad-Spectrum Antimicrobials: Rational Design, Synthesis, and Biological Evaluation

“…The SYTOX Green uptake assay was used to determine the permeability of the most promising compound 51 toward the cell membrane of Gram-positive bacteria S. aureus ATCC 29213, MRSA NCTC 10442, and Gram-negative bacteria E. coli ATCC 25922. On the one hand, it can penetrate the damaged bacterial cell membrane and combine with the DNA to result in significantly enhanced fluorescence . On the other hand, in the case of extensive damage or even complete disintegration of the bacteria, bacterial intracellular nucleic acid can leak outside the cell and SYTOX Green in the mixture binds the intracellular nucleic acid producing a signal .…”

“…On the one hand, it can penetrate the damaged bacterial cell membrane and combine with the DNA to result in significantly enhanced fluorescence . On the other hand, in the case of extensive damage or even complete disintegration of the bacteria, bacterial intracellular nucleic acid can leak outside the cell and SYTOX Green in the mixture binds the intracellular nucleic acid producing a signal . As shown in Figure a–c, after the treatment of compound 51 with S. aureus ATCC 29213, MRSA NCTC 10442, and E. coli ATCC 25922 at two different concentrations (4× and 8× MIC), the fluorescence intensity of the mixture was significantly enhanced, while the fluorescence intensity of the mixture treated by capsaicin and nonivamide at the same concentrations did not change (Figure S1a–c).…”

Escaping antibody responses to bacteriolytic enzymes Pal and Cpl-1 by epitope scanning and engineering

“…The Sytox Green solution from the ViaGram Red+ Bacterial Gram Stain and Viability Kit (Thermo Fisher Scientific) was used to measure bacteriolytic activity via a fluorescence plate reader in accordance to a previously published protocol (Harhala et al, 2021). In short, mixtures of bacteria, endolysin, and Sytox™ Green (ThermoFisher Scientific) were prepared.…”

“…Progress values were used to calculate lytic activity as part of the total bacterial sample lysed per unit of time [min -1 ] using a linear regression model for Cpl-1 and a one-phase association model for Pal as previously described. Lytic activity is defined as the highest rate of lysis per unit of time for the linear part of the curve (for Cpl-1) or the beginning of the reaction (for Pal) before the reaction slows down due to diminishing bacterial substrate in the sample as described (Harhala et al, 2021).…”