A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

Neil M O'Brien-Simpson,Troy J Attard,Namfon Pantarat,Katrina A Walsh,Eric C Reynolds,Miguel A R B Castanho

doi:10.1371/journal.pone.0151694

Neil M O'Brien-Simpson, Troy J Attard + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0151694

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Journal: PloS one	Publication Date: Mar 17, 2016
Citations: 46	License type: CC BY 4.0

Affiliation: University of Melbourne

Abstract

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.

Highlights

A recent report from the World Health Organisation has highlighted that antibiotic and multidrug resistant bacteria are a major and growing issue facing public health worldwide and that “fostering innovation and research and development of new tools” is vital in tackling this problem [1]
In other experiments we found that S. mutans and F. nucleatum were susceptible to alamethicin and ovispirin and that the Membrane Disruptive Concentration (MDC) value for each antimicrobial peptide (AMP) was consistent with the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) values (S2 Table)
The results presented here, not exhaustive in terms of AMPs and bacterial species tested clearly illustrate the potential of this novel method in its ability to rapidly screen and identify lead antimicrobial compounds in a semi- or fully-quantitative assay

Summary

Introduction

A recent report from the World Health Organisation has highlighted that antibiotic and multidrug resistant bacteria are a major and growing issue facing public health worldwide and that “fostering innovation and research and development of new tools” is vital in tackling this problem [1]. It is recognised that the rates and severity of infections caused by antibiotic and multi-drug resistant bacteria are increasing year by year and are becoming harder and more complicated to treat and manage [1, 2]. A Centres for Disease Control report has estimated that of the 2 million reported U.S hospital infections 70% are caused by antibiotic resistant bacteria leading to 44,000 deaths per year [3]. The majority of these antibiotic resistant bacterial infections are principally caused by a small number of species; Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, collectively termed the ESKAPE pathogens [4]. The antibiotic resistant strains of these bacteria were restricted to nosocomial infections, a higher

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Conclusion