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Lookup NU author(s): Dr John Perry
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2016 Cellier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In clinical microbiology the speed with which pathogenic microorganisms may be detected has a direct impact on patient health. One important strategy used in the laboratory is the growth of cultures in the presence of an enzymatic substrate which, once transformed by the appropriate microbial enzyme, generates a detectable colour or fluorescence output. Such substrates have previously been prepared by our group and others and are available as commercial diagnostic kits, however they all suffer from some degree of diffusion when used in a solid growth medium. This diffusion complicates the detection and differentiation of species in polymicrobial cultures and so we sought to improve on our previous work. In this work we have prepared and evaluated a series of novel fluorogenic enzyme substrates based on N-substituted-2-aminoacridones. All of the prepared substrates were found to be suitable for the detection and differentiation of certain microorganisms, however those based on the 2-amino-10-benzylacridone core in particular showed no apparent diffusion when incorporated into solid growth media. On transformation these substrates generated brightly fluorescent colonies that are clearly contrasted with the background medium due to the difference in emission wavelength (λem 445-450 nm for the substrate, λem 550 nm for the product). Here we have shown that our L-alanyl aminopeptidase substrate, 2-(N-L-alanylamino)-10-benzylacridone, is particularly suited to the detection of Gram-negative bacteria, and our β-alanyl aminopeptidase substrate, 2-(N-β-alanylamino)-10-benzylacridone, to the detection of Pseudomonas aeruginosa and Serratia marcescens when grown on solid media incorporating these substrates. The resulting fluorophore shows no apparent diffusion from the colonies of interest, and the enhanced sensitivity offered by fluorescent emission may allow for the detection of these organisms as microcolonies using automated fluorescence microscopy.
Author(s): Cellier M, James AL, Orenga S, Perry JD, Turnbull G, Stanforth SP
Publication type: Article
Publication status: Published
Journal: PLoS ONE
Year: 2016
Volume: 11
Issue: 7
Online publication date: 08/07/2016
Acceptance date: 15/05/2016
Date deposited: 05/04/2017
ISSN (print): 1932-6203
Publisher: Public Library of Science
URL: http://doi.org/10.1371/journal.pone.0158378
DOI: 10.1371/journal.pone.0158378
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