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Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps

Bacterial resistance to the antiseptic chlorhexidine (CHX), is a growing problem, recently shown to be caused by deleterious mutations to the phospholipid transport system component (mlaA) as well as efflux pump overexpression. Comparisons of CHX resistance mechanisms, such as porin deletions (ompCF...

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Autores principales: Gregorchuk, Branden S. J., Reimer, Shelby L., Slipski, Carmine J., Milner, Kieran A., Hiebert, Shannon L., Beniac, Daniel R., Booth, Timothy F., Zhanel, George G., Bay, Denice C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9287405/
https://www.ncbi.nlm.nih.gov/pubmed/35840757
http://dx.doi.org/10.1038/s41598-022-15775-6
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author Gregorchuk, Branden S. J.
Reimer, Shelby L.
Slipski, Carmine J.
Milner, Kieran A.
Hiebert, Shannon L.
Beniac, Daniel R.
Booth, Timothy F.
Zhanel, George G.
Bay, Denice C.
author_facet Gregorchuk, Branden S. J.
Reimer, Shelby L.
Slipski, Carmine J.
Milner, Kieran A.
Hiebert, Shannon L.
Beniac, Daniel R.
Booth, Timothy F.
Zhanel, George G.
Bay, Denice C.
author_sort Gregorchuk, Branden S. J.
collection PubMed
description Bacterial resistance to the antiseptic chlorhexidine (CHX), is a growing problem, recently shown to be caused by deleterious mutations to the phospholipid transport system component (mlaA) as well as efflux pump overexpression. Comparisons of CHX resistance mechanisms, such as porin deletions (ompCF), and over-expressed efflux pumps (acrB, qacE, aceI), are lacking and may be distinguishable using antiseptic rapid fluorescent dye testing assays. Using E. coli K-12 CHX adapted isolates (CHXR1), gene deletion mutants, and over-expressed transformants the phenotypes of these CHX resistance genes were compared using antimicrobial susceptibility tests (AST), rapid fluorescent propidium iodide dye-based membrane integrity assays (RFDMIA), and scanning electron microscopy (SEM). AST findings showed CHXR1, ΔacrB, ΔompCF, and transformants pCA24N-aceI and pCA24N-mlaA conferred greater (two to fourfold) MIC changes when compared to matched controls. Examination of these mutants/transformants using CHX RFDMIA showed that porin dual-deletions (ΔompCF) and mlaA alterations (ΔmlaA; pCA24N-mlaA, CHXR1) were distinguishable from controls. Results for over-expressed (pMS119EH-aceI) and deleted (ΔacrB) efflux pump RFDMIA could not be distinguished with propidium iodide, only with ethidium bromide, suggesting propidium iodide is better suited for detecting porin and mlaA associated CHX resistance mechanisms. SEM of CHXR1 and unadapted E. coli cells exposed to increasing CHX concentrations revealed that CHX does not visibly damage cell envelope integrity at any tested concentration but did identify elongated CHXR1 cells. ΔmlaA confers similar levels of CHX resistance as efflux overexpression and porin deletions, however, only outer membrane-altering porin and mlaA deletions can be reliably distinguished using RFDMIA.
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spelling pubmed-92874052022-07-17 Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps Gregorchuk, Branden S. J. Reimer, Shelby L. Slipski, Carmine J. Milner, Kieran A. Hiebert, Shannon L. Beniac, Daniel R. Booth, Timothy F. Zhanel, George G. Bay, Denice C. Sci Rep Article Bacterial resistance to the antiseptic chlorhexidine (CHX), is a growing problem, recently shown to be caused by deleterious mutations to the phospholipid transport system component (mlaA) as well as efflux pump overexpression. Comparisons of CHX resistance mechanisms, such as porin deletions (ompCF), and over-expressed efflux pumps (acrB, qacE, aceI), are lacking and may be distinguishable using antiseptic rapid fluorescent dye testing assays. Using E. coli K-12 CHX adapted isolates (CHXR1), gene deletion mutants, and over-expressed transformants the phenotypes of these CHX resistance genes were compared using antimicrobial susceptibility tests (AST), rapid fluorescent propidium iodide dye-based membrane integrity assays (RFDMIA), and scanning electron microscopy (SEM). AST findings showed CHXR1, ΔacrB, ΔompCF, and transformants pCA24N-aceI and pCA24N-mlaA conferred greater (two to fourfold) MIC changes when compared to matched controls. Examination of these mutants/transformants using CHX RFDMIA showed that porin dual-deletions (ΔompCF) and mlaA alterations (ΔmlaA; pCA24N-mlaA, CHXR1) were distinguishable from controls. Results for over-expressed (pMS119EH-aceI) and deleted (ΔacrB) efflux pump RFDMIA could not be distinguished with propidium iodide, only with ethidium bromide, suggesting propidium iodide is better suited for detecting porin and mlaA associated CHX resistance mechanisms. SEM of CHXR1 and unadapted E. coli cells exposed to increasing CHX concentrations revealed that CHX does not visibly damage cell envelope integrity at any tested concentration but did identify elongated CHXR1 cells. ΔmlaA confers similar levels of CHX resistance as efflux overexpression and porin deletions, however, only outer membrane-altering porin and mlaA deletions can be reliably distinguished using RFDMIA. Nature Publishing Group UK 2022-07-15 /pmc/articles/PMC9287405/ /pubmed/35840757 http://dx.doi.org/10.1038/s41598-022-15775-6 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Gregorchuk, Branden S. J.
Reimer, Shelby L.
Slipski, Carmine J.
Milner, Kieran A.
Hiebert, Shannon L.
Beniac, Daniel R.
Booth, Timothy F.
Zhanel, George G.
Bay, Denice C.
Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps
title Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps
title_full Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps
title_fullStr Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps
title_full_unstemmed Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps
title_short Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps
title_sort applying fluorescent dye assays to discriminate escherichia coli chlorhexidine resistance phenotypes from porin and mlaa deletions and efflux pumps
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9287405/
https://www.ncbi.nlm.nih.gov/pubmed/35840757
http://dx.doi.org/10.1038/s41598-022-15775-6
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