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Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay
Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10669664/ https://www.ncbi.nlm.nih.gov/pubmed/37998843 http://dx.doi.org/10.3390/antibiotics12111641 |
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author | Khire, Tejas Suresh Gao, Wei Bales, Brian Hsieh, Kuangwen Grossmann, Greg Park, Dong Jin M. O’Keefe, Christine Brown-Countess, Arnyah Peterson, Sara Chen, Fan-En Lenigk, Ralf Trick, Alex Wang, Tza-Huei Puleo, Christopher |
author_facet | Khire, Tejas Suresh Gao, Wei Bales, Brian Hsieh, Kuangwen Grossmann, Greg Park, Dong Jin M. O’Keefe, Christine Brown-Countess, Arnyah Peterson, Sara Chen, Fan-En Lenigk, Ralf Trick, Alex Wang, Tza-Huei Puleo, Christopher |
author_sort | Khire, Tejas Suresh |
collection | PubMed |
description | Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3–4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx). |
format | Online Article Text |
id | pubmed-10669664 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-106696642023-11-19 Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay Khire, Tejas Suresh Gao, Wei Bales, Brian Hsieh, Kuangwen Grossmann, Greg Park, Dong Jin M. O’Keefe, Christine Brown-Countess, Arnyah Peterson, Sara Chen, Fan-En Lenigk, Ralf Trick, Alex Wang, Tza-Huei Puleo, Christopher Antibiotics (Basel) Article Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3–4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx). MDPI 2023-11-19 /pmc/articles/PMC10669664/ /pubmed/37998843 http://dx.doi.org/10.3390/antibiotics12111641 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Khire, Tejas Suresh Gao, Wei Bales, Brian Hsieh, Kuangwen Grossmann, Greg Park, Dong Jin M. O’Keefe, Christine Brown-Countess, Arnyah Peterson, Sara Chen, Fan-En Lenigk, Ralf Trick, Alex Wang, Tza-Huei Puleo, Christopher Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay |
title | Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay |
title_full | Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay |
title_fullStr | Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay |
title_full_unstemmed | Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay |
title_short | Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay |
title_sort | rapid minimum inhibitory concentration (mic) analysis using lyophilized reagent beads in a novel multiphase, single-vessel assay |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10669664/ https://www.ncbi.nlm.nih.gov/pubmed/37998843 http://dx.doi.org/10.3390/antibiotics12111641 |
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