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...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-11-01
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| Series: | Antibiotics |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2079-6382/12/11/1641 |
| _version_ | 1797460412161064960 |
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| author | Tejas Suresh Khire Wei Gao Brian Bales Kuangwen Hsieh Greg Grossmann Dong Jin M. Park Christine O’Keefe Arnyah Brown-Countess Sara Peterson Fan-En Chen Ralf Lenigk Alex Trick Tza-Huei Wang Christopher Puleo |
| author_facet | Tejas Suresh Khire Wei Gao Brian Bales Kuangwen Hsieh Greg Grossmann Dong Jin M. Park Christine O’Keefe Arnyah Brown-Countess Sara Peterson Fan-En Chen Ralf Lenigk Alex Trick Tza-Huei Wang Christopher Puleo |
| author_sort | Tejas Suresh Khire |
| collection | DOAJ |
| 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). |
| first_indexed | 2024-03-09T17:04:42Z |
| format | Article |
| id | doaj.art-99eee922294749f0b88b07adb5baf3f8 |
| institution | Directory Open Access Journal |
| issn | 2079-6382 |
| language | English |
| last_indexed | 2024-03-09T17:04:42Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj.art-99eee922294749f0b88b07adb5baf3f82023-11-24T14:25:28ZengMDPI AGAntibiotics2079-63822023-11-011211164110.3390/antibiotics12111641Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel AssayTejas Suresh Khire0Wei Gao1Brian Bales2Kuangwen Hsieh3Greg Grossmann4Dong Jin M. Park5Christine O’Keefe6Arnyah Brown-Countess7Sara Peterson8Fan-En Chen9Ralf Lenigk10Alex Trick11Tza-Huei Wang12Christopher Puleo13GE Global Research, Niskayuna, NY 12309, USAGE Global Research, Niskayuna, NY 12309, USAGE Global Research, Niskayuna, NY 12309, USADepartment of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USAGE Global Research, Niskayuna, NY 12309, USADepartment of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USADepartment of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USAGE Global Research, Niskayuna, NY 12309, USAGE Global Research, Niskayuna, NY 12309, USADepartment of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USAGE Global Research, Niskayuna, NY 12309, USADepartment of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USADepartment of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USAGE Global Research, Niskayuna, NY 12309, USAAntimicrobial 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).https://www.mdpi.com/2079-6382/12/11/1641antimicrobial susceptibility testing (AST)antimicrobial resistance (AMR)lyophilized reagentspolymerase chain reaction (PCR)microfluidicsdiagnostics |
| spellingShingle | Tejas Suresh Khire Wei Gao Brian Bales Kuangwen Hsieh Greg Grossmann Dong Jin M. Park Christine O’Keefe Arnyah Brown-Countess Sara Peterson Fan-En Chen Ralf Lenigk Alex Trick Tza-Huei Wang Christopher Puleo Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay Antibiotics antimicrobial susceptibility testing (AST) antimicrobial resistance (AMR) lyophilized reagents polymerase chain reaction (PCR) microfluidics diagnostics |
| 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 | antimicrobial susceptibility testing (AST) antimicrobial resistance (AMR) lyophilized reagents polymerase chain reaction (PCR) microfluidics diagnostics |
| url | https://www.mdpi.com/2079-6382/12/11/1641 |
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