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Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay

Authors :
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
Source :
Antibiotics, Vol 12, Iss 11, p 1641 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

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).

Details

Language :
English
ISSN :
20796382
Volume :
12
Issue :
11
Database :
Directory of Open Access Journals
Journal :
Antibiotics
Publication Type :
Academic Journal
Accession number :
edsdoj.99eee922294749f0b88b07adb5baf3f8
Document Type :
article
Full Text :
https://doi.org/10.3390/antibiotics12111641