Combining Phenotypic and Genotypic Methods for the Rapid Detection of Antibiotic-Resistant Bacteria in Wastewater Using a Microfluidic Centrifugal Disc

Abstract

Antimicrobial resistance (AMR) has emerged as a critical global health threat, leading to untreatable infectious diseases and significantly increasing human morbidity and mortality. Rapid, portable, and user-friendly detection of antibiotic-resistant bacteria (ARB) in the environment plays a key role in controlling the spread of diseases and combating the emergence of AMR. In this study, we report a portable microfluidic centrifugal disc (CD) assay incorporating both phenotypic and genotypic methods for ARB detection. This assay significantly reduces the time required for bacterial culturing from days to hours while maintaining the reliability of phenotypic methods. Moreover, the CD assay eliminates the need for a centralized laboratory and well-trained personnel. The on-CD ARB detection can be completed in less than 3 h after sample and reagent loading. The results demonstrated that the on-CD assay provided performance comparable to that of the benchtop ARB detection assay. Analysis of variance (ANOVA) comparing the benchtop and on-CD assays yielded a p-value of 0.08 for the bacterial growth rate and a p-value of 0.73 for the cell recovery rate after the sample purification, confirming no significant differences between the two approaches. The ARB detection assay was able to identify ampicillin-resistant spiked in wastewater at concentrations as low as 10 CFU/μL. Additionally, the CD assay detected indigenous ampicillin-resistant in wastewater, demonstrating the potential of this platform for environmental applications.

Keywords: ARB; ampicillin-resistant E. coli; microfluidic centrifugal disc; sample-to-answer.

1

Overview of the assay development steps.

2

Design of microfluidic chambers and channels in a functional microfluidic unit.

3

Centrifugal disc (CD) components. (a) Schematic illustrations of four main layers of CD assembly and (b) an image of actual fabricated and assembled CD.

4

Comparison of the effect of culture enrichment for selection of AR in test tubes using the LAMP assay: (a) ampicillin-resistant Famp and (b) ampicillin-sensitive CN13. Three replicates were tested for each data point.

5

Photo images demonstrate the fluid handling on CD. The color dyes were added to the fluid for visualization.

6

Side-by-side comparison of in-tube and on-CD sample enrichment and purification procedures in the ARB assay by plate counts. (a) Growth of Famp and CN13 using on-CD and in-tube incubation. Shading represents the standard deviations of triplicate assays. (b) Recovery of Famp at various steps of the sample purification process. The standard deviations are plotted as error bars.

7

Comparison of cell lysis efficiency using on-CD and in-tube bead-beating methods detected by LAMP assay. The concentration below the detection limit was assumed to be at a lower detection threshold of 10^–1 CFU/μL.

8

On-CD phenotypic-genotypic detection of indigenous ampicillin-resistantin wastewater. The concentration below the detection limit was assumed to be at the lower detection threshold of 10^–1 CFU/μL.