Simple optical nanomotion method for single-bacterium viability and antibiotic response testing

Abstract

Antibiotic resistance is nowadays a major public health issue. Rapid antimicrobial susceptibility tests (AST) are one of the options to fight this deadly threat. Performing AST with single-cell sensitivity that is rapid, cheap, and widely accessible, is challenging. Recent studies demonstrated that monitoring bacterial nanomotion by using atomic force microscopy (AFM) upon exposure to antibiotics constitutes a rapid and highly efficient AST. Here, we present a nanomotion detection method based on optical microscopy for testing bacterial viability. This novel technique only requires a very basic microfluidic analysis chamber, and an optical microscope equipped with a camera or a mobile phone. No attachment of the microorganisms is needed, nor are specific bacterial stains or markers. This single-cell technique was successfully tested to obtain AST for motile, nonmotile, gram-positive, and gram-negative bacteria. The simplicity and efficiency of the method make it a game-changer in the field of rapid AST.

A simplified version of rapid susceptibility testing of bacteria and yeasts using optical nanomotion detection

“…The microfluidic device used as an analysis chamber can be manufactured on the spot in less than 5 min and does not require any specialized instrumentation (nor a clean room), except the 5-μm-thick double-faced rubber tape and a desk paper punch. Very basic low-cost optical microscopes can be used as imaging devices ( Villalba et al, 2023 ), and if required, the imaging camera can be replaced by a mobile phone. Contrary to the previous ONMD sensitivity testing method, no sophisticated cell tracking computer software is required.…”

“…These two parameters seriously limit the applicability of the technique on a larger scale. In order to overcome these limitations, we recently demonstrated that simple optical microscopes can achieve nanomotion detection by using sophisticated image processing algorithms that monitor fungal or bacterial displacements with a sub-pixel resolution ( Willaert et al, 2020 ; Villalba et al, 2023 ). In this case, single bacterium or yeast cell displacements are tracked using optical microscopy in a timeframe of approximately 10 s as a function of time after exposure to chemicals.…”

“…The drug’s effect on the organisms is assessed by highlighting the global population displacements between two consecutive frames. This methodology was briefly mentioned in a previous study ( Villalba et al, 2023 ) but was never optimized or systematically tested with different types of organisms. In this study, we optimized the technique and applied it to motile, non-motile, Gram-positive, Gram-negative, rapid and slow-growing bacteria, and two different yeast species.…”

Machine learning method for the classification of the state of living organisms’ oscillations

“…One of the primary and most straightforward applications is the development of rapid and label-free antimicrobial sensitivity tests for pathogenic bacteria (or yeasts) ( Stupar et al, 2017 ; Mustazzolu et al, 2019 ; Villalba et al, 2022 ; 2023 ; Radonicic et al, 2023 ). In this approach, the organism of interest is exposed to different antibiotics, and its nanomotion is monitored over time.…”

Mechanical Properties and Nanomotion of BT-20 and ZR-75 Breast Cancer Cells Studied by Atomic Force Microscopy and Optical Nanomotion Detection Method

“…Cellular nanomotion was highlighted for the first time by attaching living cells to an AFM cantilever and monitoring its oscillations. More recently, our team demonstrated that traditional optical microscopes equipped with a video camera and dedicated software can also detect bacterial [31], fungal [32,33], and mitochondrial [34] nanomotion. This type of nanomotion detection is referred to as optical nanomotion detection (ONMD) [32].…”