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.

Candida albicans is an emerging multidrug-resistant opportunistic pathogen representing an important source of invasive disease in humans and generating high healthcare costs worldwide. The development of a rapid and simple antifungal susceptibility test (AFST) could limit the spread of this disease, increase the efficiency of treatment, and lower the risk of developing resistant strains. We developed a microfluidic chip containing an array of microwells that were designed to trap the cells and perform rapid antifungal susceptibility tests using optical nanomotion detection (ONMD). Yeast cell entrapment in a microwell allows for a very rapid exchange of growth medium with the antifungal, which enables performing single-cell ONMD measurements on the same cell before and after antifungal treatment. The exposure to a low concentration of the antifungal caspofungin or flucanozole induced a significant decrease in the nanomotion signal, demonstrating the high sensitivity of this technique. We used this chip to quantify the real-time response of individual C. albicans cells to the antifungal treatment in as fast as 10 min. This simple and label-free technique could be further developed into a simple-to-use device that allows the performance of fast AFST as part of a routine hospital procedure in developed and also eventually developing world countries.

The fast emergence of multi-resistant pathogenic yeasts is caused by the extensive—and sometimes unnecessary—use of broad-spectrum antimicrobial drugs. To rationalise the use of broad-spectrum antifungals, it is essential to have a rapid and sensitive system to identify the most appropriate drug. Here, we developed a microfluidic chip to apply the recently developed optical nanomotion detection (ONMD) method as a rapid antifungal susceptibility test. The microfluidic chip contains no-flow yeast imaging chambers in which the growth medium can be replaced by an antifungal solution without disturbing the nanomotion of the cells in the imaging chamber. This allows for recording the cellular nanomotion of the same cells at regular time intervals of a few minutes before and throughout the treatment with an antifungal. Hence, the real-time response of individual cells to a killing compound can be quantified. In this way, this killing rate provides a new measure to rapidly assess the susceptibility of a specific antifungal. It also permits the determination of the ratio of antifungal resistant versus sensitive cells in a population.

Candida albicans is an emerging pathogen that poses a significant challenge due to its multidrug-resistant nature. There are two types of antifungal agents, fungicidal and fungistatic, with distinct mechanisms of action against fungal pathogens. Fungicidal agents kill fungal pathogens, whereas fungistatic agents inhibit their growth. The growth can be restored once the agent is removed and favorable conditions are established. Recognizing this difference is crucial as it influences treatment selection and infection prognosis. We present a technique based on optical nanomotion detection (ONMD) (i.e., observing the movement of the cells using an optical microscope) to discriminate rapidly between fungicidal (caspofungin) and fungistatic (fluconazole) drugs. The technique is based on the change in a yeast cell’s nanomotion as a function of time during a two-hour treatment with the antifungal of interest followed by a one-hour growth period. The cells are entrapped in microwells in a microfluidic chip, which allows a quick exchange of growth medium and antifungal agent, enabling ONMD measurements on the same individual cells before and after treatment. This procedure permits to discriminate between fungicidal and fungistatic antifungals in less than 3 h, with single-cell resolution by observing if the nanomotion recovers after removing the treatment and reintroducing growth medium (YPD), or continues to drop. The simplicity of the approach holds promise for further development into a user-friendly device for rapid antifungal susceptibility testing (AFST), potentially being implemented in hospitals and medical centers worldwide in developed and developing countries.

Yeast vitality during storage is essential for maintaining consistent fermentation performance. This study compares the physiological responses of top- and bottom-cropped Saccharomyces cerevisiae stored at 4 °C for 20 days and evaluates Optical Nanomotion Detection (ONMD) as a rapid, label-free vitality assessment tool. Classical assays (FUN-1, methylene blue, propidium iodide, glucose acidification power, glycogen content, and ethanol tolerance) were used to monitor metabolic activity, membrane integrity, and stress resilience. Bottom-cropped yeast retained metabolic activity, membrane stability, and energy reserves longer than top-cropped cells. ONMD revealed distinct single-cell nanomotion signatures and detected mechanically active subpopulations even when traditional vitality indicators declined. Analysis of nanomotion slopes showed an increasingly negative trend in the decline over storage time in top-cropped cells, indicating reduced temporal stability of nanomechanical activity during the 180 min recordings. Ethanol-challenge experiments confirmed the vitality dependence and stress-sensitivity of the ONMD signal. Together, these findings demonstrate that ONMD resolves cold-storage-induced changes in yeast nanomechanical vitality and provides complementary information beyond conventional vitality and viability assays. ONMD offers a fast, reagent-free method for monitoring brewing yeast physiology and represents a promising basis for future development toward brewery quality-control applications.

Antimicrobial resistance in microorganisms associated with fermented foods is increasingly recognized, yet rapid methods to characterize antibiotic response dynamics remain limited. This study evaluates antibiotic susceptibility and physiological response patterns of kombucha-associated acetic acid bacteria and motile Escherichia coli using optical nanomotion detection (ONMD), a label-free technique that quantifies single-cell mechanical activity. Two cellulose-producing species (Komagataeibacter xylinus and K. rhaeticus), one non-cellulose-producing species (K. melaceti), and E. coli were exposed to ampicillin, ciprofloxacin, and chloramphenicol. Minimum inhibitory concentrations (MICs) were determined prior to time-resolved ONMD analysis. Susceptible strains exhibited progressive suppression of confined nanomotion consistent with MIC-defined susceptibility, whereas resistant profiles maintained sustained mechanical activity. Chloramphenicol initially induced persistent or increased nanomotion at 120 min; however, extending the observation to 180 min revealed delayed suppression in susceptible strains, demonstrating that bacteriostatic antibiotics require longer observation windows for accurate ONMD classification. In motile E. coli, ONMD revealed both intracellular nanomotion puncta and swimming trajectories, which were progressively attenuated following antibiotic exposure. These findings demonstrate that ONMD complements conventional susceptibility testing by resolving time-dependent suppression of both translational motility and intracellular nanomechanical activity at the single-cell level.