New technology, fast and affordable

News Medical speaks with Dr. Sandor Casas, principal investigator at Ecole Polytechnique Fédérale de Lausanne in Switzerland. Here we discuss his recent development of a novel, high-efficiency method for rapid antibiotic susceptibility testing using optical microscopy.

The new technology, known as optical nanoscale motion detection (ONMD), is an extremely fast, label-free, sensitive single-cell method for antibiotic susceptibility testing. ONMD requires a conventional optical microscope with a camera or a mobile phone. The simplicity and efficiency of the technology could be a game-changer in the field of antibiotic resistance.

Please, could you introduce yourself, tell us about your professional background, and what inspired you in your career in biology and medicine?

I graduated in medicine, but I did not practice medicine in hospitals or medical centers. After my studies, I started working as an assistant in histology at the University of Friborg in Switzerland. My first research projects included image processing, scanning tunneling, and atomic force microscopy.

Later, and for most of the other people I work with, I focused primarily on the biological applications of AFM. For the past ten years, my research interest has been on nanokinesis, that is, the study of nanometer-scale oscillations of living organisms.

You started working on the biological applications of atomic force microscopy (AFM) in 1992. In your view, how has the landscape of antibiotic resistance changed over the past two decades? What role has technological progress played in enhancing our understanding?

In the early 1990s, AFM was mainly used for shooting. Later, AFM microscientists noticed that the tool could also be used to investigate the mechanical properties of living organisms. Recently, several ‘exotic’ applications of AFM have emerged, such as its use for weighing single cells or studying their oscillations on the nanometer scale. In the 1990s, antibiotic resistance wasn’t as serious a problem as it is today, but several teams were already using AFM to assess the effects of antibiotics on bacterial morphology.

The first investigations were limited to structural changes, but later, as the application fields of AFM expanded, the tool made it possible to monitor the mechanical properties of the bacterial cell wall upon exposure to antibiotics. In 2010, with G. Longo and G. Dietler, we showed that AFM can also track nanoscale oscillations of organisms. The first application we had in mind was using the tool for a rapid antibiotic susceptibility test.

We have therefore developed devices based on AFM technology intended for fast AST performance (ie in 2-4 hours). AFM-based nanoscale motion detection tools have already been implemented in medical centers in Switzerland, Spain and Austria. However, this type of device has some drawbacks, including the need to fix the organism of interest on a cantilever. To overcome this limitation, together with R. Willaert, we developed a nanoscale motion detector based on an optical microscope.

My latest research has led to the development of a new, highly efficient technique for rapid antibiotic susceptibility testing using optical microscopy. Please, can you tell us why it is so important to develop fast, efficient and affordable testing methods in the world of antimicrobial resistance?

Rapid antibiotic susceptibility testing can reduce the use of broad-spectrum antibiotics. Conventional transcription rate-dependent ASTs require 24 hours (but up to 1 month in the case of TB) to select the most effective antibiotic. Doctors prescribe broad-spectrum antibiotics between a patient’s admission to the medical center and the results of an AST test.

These drugs rapidly improve patients’ conditions, but unfortunately promote resistance. A rapid AST that can identify the most appropriate antibiotic within 2-4 hours would eliminate broad-spectrum antibiotics, increase treatment efficiency, and reduce the development of resistant bacterial strains. Since bacterial resistance is a global problem, rapid ASTs should also be implemented in developing countries. Therefore, affordable and easy-to-use tests are needed.

Image credit: Fahroni/Shutterstock.com

Did you encounter any limitations or obstacles in the research process? If so, how did you overcome them?

The detection of antibiotic sensitivity with the ONMD is very similar to the AFM-based technique. As long as the bacteria are alive they oscillate. If the antibiotic is effective, it kills the microorganism and stops its vibrations. The first limitation we encountered when developing ONMD was the depth of field of view of our microscopes. To prevent bacteria from leaving the focal plane of the optical microscope during measurement, we had to confine the microbes to microfluidic channels a few micrometers high.

Microfabrication of such devices is relatively easy in an academic environment, but we have been looking for simpler solutions. One option for constructing such a device is to use a 10-micron double-sided rubber band. It allows you to “build” a microfluidic chamber in 5 minutes with two cover glass and a drill.

Another challenge was motion detection at the nanoscale. For this purpose, we used freely available cross-correlation algorithms that achieve sub-pixel accuracy. (eg, a few nanometers). We first developed ONMD for larger organisms, such as yeast cells, and extended the method to bacteria. This further development took about two years.

I have worked alongside Dr. Ronnie Willart, Professor of Structural Biology at Vrije Universiteit Brussel, on the development of the new rapid AST technique. How do your areas of expertise and research background complement each other in the development of ONMD?

R. Willaert is an expert in yeast microbiology and microfluidics, while our team in Lausanne is primarily involved in AFM-based nanomotion detection and the application of AFM to clinically relevant problems. The two teams were supported by a joint grant from the Swiss National Science Foundation and the Flanders Research Foundation (FWO) that enabled the development of the method.

The field of antimicrobial resistance requires a high level of international collaboration, with everyone working together towards a common goal. With antimicrobial resistance rising to dangerously high levels worldwide, how important is collaboration in this area?

Our project required expertise in various fields, such as microbiology, microscopy, microfluidics, programming, and data processing. In the development of rapid AST tools and many others, a multidisciplinary approach and close collaboration between teams with complementary expertise is today the only way to success.

You and Dr. Willaert said, “The method’s simplicity and effectiveness make it a game-changer in the field of AST.” Could you please expand on what makes ONMD a game changer in the field of AST and what implications this research could have in clinical and research settings?

As mentioned earlier, bacterial resistance is a global health problem. Rapid AST should also be readily implemented in developing countries to reduce the spread of resistant strains. The cheaper and simpler the technology, the more likely it is to be widely used. We are convinced that the ONMD approach can meet these requirements. ONMD can also be used for drug discovery or basic research.

While we recognize the importance of rapid AST, what are the next steps that need to be taken before this technology can be used globally in research and clinical landscapes?

For basic research, there are no other significant developments to be made. Any reasonably equipped research center can implement and use the technology. Regarding the implementation of the technique in developing countries or harsh environments, stand-alone devices must be used, which have not yet been manufactured.

There is a rapidly increasing need for an efficient AST globally; However, the need for simple, accessible and affordable technologies is of great importance in developing countries that are disproportionately affected by antibiotic resistance due to existing global health disparities. Could this rapid AST technology be used in lower-middle-income countries to slow the increasing spread of multi-resistant bacteria? What does this mean for global health?

We are confident that the ONMD-based AST test can soon be implemented in research centers in both developed and developing countries. However, accreditation by health authorities is necessary for its use as a standard diagnostic tool; This process can take several years, depending on government health policy.

What’s next for you and your research? Are you involved in any exciting upcoming projects?

We want to develop an autonomous device for harsh environments. It will consist of a small microscope with a camera and a data processing unit. The microfluidic part of the device can have different antibiotics ready for testing.

ONMD technology can also monitor pollution levels in closed environments such as submarines, spacecraft, and space stations. One of our recent projects has been funded by the European Space Agency (ESA) to develop a rapid antifungal susceptibility test that can work in microgravity. In addition, ONMD could be used for more exciting projects, such as chemistry-independent life detection in the search for extraterrestrial life.

Where can readers get more information?

• Villalba MI, Rossetti E, Bonvallat A, Yvanoff C, Radonicic V, Willaert RG*, Kasas S. *A simple optical nanoaction method for the viability of single bacteria and antibiotic response testing. PNAS 2023, May 2; 120 (18): e2221284120. doi: 10.1073/pnas.2221284120. Epub 2023 Apr 24. PMID: 37094120. * Contribute equally. https://doi.org/10.1073/pnas.2221284120
• Radonic, v.; Ivanov, C; Villalba, Michigan; DeVries, b. Casas, S.; Willaert, R.G. Motion of single-cell photonic nanoparticles of Candida albicans in Microwells for rapid antifungal susceptibility testing. Fermentation 2023, 9: 365. https://doi.org/10.3390/fermentation9040365
• Parmar P, Villalba MI, Horii Huber AS, Kalauzi A, Bartolić D, Radotić K, Willaert RG, MacFabe DF, Kasas S. Mitochondrial nanomotion measured by light microscopy. Before. microbiol. 2023, 14: 1133773. https://doi.org/10.3389/fmicb.2023.1133773
• Starodubtseva MN, Irina A. Chelnokova IA, Shkliarava NM, Villalba MI, Tapalski DV, Kasas S, Willaert RG. Modulation of the rate of nanomovement of Candida albicans ovaries by X-rays. Before. microbiol. 2023, 14: 1133027. https://doi.org/10.3389/fmicb.2023.1133027
• Radonik V, Ivanov C, Villalba MI, Casas S, and Willarit RG. Dynamics of motion behavior of single-cell nanoparticles of saccharomyces cerevisiae in a microfluidic slide for rapid antifungal susceptibility testing. fermentation. 2022; 8 (5): 195. https://doi.org/10.3390/fermentation8050195

when. Sandor Casas

Nanomotion is a great new way to observe living things.

Our team focuses almost exclusively on recording the nanoscale motion of bacterial mitochondria and mammalian cells using optical and AFM-based devices.

We have recently demonstrated that this technique can be used not only for rapid antimicrobial susceptibility testing but also for probing the metabolism of single-celled organisms. We hope that our efforts will allow us to expand the areas of application of ONMD.