News Medical speaks with Dr. Sandor Kasas, a lead researcher at Ecole Polytechnique Fédérale de Lausanne in Switzerland. Here we discuss his recent development of a novel and highly efficient method for rapid antibiotic susceptibility testing using optical microscopy.

The brand new technique, referred to as Optical Nanomotion Detection (ONMD), is an especially rapid, label-free, and single-cell sensitive method to check for antibiotic sensitivity. ONMD requires only a standard optical microscope equipped with a camera or cell phone. The simplicity and efficiency of the technique could prove to be a game changer in the sector of antibiotic resistance.

Please are you able to introduce yourself, tell us about your profession background, and what inspired your profession in biology and medicine?

I graduated in medicine but never practiced in hospitals or medical centers. After my studies, I began working as an assistant in histology on the University of Fribourg in Switzerland. My first research projects included image processing, scanning tunneling, and atomic force microscopy.

Later, and for many of the remainder of my scientific carrier, I focused totally on the biological applications of AFM. For the past ten years, my research interest is about nanomotion, i.e., the study of oscillations at a nanometric scale of living organisms.

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You began working on biological applications of the atomic force microscope (AFM) in 1992. Out of your perspective, how has the antibiotic resistance landscape modified during the last 20 years? What role has the advancement in technology played in furthering our understanding?

Within the early ’90s, the AFM was mainly used for imaging. Later, AFM microscopists noticed that the instrument may be used to explore the mechanical properties of living organisms. More recently, many “exotic” applications of the AFM have emerged, similar to its use to weigh single cells or study their oscillations on the nanometric scale. Within the Nineteen Nineties, antibiotic resistance was not as serious an issue as today, but several teams were already using AFM to evaluate the results of antibiotics on bacterial morphology.

The primary investigations were limited to structural changes, but later, because the fields of application of AFM expanded, the instrument made it possible to observe the mechanical properties of the bacterial cell wall upon exposure to antibiotics. Within the 2010s, with G. Longo and G. Dietler, we demonstrated that AFM could also track nanoscale oscillations of living organisms. The very first application we had in mind was using the instrument to perform rapid antibiotic susceptibility testing.

We’ve due to this fact developed devices based on dedicated AFM technology to perform fast AST (i.e., in 2-4h). AFM-based nanomotion detection instruments are already implemented in medical centers in Switzerland, Spain, and Austria. Nonetheless, any such device has some drawbacks, including the necessity to fix the organism of interest on a cantilever. To beat this limitation, we now have developed with R. Willaert a nanomotion detector based on an optical microscope.

Your most up-to-date research has led to the event of a novel and highly efficient technique for rapid antibiotic susceptibility testing using optical microscopy. Please could you tell us why the event of rapid, reasonably priced, and efficient testing methods is so vital on this planet of antimicrobial resistance?

Rapid antibiotic susceptibility testing could reduce using broad-spectrum antibiotics. Traditional ASTs based on replication rate require 24 hours (but as much as 1 month within the case of tuberculosis) to discover essentially the most effective antibiotic. Doctors prescribe broad-spectrum antibiotics between the patient’s admission to a medical center and the outcomes of the AST.

These drugs quickly improve patients’ conditions but, unfortunately, promote resistance. A rapid AST that might discover essentially the most suitable antibiotic inside 2-4 hours would eliminate broad-spectrum antibiotics and increase treatment efficiency and reduce the event of resistant bacterial strains. Since bacterial resistance is a world problem, rapid ASTs must also be implemented in developing countries. Due to this fact, reasonably priced and simple-to-use tests are needed.

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Were there any limitations and obstacles you faced within the research process? In that case, how did you overcome them?

Antibiotic sensitivity detection with ONMD may be very just like the AFM-based technique. So long as the bacterium is alive, it oscillates; if the antibiotic is effective, it kills the micro-organism, and its oscillations stop. The primary limitation we faced when developing the ONMD was our microscopes’ depth of field of view. To stop the bacteria from leaving the focal plane of the optical microscope throughout the measurement, we needed to constrain the microbes into microfluidic channels just a few micrometers high.

Microfabrication of such devices is comparatively straightforward in a tutorial environment, but we were in search of simpler solutions. One option for constructing such a tool is to make use of 10-micron double-sided rubber tape. It means that you can “construct” a microfluidic chamber in 5 minutes with two glass coverslips and a puncher.

One other challenge was nanoscale motion detection. For this purpose, we used freely available cross-correlation algorithms that achieve sub-pixel resolution. (i.e., just a few nanometers). We first developed the ONMD for larger organisms, similar to yeast cells, and expanded the strategy to bacteria. This further development took us around two years.

You worked alongside Dr. Ronnie Willaert, a professor of structural biology at Vrije Universiteit Brussel, on developing this latest rapid AST technique. How did your areas of experience and research backgrounds complement one another in developing ONMD?

R. Willaert is an authority in yeast microbiology and microfluidics, while our team in Lausanne is primarily involved in AFM-based nanomotion detection and applying AFM to clinically relevant problems. The 2 teams were supported by a joint grant from the Swiss National Science Foundation and the Research Foundation Flanders (FWO) which enabled the event of the strategy.

The sphere of antimicrobial resistance requires a high level of international collaboration, with everyone working together to attain a standard goal. With antimicrobial resistance rising to dangerously high levels in all parts of the world, how vital is collaboration on this field?

Our project required expertise in various fields, similar to microbiology, microscopy, microfluidics, programming, and data processing. In the event of rapid AST instruments and plenty of others, only a multidisciplinary approach and shut collaboration between teams with complementary expertise is today the one path to success.

You and Dr. Willaert have said, ‘The simplicity and efficiency of the strategy make it a game-changer in the sector of AST.’ Are you able to please expand on what makes ONMD a game changer within the AST field and what implications this research could have in clinical and research settings?

As mentioned earlier, bacterial resistance is a world health problem. Rapid AST must also be easily implemented in developing countries to limit the spread of resistant strains. The cheaper and simpler the technique, the more likely it’s for use on a big scale. We’re convinced that the ONMD approach can meet these requirements. ONMD may be used for drug discovery or basic research.

While we recognize the importance of rapid AST, what next steps should be taken before this system will be used globally in research and clinical landscapes?

For fundamental research, there are not any other vital developments to be made. Any reasonably equipped research center can implement the technique and use it. Regarding implementing the technique in developing countries or extreme environments, stand-alone devices should be used, which have yet to be manufactured.

There may be a rapidly expanding need for efficient AST globally; nonetheless, the necessity for reasonably priced, accessible, and straightforward techniques are of grave importance in developing countries disproportionately affected by antibiotic resistance because of existing global health disparities. Could this rapid AST technique be utilized in low-middle-income countries to slow the growing spread of multi-resistant bacteria? What would this mean for global health?

We’re confident that ONMD-based AST testing can soon be implemented in research centers in each developed and developing countries. Nonetheless, accreditation by the health authorities is mandatory to make use of it as a typical diagnostic tool; this process can take several years, depending on the federal government health policy.

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

We wish to develop a self-contained device for extreme environments. It could consist of a small microscope equipped with a camera and an information processing unit. The microfluidic a part of the device could contain different antibiotics able to be tested.

The ONMD technique could also monitor contamination levels in enclosed environments similar to submarines, spacecraft, and space stations. One in all our recent projects is funded by the European Space Agency (ESA) to develop a rapid antifungal susceptibility test that might work in microgravity. Moreover, ONMD could possibly be used for much more exciting projects, similar to chemistry-independent life detection within the seek for extraterrestrial life.

Where can readers find more information?

• Villalba MI, Rossetti E, Bonvallat A, Yvanoff C, Radonicic V, Willaert RG*, Kasas S.*.Easy optical nanomotion method for single-bacterium viability and antibiotic response testing. PNAS 2023, May 2;120(18):e2221284120. doi: 10.1073/pnas.2221284120. Epub 2023 Apr 24. PMID: 37094120. * Contributed equally. https://doi.org/10.1073/pnas.2221284120
• Radonicic, V.; Yvanoff, C.; Villalba, M.I.; Devreese, B.; Kasas, S.; Willaert, R.G. Single-Cell Optical Nanomotion 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 and Kasas S. Mitochondrial nanomotion measured by optical microscopy. Front. 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 nanoscale motion rate of Candida albicans by X-rays. Front. Microbiol. 2023, 14:1133027. https://doi.org/10.3389/fmicb.2023.1133027
• Radonicic V, Yvanoff C, Villalba MI, Kasas S, Willaert RG. The Dynamics of Single-Cell Nanomotion Behaviour of Saccharomyces cerevisiae in a Microfluidic Chip for Rapid Antifungal Susceptibility Testing. Fermentation. 2022; 8(5):195. https://doi.org/10.3390/fermentation8050195

About Dr. Sandor Kasas

Nanomotion is an enchanting and novel approach to observing living organisms.

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

Recently, we demonstrated that the technique could possibly be used not just for fast antimicrobial sensitivity testing but in addition to explore the metabolism of unicellular organisms. We hope our efforts will permit us to expand the applying domains of ONMD.