That, at its simplest, is the approach Mohamed Seleem’s lab at the Center for One Health Research has found may be a key treatment strategy in the battle against Candida auris, a frighteningly deadly fungal pathogen discovered in 2009 that is considered an urgent threat by the Centers for Disease Control and Prevention (CDC).
Candida auris, first discovered in Japan as an ear infection, has a staggering 60 percent mortality rate among those it infects, primarily people with compromised health in hospitals and nursing homes.
Recently, Seleem and Ph.D. students Yehia Elgammal and Ehab A. Salama published a paper in the American Society for Microbiology’s Antimicrobial Agents and Chemotherapy journal detailing the potential use of atazanavir, an HIV protease inhibitor drug, as a new avenue to improving the effectiveness of existing antifungals for those with a Candida auris infection.
A perfect storm of antimicrobial resistance, global warming and the COVID-19 pandemic has resulted in the rapid spread of Candida auris around the world, said Seleem, director of the center, a collaboration between the Virginia-Maryland College of Veterinary Medicine and the Edward Via College of Osteopathic Medicine.
We don’t have lots of drugs to use to treat fungal pathogens. We have only three classes of antifungal drugs. With a fungal pathogen, it’s often resistant to one class, but then we have two other options. What’s scary about Candida auris is it shows resistance to all three classes of the antifungal.
The CDC has a list of urgent threats, but on that list there is just one fungal pathogen, which is Candida auris. Because it’s urgent, we need to deal with it.”
Mohamed Seleem, the Tyler J. and Frances F. Young Chair in Bacteriology at Virginia Tech
Widespread use of fungicides in agriculture, in addition to the three classes of antifungal drugs used widely in medicine, has contributed to fungal pathogens developing more resistance, particularly Candida auris.
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Also, its rise has been linked to rising global temperatures and to easier spread through hospitals filled with COVID-19 patients in recent years during the global pandemic.
Atazanavir, an HIV protease inhibitor drug, has been found by Seleem’s lab to block the ability of Candida auris to excrete antifungals through its efflux pumps.
Think of a boat taking on water and hoses siphoning that water out of the boat to keep it afloat. Atazanavir stops up the hoses.
That allows the azole class of antifungal drugs to not be expelled as easily and perform better against Candida auris, the Seleem lab’s research has found.
The research on atazanavir builds on work three years ago by Seleem’s lab, then at Purdue University, finding potentially similar benefit in lopinavir, another HIV protease inhibitor.
HIV protease drugs are already in wide use among HIV patients, who can also be extra susceptible to Candida auris. Some HIV patients have likely been taking HIV protease drugs and azole-class antifungals in tandem for separate purposes, providing a potential source of already existing data that can be reviewed on whether those patients had Candida auris and what effects the emerging pathogen had on them.
Repurposing drugs already on the market for new uses can allow those treatments to reach widespread clinical use much more rapidly than would happen with the discovery of an entirely new drug, as existing drugs have already been tested and approved by the Food and Drug Administration and have years of further observation of effects in prescriptive use.
In 2022, the Center for One Health Research received a $1.9 million grant from the National Institutes of Health for the Seleem lab’s research on repurposing already approved drugs for treating gonorrhea.
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Thought LeadersDr. Sandor KasasResearch LeadEcole Polytechnique Fédérale de Lausanne
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 new technique, known as Optical Nanomotion Detection (ONMD), is an extremely rapid, label-free, and single-cell sensitive method to test for antibiotic sensitivity. ONMD requires only a traditional optical microscope equipped with a camera or mobile phone. The simplicity and efficiency of the technique could prove to be a game changer in the field of antibiotic resistance.
Please can you introduce yourself, tell us about your career background, and what inspired your career in biology and medicine?
I graduated in medicine but never practiced in hospitals or medical centers. After my studies, I started working as an assistant in histology at the University of Fribourg in Switzerland. My first research projects included image processing, scanning tunneling, and atomic force microscopy.
Later, and for most of the rest of my scientific carrier, I focused primarily 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.
Image Credit: dominikazara/Shutterstock.com
You started working on biological applications of the atomic force microscope (AFM) in 1992. From your perspective, how has the antibiotic resistance landscape changed over the last two decades? What role has the advancement in technology played in furthering our understanding?
In the early ’90s, the AFM was mainly used for imaging. Later, AFM microscopists noticed that the instrument could also be used to explore the mechanical properties of living organisms. More recently, many “exotic” applications of the AFM have emerged, such as its use to weigh single cells or study their oscillations at the nanometric scale. In the 1990s, antibiotic resistance was not as serious a problem as 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 fields of application of AFM expanded, the instrument made it possible to monitor the mechanical properties of the bacterial cell wall upon exposure to antibiotics. In 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 have therefore 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. However, this type of device has some drawbacks, including the need to fix the organism of interest on a cantilever. To overcome this limitation, we have developed with R. Willaert a nanomotion detector based on an optical microscope.
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Your most recent research has led to the development of a novel and highly efficient technique for rapid antibiotic susceptibility testing using optical microscopy. Please could you tell us why the development of rapid, affordable, and efficient testing methods is so important in the world of antimicrobial resistance?
Rapid antibiotic susceptibility testing could reduce the use of broad-spectrum antibiotics. Traditional ASTs based on replication rate require 24 hours (but up to 1 month in the case of tuberculosis) to identify the most effective antibiotic. Doctors prescribe broad-spectrum antibiotics between the patient’s admission to a medical center and the results of the AST.
These drugs quickly improve patients’ conditions but, unfortunately, promote resistance. A rapid AST that could identify the most suitable antibiotic within 2-4 hours would eliminate broad-spectrum antibiotics and 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 simple-to-use tests are needed.
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Were there any limitations and obstacles you faced in the research process? If so, how did you overcome them?
Antibiotic sensitivity detection with ONMD is very similar to the AFM-based technique. As long as the bacterium is alive, it oscillates; if the antibiotic is effective, it kills the micro-organism, and its oscillations stop. The first limitation we faced when developing the ONMD was our microscopes’ depth of field of view. To prevent the bacteria from leaving the focal plane of the optical microscope during the measurement, we had to constrain the microbes into microfluidic channels a few micrometers high.
Microfabrication of such devices is relatively straightforward in an academic environment, but we were looking for simpler solutions. One option for constructing such a device is to use 10-micron double-sided rubber tape. It allows you to “build” a microfluidic chamber in 5 minutes with two glass coverslips and a puncher.
Another challenge was nanoscale motion detection. For this purpose, we used freely available cross-correlation algorithms that achieve sub-pixel resolution. (i.e., a few nanometers). We first developed the ONMD for larger organisms, such as yeast cells, and expanded the method 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 new rapid AST technique. How did your areas of expertise and research backgrounds complement each other in developing 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 applying AFM to clinically relevant problems. The two teams were supported by a joint grant from the Swiss National Science Foundation and the Research Foundation Flanders (FWO) which enabled the development of the method.
The field of antimicrobial resistance requires a high level of international collaboration, with everyone working together to achieve a common goal. With antimicrobial resistance rising to dangerously high levels in all parts of the world, how important is collaboration in this field?
Our project required expertise in various fields, such as microbiology, microscopy, microfluidics, programming, and data processing. In the development of rapid AST instruments and many others, only a multidisciplinary approach and close collaboration between teams with complementary expertise is today the only path to success.
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You and Dr. Willaert have said, ‘The simplicity and efficiency of the method make it a game-changer in the field of AST.’ Can you please expand on what makes ONMD a game changer in the AST field 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 easily implemented in developing countries to limit the spread of resistant strains. The cheaper and simpler the technique, the more likely it is to be used on a large scale. We are convinced that the ONMD approach can meet these requirements. ONMD could also be used for drug discovery or basic research.
While we recognize the importance of rapid AST, what next steps must be taken before this technique can be used globally in research and clinical landscapes?
For fundamental research, there are no other important 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 have to be used, which have yet to be manufactured.
There is a rapidly expanding need for efficient AST globally; however, the need for affordable, accessible, and simple techniques are of grave importance in developing countries disproportionately affected by antibiotic resistance due to 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 are confident that ONMD-based AST testing can soon be implemented in research centers in both developed and developing countries. However, accreditation by the health authorities is necessary to use it as a standard diagnostic tool; this process can take several years, depending on the government health policy.
What’s next for you and your research? Are you involved in any exciting upcoming projects?
We want to develop a self-contained device for extreme environments. It would consist of a small microscope equipped with a camera and a data processing unit. The microfluidic part of the device could contain different antibiotics ready to be tested.
The ONMD technique could also monitor contamination levels in enclosed environments such as submarines, spacecraft, and space stations. One of our recent projects is funded by the European Space Agency (ESA) to develop a rapid antifungal susceptibility test that could work in microgravity. Additionally, ONMD could be used for even more exciting projects, such as chemistry-independent life detection in the search for extraterrestrial life.
Where can readers find more information?
Villalba MI, Rossetti E, Bonvallat A, Yvanoff C, Radonicic V, Willaert RG*, Kasas S.*.Simple 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 a fascinating 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 be used not only for fast antimicrobial sensitivity testing but also to explore the metabolism of unicellular organisms. We hope our efforts will permit us to expand the application domains of ONMD.
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In a recent study published in the journal Clinical Microbiology and Infection, researchers in Spain, summarized the current understanding of the emergence and ecologic niches of Candida auris.
C. auris was first identified in a Japanese inpatient in 2009. The United States (US) Centers for Disease Control and Prevention (CDC) categorized the pathogen as an urgent threat. Moreover, C. auris has been designated as a fungal pathogenic species of critical concern by the World Health Organization (WHO) in their fungal priority list in October 2022.
Five clonally distinct clades of this fungus emerged independently and concurrently on three continents. Whole-genome sequencing of 47 isolates identified many single nucleotide polymorphisms (SNPs) with minimal intra-regional genetic diversity, suggesting a near-contemporary emergence in distinct geographic locations.
In the present study, the authors discussed the likely pathways of the emergence of C. auris and the role of inter-species transmission. In doing so, the study postulates that climate change has played a major role in high thermotolerant C. auris emergence. Thus, hypothesizing that climate change induced an environmental ancestor to become pathogenic through thermal adaptation.
Hypothetical emergence due to global warming
Global warming is proposed as the likely explanation for the independent and contemporary emergence of distinct C. auris clades. Few fungal species are pathogenic in endothermal animals and humans; very few fungi thrive at mammals’ high basal temperatures, creating a thermal barrier preventing infections.
Several reports suggest that increasing environmental temperatures due to climate change may result in the selection of thermotolerant fungal lineages that can circumvent the thermal barrier and infect/colonize endothermic animals.
One study showed that C. auris could grow at elevated temperatures than its close phylogenetic relatives. In addition, the remarkable halotolerance exhibited by this fungus suggested that it could have previously existed as an environmental species in wetlands/marshes.
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These ancestors might have become pathogens in humans after gaining thermotolerance due to climate change adaptation. Nevertheless, this hypothesis cannot explain the geographic dispersion of the independently evolved clades of C. auris.
Ecological niche(s) of C. auris
The first environmental isolates of C. auris were reported in 2021 from a salt marsh in the Andaman Islands and recently in Colombian estuaries. Notably, one of the isolates was less multidrug-resistant and less heat-tolerant.
It was also significantly different from clinical isolates suggesting a higher similarity to its wild ancestors from marine ecosystems.
C. auris also exhibits high-stress resistance, allowing for continued survival in stressful environments. This plasticity might contribute to its emergence and growing prevalence. Further, this fungus was detected in stored but not freshly pickled apples in India, suggesting a new human transmission pathway and a possible selection route for drug-resistant isolates in agriculture, storage, and supply chains.
Isolation from animal cultures or the environment has not been documented yet. Nonetheless, a study employing in silico DNA metabarcoding screened the internal transcribed spacer region in public datasets.
DNA metabarcoding identified partial matches in non-human sources, such as activated sludge, air dust, the ear canal of a dog with otitis, peanut fields, and the skin of newts. This provided evidence of the ubiquitous presence of the fungus in anthropogenic and natural environments.
One Health approach to understand and manage C. auris
One Health is an integrative, collaborative, multi/trans-disciplinary approach for sustainable balance and optimization of the health of humans, animals, and ecosystems.
Zoophilic fungi and, hypothetically, C. auris might have a dual life cycle wherein host, and environmental reservoirs may serve as durable sources of propagules. This might contribute to the global rise of emergent fungal diseases across continents.
Concluding remarks
The striking plasticity and the ability of C. auris to adapt to harsh environments could allow the fungus to thrive in sludge, wastewater, and fresh/marine waters.
Global warming, the impact of changes in the environment and human population, and indiscriminate antifungal use in agriculture might have led to C. auris evolving into a much more resistant/invasive pathogen that can infect/colonize endothermic animals.
Aquatic marine hosts could have spread primitive strains to humans. Therefore, adopting the One Health approach can help understand the relationship between animal/human health and ecological changes as factors in the emergence and transmission of fungal pathogens.
A recent study published in the Journal of Fungi used a novel OrbitrapTM high-resolution mass spectrometric technology coupled with liquid chromatography to identify geographically different clades of Candida auris(C. auris) isolates. This proof-of-concept methodology could accurately detect C. auris in the microbiology laboratory.
Over a decade ago, C. auris was first found in East Asia, causing bloodstream infections. Although this fungal infection was initially found in India, South America, South Africa, and the Middle East, it soon prevailed globally.
C. auris soon became a common nosocomial fungal pathogen, particularly among intensive care unit (ICU) patients. As a result, the Centers for Disease Control and Prevention (CDC) has classified C. auris as an urgent threat pathogen.
An important factor that allows C. auris outbreaks worldwide is the improper identification of yeast pathogens in hospital laboratories. Hence, there is an urgent need for accurate and rapid identification of C. auris in hospital laboratories, which can reduce their transmission in healthcare facilities.
Genomic analysis of worldwide C. auris isolates has indicated that around five clades have emerged in the last 20 years, independently and simultaneously. These five distinct geographically restricted clades are clade I: South Asia, clade II: East Asia, clade III: Africa, clade IV: South America, and clade V: Iran. Each clade differs from the other by around ten thousand single-nucleotide polymorphisms.
Each clade has differential resistance to antifungal agents; for example, clade I is more resistant to fluconazole, while clade II exhibits susceptibility. Currently, C. auris isolates belonging to these clades have been introduced to many countries worldwide. Scientists have highlighted the importance of quickly identifying and monitoring these clades to restrict further spread.
C. auris possesses several structurally unique sphingolipids and mannoproteins, enabling it to adhere to medical devices and hospital environments persistently. These proteins also aid in biofilm formation and prevent elimination by common disinfectants.
Several studies have indicated that molecular techniques fail to identify C. auris, whereas matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) technology can accurately identify this fungus at the species level.
The Study and its Findings
102 clinical C. auris strains were selected, representing all five clades. These clades were determined based on a short tandem repeat (STR) typing assay, which was subsequently compared to whole-genome sequencing results.
The current study applied OrbitrapTM high-resolution mass spectrometric technology to identify C. auris based on protein analysis methods. This technique was combined with liquid chromatography (LC) for initial separation. In this method, electrospray ionization (ESI) transfers proteins into the gas phase for ionization and is subsequently introduced to the mass spectrometer (LC-MS).
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Mass analysis is conducted by either fragment ions or intact mass (MS) through tandem mass spectrometry (MS/MS). Some of the key features of the OrbitrapTM mass analyzer are a high resolution of up to 200,000, a high mass-to-charge ratio of 6,000, high mass accuracy between 2 and 5 ppm, and a dynamic range greater than 104.
C. auris clade differentiation using monoisotopic mass measurements depicted as heat map. Color scale ranges from blue (max signal) to dark red (no signal), representing abundance of measured monoisotopic masses in each strain. Clade specific differential protein masses are visible from the rectangular vertical boxes indicating the geographic affiliation and clade assignment and its vertically associated dendrogram indicating observed protein masses (columns vs. rows). X-axis indicating clade assignment and y-axis indicating observed MS1 protein masses.
In addition, this method is highly sensitive and can measure the exact mass of a compound. It can also identify minor structural changes due to a translated single nucleotide polymorphism into an amino acid change.
Importantly, this newly developed technology could identify all C. auris isolates with high confidence. Furthermore, it could differentiate C. auris across clades. Even though a limited number of isolates were present from each clade, this spectrometric technology identified C. auris clades with 99.6% identification accuracy.
Based on a principal component analysis (PCA) and a subsequent affinity clustering study, the South Asian, East Asian, and Iranian C. auris clades were more proteomically closely related. Long branches in the affinity clustering analysis suggested that the C. auris strains were present as outliers that required more attention, regardless of the detection technique.
Proteomic typing results indicated the capacity to track strains of the same origin isolated from diverse geographical locations. In the future, more precise matching and alignment of typing schemes (based on next-generation sequencing) is required to build on these results. This would significantly reduce false identifications and classifications of unknown strains associated with new clades or lineage.
Conclusions
Although the workflow linked to mass spectrometry and next-generation sequencing are not directly comparable, their results are similar, i.e., identifying unknown clinical microbes. The standard next-generation sequencing method is a highly time-consuming process that requires many delicate time-intensive quality-control steps, particularly during multiplexed sample runs.
In contrast, the newly developed methodology can provide results within 60 minutes. Therefore, applying the high-resolution OrbitrapTM mass spectrometer to accurately and rapidly identify C. auris clades is an attractive alternative to conventional platforms.
Journal reference:
Jamalian, A. et al. (2023) “Fast and Accurate Identification of Candida auris by High Resolution Mass Spectrometry”, Journal of Fungi, 9(2), p. 267. doi: 10.3390/jof9020267, https://www.mdpi.com/2309-608X/9/2/267
Microbiome: From Research and Innovation to Market
