When I joined the pathogenic fungi research community to conduct my PhD at the laboratory of Molecular Cell Biology at KU Leuven, the fungus Candida auris was in the midst of its infamous stardom. This new bug was discovered just 10 years earlier, in 2009, in an ear infection of a 70-year-old Japanese woman. Back then, nobody could have predicted that it would be the first fungus on the CDC list of urgent antimicrobial threats, ten years later. Although initially, I was working on polymicrobial biofilms, this “new kid on the block” intrigued me, and soon, a “small side project” was my major focus. In our three latest Springer Nature articles, we take a closer look at how experimental evolution can be used to study antifungal resistance in MDR fungi like Candida auris. 

Candida auris behaves like a true multidrug-resistant nosocomial pathogen, comparable to ‘hospital bugs’ like MRSA

The word “auris” refers to the Latin word for “ear”, a name somehow misleading as in the majority of case reports since its formal description, C. auris infections manifest in invasive infections and candidemia with mortality rates often surpassing 50%. In less than ten years since its description, C. auris infections had been reported on all inhabited continents. C. auris appears to have simultaneously emerged on different continents with no clear environmental origin, and its sudden emergence has remained the topic of much speculation. C. auris is substantially different from any other pathogenic fungus or Candida species: it behaves like a true multidrug-resistant nosocomial pathogen, comparable to “hospital bugs” like MRSA. Besides an extreme ability to persist in health care environments – it has been coined “a fungal barnacle” – it is able to develop antifungal multidrug resistance to an extent that has not been observed before. 

But how? How can C. auris evolve resistance to all major antifungal drug classes – there are only four – and remain fit enough to infect patients? Does it employ novel mechanisms of resistance? Or is it just very stress tolerant? Is it truly special? These were – and still are – the questions that dominate my research focus since 2019. Back then, the majority of molecular antifungal drug resistance research employed GWAS (genome-wide association studies) or sequencing consecutive clinical isolates over time, to pinpoint mutations that drive antifungal drug resistance. But a handful of groups started using experimental evolution coupled to whole genome sequencing. Experimental evolution is a straightforward way of obtaining resistant isolates from susceptible parents. As such, resistant isolates can be directly compared to their susceptible origins and quantitative analyses can be done. In addition, experimental evolution offers a controlled and repeatable method to study antifungal drug resistance, allowing insights into the molecular mechanisms of resistance, but also the eco-evolutionary dynamics of resistance. Nevertheless, it also oversimplifies real-world complexity, such as host interactions and microbial community dynamics — every method has its trade-off.

We saw that experimental evolution had a lot of potential, and still think it can offer a mainly untapped source of insight into antifungal drug resistance dynamics. This motivated us to write a review on the potential of this method in molecular mycology, now published in NPJ Antimicrobials and Resistance [1].

We started evolving Candida auris to become resistant to all major antifungal drug classes consecutively. We evolved them for 30 days to drug A, then switched to drug B, then to drug C, or the other way around. Sometimes, consecutive evolution experiments would fail – the culture died out. We thought this was just bad luck.   

We were also focused on the mechanisms of resistance [2], not on the dynamics hereof. Luckily, near the same period late 2019, I attended a presentation of Dr. Leila Imamovic, about how Escherichia coli showed collateral sensitivity – whilst acquiring resistance to one drug, it simultaneously got more sensitive to one or more other drugs [3]. A fitness trade-off of potentially immense importance in treatment design. This collateral sensitivity could maybe explain why some of our evolution experiments failed? Or why we didn’t succeed in creating a pan-resistant C. auris strain?

Collateral sensitivity can be a powerful force in counteracting resistance evolution, opening new therapeutic avenues to mitigate antifungal resistance

Next, we set up a large experiment, in which we performed experimental evolution in 60 replicates per drug for 10 different drugs, and obtained hundreds of resistant lineages. Next, we systematically mapped their susceptibility to a range of antifungal drugs, including repurposed antifungal agents. We observed how isolates resistant to one drug, for example amphotericin B, were more sensitive to another drug, like caspofungin. But also the opposite was observed: cross-resistance – isolates resistant to amphotericin B were, for example, also resistant to azoles. We looked into the robustness of these phenotypes by investigating the mutations that confer resistance and collateral sensitivity, and by testing resistant strains in other backgrounds from other C. auris clades. For some of the trends, a conservative and robust collateral sensitivity or cross-resistance trend was evident. Next, we used experimental evolution and mathematical modelling to demonstrate that switching or combining drug pairs that exert collateral sensitivity can actively prevent the evolution of resistance. In addition, competition experiments showed that a switch to a drug to which resistant cells are collateral sensitive can actively eliminate resistant subpopulations. These findings, published in Nature Microbiology [4], demonstrate that collateral sensitivity can be a powerful force in counteracting resistance evolution, opening new therapeutic avenues to mitigate antifungal resistance. This is however, just the beginning. The exploitation of collateral sensitivity dynamics in clinical and in vivo environments, has yet to be shown, while further robustness studies, including other fungal species, will gain major insights into the applicability of collateral sensitivity-based treatments to counteract antifungal drug resistance. 

Among the repertoire of antifungal drugs we used in our evolution experioments, one drug always stood out. It's old, it's toxic, it’s very potent and over 50% of clinical isolates of C. auris show resistance to it: amphotericin B.

By using both in vitro and in vivo experimental evolution, we found that amphotericin B resistance can be acquired by four major mechanisms, but most come at a high cost: resistant strains exhibited severe fitness trade-offs, often growing more slowly and showing increased environmental stress sensitivity. However, we also observed that strains can mitigate these costs through compensatory evolution, regaining fitness while retaining resistance. This mirrors evolutionary patterns observed in bacterial resistance, highlighting the adaptability of C. auris. Our study also identified novel sterol biosynthesis mutations and chromosomal alterations driving amphotericin B resistance, shedding light on previously uncharacterised mechanisms of resistance evolution in this emerging pathogen [5].

Together, these studies underscore the power of experimental evolution in unraveling the complex interplay between antifungal resistance, fitness trade-offs, and treatment strategies. By systematically evolving C. auris under controlled conditions, we were able to dissect how resistance emerges, which constraints it faces, and how we might use these insights to design better therapies. While C. auris remains a formidable adversary, our findings suggest that evolution itself can be leveraged as a tool against resistance. Currently, we are expanding this research by further investigating the evolutionary dynamics of resistance in C. auris and other multidrug-resistant fungal species, with a focus on the complex in vivo environment, the potential of repurposed drugs, influence of drug tolerance and more. By strategically exploiting collateral sensitivity and understanding resistance dynamics in complex environments, we might turn fungal adaptability against itself – an evolutionary paradox that could shape future antifungal treatment strategies.

[poster image created by ChatGPT4o - OpenAI].