Raman Study of Pathogenic Candida auris: Imaging Metabolic Machineries in Reaction to Antifungal Drugs

Abstract

The multidrug-resistant Candida auris often defies treatments and presently represents a worldwide public health threat. Currently, the ergosterol-targeting Amphotericin B (AmB) and the DNA/RNA-synthesis inhibitor 5-flucytosine (5-FC) are the two main drugs available for first-line defense against life-threatening Candida auris infections. However, important aspects of their mechanisms of action require further clarification, especially regarding metabolic reactions of yeast cells. Here, we applied Raman spectroscopy empowered with specifically tailored machine-learning algorithms to monitor and to image in situ the susceptibility of two Candida auris clades to different antifungal drugs (LSEM 0643 or JCM15448T, belonging to the East Asian Clade II; and, LSEM 3673 belonging to the South African Clade III). Raman characterizations provided new details on the mechanisms of action against Candida auris Clades II and III, while also unfolding differences in their metabolic reactions to different drugs. AmB treatment induced biofilm formation in both clades, but the formed biofilms showed different structures: a dense and continuous biofilm structure in Clade II, and an extra-cellular matrix with a “fluffy” and discontinuous structure in Clade III. Treatment with 5-FC caused no biofilm formation but yeast-to-hyphal or pseudo-hyphal morphogenesis in both clades. Clade III showed a superior capacity in reducing membrane permeability to the drug through chemically tailoring chitin structure with a high degree of acetylation and fatty acids networks with significantly elongated chains. This study shows the suitability of the in situ Raman method in characterizing susceptibility and stress response of different C. auris clades to antifungal drugs, thus opening a path to identifying novel clinical solutions counteracting the spread of these alarming pathogens.

Raman Spectroscopy of Oral Candida Species: Molecular-Scale Analyses, Chemometrics, and Barcode Identification

“…In support of this assertion, one could notice that C. krusei, which is also studied here, has been found capable to induce severe levels of candidemia after developing echinocandin resistance during caspofungin therapy [102]. Moreover, Raman analyses similar to those proposed in this study have previously been applied to C. auris clades II and III in comparison with C. albicans, and showed capable to reveal structural details at the molecular scale, which relate to both virulence and drug resistance characteristics [32,33].…”

“…Chemometrics was then applied to probe for statistical validity and a Raman barcode approach employed to thoroughly classify different species. The analytical approaches followed in this study were in line with our recently published Raman studies of Candida auris clades [32,33], and bacteria and virus variants [34,35]. They also lie in the stream of Raman studies by other authors about the characterization of Candida isolates and related biofilms, which were performed with the aim to link the observed Raman spectra to genomic diversity [36][37][38][39].…”

Raman Metabolomics of Candida auris Clades: Profiling and Barcode Identification

“…The relative intensity ratio, R i/i = I 1644 / I 1660 , namely, the Raman ratio of intermolecular-to-intramolecular hydrogen bonds, could be taken as a parameter measuring the degree of acetylation of α-chitin: the higher the ratio, the more acetylated the structure. As discussed later, a highly acetylated structure of chitin serves to reduce membrane permeability against external molecules, including drugs [ 23 ].…”

“…In our previous Raman studies of Candida species/clades [ 22 , 23 , 24 ], we have interrogated spectral profiles in search of peculiar chemical fingerprints of speciation, virulence, and drug resistance. In addition to using machine-learning procedures, we have also proposed a new bioinformatics approach referred to as “Raman barcoding”.…”

In Situ Raman Analysis of Biofilm Exopolysaccharides Formed in Streptococcus mutans and Streptococcus sanguinis Commensal Cultures

“…Since no dehydration or stain/markers are needed to obtain Raman spectra, Raman measurements can be performed in situ with a procedure compatible with bacterial life. In previous studies [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ], we have performed Raman spectroscopic analyses of cells, bacteria, pathogenic yeasts, and viruses upon exploiting a machine-learning algorithm linked to a library of Raman spectra from elementary molecules. The analytical approach followed in this study is in line with those previous studies and represents a first attempt to their further extension to the analysis of bacterial biofilms.…”