Comparative genomics for the elucidation of multidrug resistance (MDR) in Candida lusitaniae

Abstract

Multidrug resistance (MDR) has emerged in hospitals due to the use of several agents administered in combination or sequentially to the same individual. We reported earlier MDR in Candida lusitaniae during therapy with amphotericin B (AmB), azoles and candins. We used here comparative genomic approaches between the initial susceptible isolate and 4 other isolates with different MDR profiles. From a total of 18 non-synonymous SNPs (NSS) in genome comparisons with the initial isolate, six could be associated with MDR. One of the SNPs occurred in a putative transcriptional activator (MRR1) resulting in a V668G substitution in isolates resistant to azoles and 5-fluorocytosine (5-FC). We demonstrated by gene editing that MMR1 acted by upregulation of MFS7 (a multidrug transporter) in the presence of the V668G substitution. MFS7 itself mediated not only azole resistance but also 5-FC resistance, which represents a novel resistance mechanism for this drug class. Three other distinct NSS occurred in FKS1 (a glucan synthase that is targeted by candins) in three candin-resistant isolates. Lastly, two other NSS in ERG3 and ERG4 (ergosterol biosynthesis) resulting in non-sense mutations were revealed in AmB-resistant isolates, one of which accumulated the 2 ERG NSS. AmB-resistant isolates lacked ergosterol and exhibited sterol profiles consistent with ERG3 and ERG4 defects. In conclusion, this genome analysis combined with genetics and metabolomics helped to decipher the resistance profiles identified in this clinical case. MDR isolates accumulated 6 different mutations conferring resistance to all antifungal agents used in medicine. This case study illustrates the capacity of C. lusitaniae to rapidly adapt under drug pressure within the host.\n\nImportanceAntifungal resistance is an inevitable phenomenon when fungal pathogens get exposed to antifungal drugs. These drugs can be grouped in 4 distinct classes (azoles, candins, polyenes, pyrimidine analogs) and are used in different clinical settings. Failures in therapy implicates the sequential or combined use of these different drug classes, which can result in some cases in the development of multidrug resistance (MDR). MDR is particularly challenging in the clinic since it drastically reduces possible treatment alternatives. In this study, we report the rapid development of MDR in Candida lusitaniae in a patient, which became resistant to all known antifungal agents used up to now in medicine. To understand how MDR developed in C. lusitaniae, whole genome sequencing followed by comparative genome analysis was undertaken in sequential MDR isolates. This helped to detect all specific mutations linked to drug resistance and explained the different MDR patterns exhibited by the clinical isolates.