Antifungal susceptibility profiles and drug resistance mechanisms of clinical lomentospora prolificans isolates

Lomentospora prolificans is an opportunistic fungal pathogen with low susceptibility to current antifungal drugs. Here, we tested the in vitro susceptibility of 8 drugs against 42 clinical L. prolificans isolates. All isolates showed high MICs to voriconazole (MIC₉₀>16 μg/ml), itraconazole (MIC₉₀>16 μg/ml), posaconazole (MIC₉₀>16 μg/ml), isavuconazole (MIC₉₀>16 μg/ml), amphotericin B (MIC₉₀>16 μg/ml), and terbinafine (MIC₉₀>64 μg/ml) and high minimum effective concentrations (MECs) to micafungin (MEC₉₀>8 μg/ml), with the exception of miltefosine showing an MIC₉₀ value of 4 μg/ml. We examined six different in vitro drug combinations and found that the combination of voriconazole and terbinafine achieved the most synergistic effort against L. prolificans. We then annotated the L. prolificans whole genome and located its Cyp51 and Fks1 genes. We completely sequenced the two genes to determine if any mutation would be related to azole and echinocandin resistance in L. prolificans. We found no amino acid changes in Cyp51 protein and no tandem repeats in the 5= upstream region of the Cyp51 gene. However, we identified three intrinsic amino acid residues (G138S, M220I, and T289A) in the Cyp51 protein that were linked to azole resistance. Likewise, two intrinsic amino acid residues (F639Y, W695F) that have reported to confer echinocandin resistance were found in Fks1 hot spot regions. In addition, three new amino acid alterations (D440A, S634R, and H1245R) were found outside Fks1 hot spot regions, and their contributions to echinocandin resistance need future investigation. Overall, our findings support the notion that L. prolificans is intrinsically resistant to azoles and echinocandins.