Determinants of fluconazole resistance and echinocandin tolerance in C. parapsilosis isolates causing a large clonal candidemia outbreak among COVID-19 patients in a Brazilian ICU

Figures & data

Table 1. Oligonucleotides used to generate mutant C. parapsilosis isolates carrying TAC1^L518F and FKS1^E1393G.

Production of the guide RNA The protospacer sequence/PAM site is indicated, followed by the 23-mers (TOP and BOT oligos) for generating the guide RNA. Overhangs are highlighted in bold
TAC1-g1 (sequence)	5^′-GTGGCTGATGAGGCATTACT/TGG-3^′
TAC1-g1-TOP	5^′-CCAGTGGCTGATGAGGCATTACT-3^′	Annealing of the 23-mer to produce the guide RNA that was cloned into SapI-digested pCP-tRNA
TAC1-g1-BOT	5^′-AACAGTAATGCCTCATCAGCCAC-3^′
FKS1-g1 (sequence)	5^′-AGTTGATTGAAAGAGGTGTG/TGG-3^′
FKS1-g1-TOP	5^′-CCAAGTTGATTGAAAGAGGTGTG-3^′	Annealing of the 23-mer to produce the guide RNA that was cloned into SapI-digested pCP-tRNA
FKS1-g1-BOT	5^′-AACCACACCTCTTTCAATCAACT-3^′
Synthesis of RTs The sequence of each RT is indicated, followed by the long oligos (TOP and BOT) used for primer extension. The SNPs are highlighted in bold, and described on the right (the numbering refers to the ORF); syn = synonymous
TAC1-RT-1 (sequence)	5^′-GGCGACGAATTGGATCGTCAAATGTCGATTGCAGTGGCTGATGAGGCATTGTTTGGAGATCCAGCACTACCACTCAGTTTTCGATTGTTGAAAAAGTTGA-3^′	SNPs introduced: A1551G [syn SNP]; C1552T [non syn SNP]
TAC1-RT-1-TOP	5^′-GGCGACGAATTGGATCGTCAAATGTCGATTGCAGTGGCTGATGAGGCATTGTTTGGAGAT-3^′
TAC1-RT-1-BOT	5^′-TCAACTTTTTCAACAATCGAAAACTGAGTGGTAGTGCTGGATCTCCAAACAATGCCTCAT-3^′
FKS1-RT-1 (sequence)	5^′-TCTTCATTTCGTTCATTCCATTGGTTGTTCAAGGGTTGATTGAAAGAGGAGTCTGGAAAGCTTGTCAAAGATTTGTTAGACATTTCATTTCGTTGTCACC-3^′	A4178G [non syn SNP]; G4197C [syn SNP]; T4194A [syn SNP]
FKS1-RT-1-TOP	5^′-TCTTCATTTCGTTCATTCCATTGGTTGTTCAAGGGTTGATTGAAAGAGGAGTCTGGAAAG-3^′
FKS1-RT-1-BOT	5^′-GGTGACAACGAAATGAAATGTCTAACAAATCTTTGACAAGCTTTCCAGACTCCTCTTTCA-3^′
Sequencing of mutated loci
sTAC1-F	5^′-GGTATGCTCAGGAGATTGGA-3^′	Sequencing of TAC1 edited site
sTAC1-R	5^′-ATAGTTCCACGTTCAGGCTC-3^′
sFKS1-F	5^′-CGGACATCCTGGTTTCCATA-3^′	Sequencing of FKS1 edited site
sFKS1-R	5^′-CAATGAAGACAACGAAGCCC-3^′

Figure 1. Expression profile of ERG11 (A), MDR1 (B), and CDR1 (C) from a selected number of C. parapsilosis isolates (n = 8) after exposure to fluconazole, which showed that fluconazole-resistant isolates (FLZR) significantly overexpressed CDR1 relative to susceptible ones. C. parapsilosis isolates grown at logarithmic phase were subjected to one dilution below MIC of fluconazole for 90 min, and after RNA extraction, relative gene expression was assessed as described in methods section.

Table 2. Minimum inhibitory concentration of antifungal agents used against Candida parapsilosis isolates (n = 60). The number of isolates for each concentration of a given drug is indicated.

Antifungal agent	Minimum inhibitory concentration (µg/ml)													MIC50	MIC90	GM
	0.016	0.032	0.06	0.12	0.25	0.5	1	2	4	8	16	32	64
Fluconazole						4	3			53^a				8	16–32	6.64
Voriconazole	4	1	2		53									0.032	0.5	0.192
Micafungin						3	4	53						4	8	3.52
Anidulafungin						1	6	53						4	8	3.56
Amphotericin B					1	47	12							0.5	1	0.567

^a Note that 19 strains carrying K143R in Erg11 and L518F in Tac1 had fluconazole MICs >8 µg/ml.

Table 3. The characteristics of Candida parapsilosis isolates selected for sequencing and gene expression analysis. The expression profile values of the genes studied are based on the average ± standard deviation.

Strain #	FLZ (µg/ml)	VOR (µg/ml)	MICA (µg/ml)	ANI (µg/ml)	HS2-Fks1	Erg11	CDR1 expression	ERG11 expression	MDR1 expression	Tac1	Upc2	Mrr1
1	8	0.25	2	2	E1393G	WT	2.64 ± 0.94	0.19 ± 0.004	9.32 ± 1.86	L518F	WT	WT
33	16	0.25	2	2	E1393G	WT	5.91 ± 0.80	0.11 ± 0.02	7.16 ± 1.37	L518F	WT	WT
40	8	0.25	2	2	E1393G	WT	4.57 ± 0.39	0.31 ± 0.03	3.25 ± 0.38	L518F	WT	WT
51	8	0.25	2	2	E1393G	WT	3.69 ± 0.53	0.24 ± 0.03	2.95 ± 0.98	L518F	WT	WT
7	8	0.25	2	2	E1393G	K143R	2.93 ± 1.36	0.16 ± 0.01	8.6 ± 2.3	L518F	WT	WT
27	16	0.25	2	2	E1393G	K143R	5.81 ± 0.37	0.29 ± 0.07	9.50 ± 2.2	L518F	WT	WT
10	1	0.03	1	1	WT	WT	0.52 ± 0.16	0.24 ± 0.02	7.91 ± 1.11	WT	WT	WT
20	0.5	0.015	0.5	0.5	WT	WT	0.74 ± 0.25	0.20 ± 0.205	3.83 ± 1.08	WT	WT	WT

FLZ: Fluconazole; VOR: Voriconazole; MICA: Micafungin; ANI: Anidulafungin.

Figure 2. The expression profile of CDR1 for parental strain ATCC 22019 and its mutants carrying L518F in Tac1 and the micafungin tolerance of ATCC 22019 and it mutant carrying E1393G in Fks1. Mutants carrying TAC1^L518F (from two independent mutants) had a significantly higher basal expression of CDR1, which was not induced upon fluconazole exposure (A and B). Mutants carrying FKS1^E1393G had a significantly higher tolerance to micafungin (4 µg/ml), which shows the average of two independent mutants.

Table 4. The minimum inhibitory concentration of antifungal drugs against the mutants carrying L518F in Tac1 and E1393G in Fks1 and their parental wild-type (WT) strain.

Strain	Phenotype	Fluconazole (µg/ml)	Voriconazole (µg/ml)	Micafungin (µg/ml)
WT	Susceptible	0.5	0.03	2
L518F	Fluconazole non-susceptible	4	0.125	2
E1393G	Micafungin tolerant	0.5	0.03	2

Figure 3. Minimum spanning tree of C. parapsilosis isolates.

Figure 4. Biofilm formation of fluconazole resistant (FLZR-WT and FLZR-K143R) and fluconazole susceptible isolates. The Y-axis represents biofilm production as a function of absorption at OD490.