Candida albicans, a predominant human fungal pathogen, causes both mucosal and bloodstream infections (BSIs), and fluconazole is one of the most widely prescribed antifungal agents used to treat these infections [1, 2]. Acquired fluconazole resistance (FR) in C. albicans has been reported at its highest frequency in HIV-infected patients with oropharyngeal candidiasis as well as in patients with recurrent vaginal candidiasis [2, 3]. The long-term use of fluconazole for prophylaxis or treatment of mucosal C. albicans infections can lead to selective pressure, resulting in the emergence of acquired FR in C. albicans [2, 3]. The main mechanisms responsible for acquired FR in C. albicans from mucosal infections include mutations or overexpression of ERG11, which encodes an enzyme targeting the drug (lanosterol 14α-demethylase), and the overexpression of genes encoding efflux pumps (CDR1, CDR2, and MDR1) [2, 4, 5]. The overexpression of FR-associated genes occurs mainly due to gain-of-function (GoF) mutations in the transcription factor-encoding genes TAC1 (involved in CDR1 and CDR2 regulation), MRR1 (involved in MDR1 regulation), and UPC2 (involved in ERG11 regulation) [2, 5^-7].

In contrast to mucosal isolates with FR rates of 12%–22%, the rates of FR among BSI isolates of C. albicans are low (0.06%–2.3%); this could be partly because of the relatively short-term use of antifungal agents for the treatment of candidemia [2, 8^-10]. Among 2,712 C. albicans BSI isolates obtained from Korean multicenter surveillance studies during 2006–2021, only 14 (0.5%) were determined to be fluconazole non-susceptible (FNS; minimum inhibitory concentration [MIC] ≥4 μg/mL). To date, only few studies have characterized the molecular and clinical features of BSI isolates of C. albicans with acquired FR [8]. Therefore, we investigated the gene mutations causing amino acid substitutions (AASs) in ERG11, TAC1, MRR1, and UPC2; their genotypic relationships; and the clinical features of FNS BSI isolates of C. albicans that were submitted to Chonnam National University Hospital from Korean multicenter surveillance studies over 15 years (2006–2021).

We assessed 26 BSI isolates of C. albicans: 11 FR (MIC ≥8 μg/mL), three fluconazole-susceptible dose-dependent (F-SDD; MIC, 4 μg/mL), and 12 control fluconazole-susceptible (FS; MIC, 0.25–0.5 μg/mL) isolates. All isolates were identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Bruker Biotyper library v. 4.0; Bruker Daltonics GmbH, Bremen, Germany) or by sequencing the D1/D2 domains of the 26S rRNA gene [11]. In vitro antifungal susceptibility testing was performed with the Sensititre Yeast One system (Thermo Fisher Scientific Inc., Cleveland, OH, USA). Sequence analyses of ERG11, TAC1, MRR1, and UPC2 were conducted as described previously [6, 12, 13]. All isolates were genotyped using multi-locus sequence typing (MLST); each strain was assigned a diploid sequence type (DST) reflecting the combination of the genotypes of seven genes in the MLST database (https://pubmlst.org/organisms/candida-albicans), and a dendrogram was constructed [14]. Clinical information for all 14 patients with FNS C. albicans BSI isolates was collected retrospectively [15]. This study was approved by the Institutional Review Board of Chonnam National University Hospital, Gwangju, Korea (approval No. CNUH-2014-290) that also waived the requirement for informed consent.

Table 1 shows the results of antifungal susceptibility testing and ERG11, TAC1, MRR1, and UPC2 sequencing of all 14 FNS (11 FR [R1–R11] and 3 F-SDD [D1–D3]) and 12 FS (S1–S12) C. albicans BSI isolates. No isolate was found to be resistant to amphotericin B or the three echinocandins. The sequencing results of all 14 FNS C. albicans isolates were compared with those of 12 FS control isolates and previously reported data [2, 6, 7, 16^-20]; five Erg11p, seven Tac1p, and one Mrr1p AASs were found in only the FNS isolates. Of the five Erg11p AASs that were found in only the FNS isolates, two (R264T and D428N) were potentially novel; three AAS (K143R, F145L, and G464S) that were found in eight FNS isolates were confirmed to cause FR through in vitro experiments [16]. Of the seven AASs in Tac1p that were not present in the FS isolates, three (T225A, A736T, and A736V) AASs that were found in six FNS isolates were previously described as GoF mutations [2]; another (R673L) AAS found in one FNS isolate was previously described in azole-resistant isolates [2, 18^-20]. The remaining three (Y269H, L744I, and N972K) Tac1p AASs from four FNS isolates were potentially novel, although N972K occurred at a position already described in FR isolates [19]. Only one substitution in Mrr1p (N33S), which was found in one FNS isolate, was not described previously [2, 7]. Although it is unclear whether the newly identified AASs in this study result in FR, the Erg11p, Tac1p, Mrr1p, and Upc2p AASs that were either novel or described in azole-resistant isolates were found in 8 (57.1%), 11 (78.6%), 1 (7.1%), and 0 (0%) isolates, respectively. A previous study characterized azole resistance mechanisms in five invasive FNS C. albicans isolates that had been collected in 29 countries in 2014 and 2015. The authors found that MDR1 overexpression (three isolates) was more common than CDR2 expression (one isolate) or ERG11 mutation (one isolate) [10]. In contrast, our results suggested that ERG11 mutations and CDR overexpression are likely the dominant mechanisms of FR in C. albicans BSI isolates from Korean hospitals.

Table 1 . Comparison of azole antifungal susceptibility testing and sequencing of azole-resistant related genes between fluconazole non-susceptible and fluconazole susceptible bloodstream isolates of Candida albicans

Isolate No.	MIC (μg/mL)*	Erg11p AAS found in†		Tac1p AAS found in†		Mrr1p AAS found in†		Upc2p AAS found in†
	FLC/VOR/ITRA/POSA	FNS isolates only‡	Both FNS and FS isolates	FNS isolates only‡	Both FNS and FS isolates||	FNS isolates only	Both FNS and FS isolates||	FNS isolates only	Both FNS and FS isolates
R1	> 256/> 8/> 16/>8	None	D116E, K128T	L744I§	N772K, S935L	None	L171P, L248V, V341E	None	None
R2	> 256/> 8/> 16/>8	K143R	D116E, K128T	None	I558V, N772K	None	-	None	I142S
R3	16/0.12/0.25/0.25	K143R	D116E, K128T	A736T	N772K, S935L	None	L171P, L248V, V341E	None	None
R4	16/0.12/0.25/0.25	K143R	D116E, K128T	A736T	N772K, S935L	None	L171P, L248V, V341E	None	None
R5	16/0.12/0.25/0.25	K143R	D116E, K128T	A736T	N772K, S935L	None	N159H, I160T, A162P, L171P, L248V, V341E	None	None
R6	16/0.5/0.5/0.5	R264T§, G464S	D116E, K128T	N972K§	N772K, S935L	None	V27I¶, L171P, L248V, V341E	None	None
R7	8/0.12/0.25/0.25	None	D116E, E266D, V488I	R673L	I895T, N896S	None	L171P, V341E	None	None
R8	8/0.12/0.06/0.03	None	D116E, E266D, V488I	None	I895T, N896S	None	L171P, V341E	None	None
R9	8/0.12/0.25/0.25	F145L	K342R¶	Y269H§	N896S	None	L171P, V341E	None	I142S
R10	8/0.12/0.25/0.25	None	D116E, K128T	A736V	N772K, S935L	None	N159H, I160T, A162P, L171P, L248V, V341E	None	None
R11	8/0.03/0.12/0.06	K143R	D116E, K128T	A736T	N772K, S935L	None	N159H, I160T, A162P, L171P, L248V, V341E	None	None
D1	4/0.12/0.25/0.25	F145L, D428N§	K342R¶	Y269H§	N896S	N33S§	L171P, V341E	None	I142S, P299L
D2	4/0.015/0.06/0.03	None	E266D, V437I	None	K87N, M170I, N174D, F189S	None	L171P, L248K, V341E	None	I142S
D3	4/0.06/0.25/0.25	None	E266D, V488I	T225A	K87N, M170I, N174D, F189S, N772K, N896S	None	-	None	I142S, S190N, S228N
S1	0.5/0.03/0.03/0.015	None	D153E	None	I895T, N896S	None	L171P	None	R68K, I142S, S228N
S2	0.5/0.03/0.06/0.03	None	E266D, V488I	None	N896S	None	S16I, T73K, L171P	None	None
S3	0.25/0.008/0.015/0.015	None	D116E, D153E	None	I895T, N896S	None	L171P	None	R68K, I142S, S228N
S4	0.25/0.015/0.03/0.015	None	D116E, D153E	None	I895T, N896S	None	L171P	None	I142S, S228N
S5	0.25/0.008/0.03/0.03	None	E266D, V437I	None	K87N, A90T, M170I, N174D, F189S, F222L	None	P19L, G75R, N937K, F1032L	None	R68K, I142S, S190N, S228N
S6	0.25/0.008/0.03/0.015	None	E266D, V437I	None	M170I, N174D, F189S	None	N937K, F1032L	None	R68K, I142S, S190N, S228N
S7	0.25/0.008/0.03/0.015	None	E266D, V437I	None	A90T	None	N937K, F1032L	None	R68K, I142S, S190N, S228N
S8	0.25/0.015/0.03/0.03	None	D116E, D153E	None	I895T, N896S	None	-	None	R68K, I142S, S288N, P299L, A300P
S9	0.25/0.015/0.03/0.03	None	D116E, K128T	None	N772K	None	L171P, L248V, V341E	None	None
S10	0.25/0.008/0.015/0.015	None	E266D, V488I	None	N772K, N896S	None	L171P	None	None
S11	0.25/0.008/0.015/0.03	None	E266D, V488I	None	N896S	None	L171P, S219F	None	None
S12	0.25/0.008/0.03/0.015	None	D116E	None	I895T, N896S	None	L171P	None	I142S, S228N

*Antifungal MICs were determined using the Sensititre Yeast One system (Thermo Fisher Scientific Inc., Cleveland, OH, USA); ^†The sequences of the isolates were compared and analyzed based on the reference sequences for ERG11 (GenBank accession No. X13296), TAC1 (GenBank accession No. DQ393587), MRR1 (GenBank accession no. XM711520), and UPC2 (GenBank accession No. EU583451) from C. albicans [6]; homozygous alleles are underlined; ^‡AASs that were previously detected in fluconazole-resistant C. albicans isolates are shown in bold; ^§New AASs (Erg11p R264T and D428N AASs, Tac1p Y269H, N744I, and N972K, and Mrr1p N33S) that were found in the FNS isolates of C. albicans in this study have been deposited into GenBank with accession numbers OQ161592, OQ161593, OQ161595, OQ383350, OQ161594, and OQ161598, respectively; ^||Eight common Tac1p AASs (F104V, S199N, R206H, V207A, N396S, D776N, E829Q, and L941P) and one Mrr1p ASS (E1020Q) that were found in almost all (≥23) isolates were excluded; ^¶Erg11p K342R AAS and Mrr1p V27I AAS were reported previously in FS isolates [7, 17].

Abbreviations: MIC, minimal inhibitory concentration; AAS, amino acid substitution; FLC, fluconazole; VOR, voriconazole; ITRA, itraconazole; POSA, posaconazole; FNS, fluconazole non-susceptible; FS, fluconazole-susceptible.

Of the 14 FNS isolates, 12 exhibited weak FR (MICs: 4–16 mg/L) without voriconazole resistance, whereas the remaining two isolates (R1 and R2) showed high MICs for fluconazole (>256 mg/L) and voriconazole (>8 mg/L). Isolate R1 harbored L744I in Tac1p (new AAS), which might be the major contributor to CDR-mediated azole resistance; isolate R2 harbored Erg11p K143R. Of the five Erg11p K143R isolates with variable MICs for fluconazole (8–>256 mg/L), isolate R2 showed the highest fluconazole MIC; however, it did not show FR-specific Tac1p, Mrr1p, or Upc2p AASs. Two FNS isolates (R8 and D2) did not show any of the FR-associated AASs that were evaluated in this study. CDR1/CDR2 and MDR1 overexpression can be explained by TAC1 and MRR1 GoF mutations, but ERG11 overexpression is not always associated with UPC2 GoF mutations, suggesting the existence of additional regulators [21]. Thus, these isolates might have other resistance mechanisms, such as the overexpression of ERG11, which could not be detected in this study.

The MLST results for the 14 FNS isolates showed that nine isolates had different DSTs, whereas three and two isolates belonged to the DSTs 1179 and 1539, respectively (Fig. 1). Three DST 1179 isolates (R3–R5) were isolated at three different hospitals but had the same AASs in Erg11p (K143R) and Tac1p (A736T). Two isolates of DST 1539 (R9 and D1) were also isolated at different hospitals, and they had similar Erg11p AASs (F145L and F145L+D428N) and the same Tac1p (Y269H) AAS. TAC1 is located on the left arm of chromosome 5, where ERG11 is also located, and a combination of TAC1 and ERG11 point mutations has been suggested to contribute to an increased MIC for fluconazole among azole-resistant isolates [17]. Overall, 50.0% (7/14) of the FNS isolates showed combined Erg11p and Tac1p AASs in this study.

Figure 1. Dendrogram based on a combination of seven housekeeping genes (AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b) of 14 fluconazole non-susceptible Candida albicans isolates, constructed based on the UPGMA using MEGA 11 software [¹⁴]. Three isolates of DST 1179 (R3–R5) are more closely related to four FNS isolates (R1, R6, R10, and R11), all of which share the same Erg11p (D116E and K128T), Tac1p (N772K and S935L), and Mrr1p (L171P, L248V, and V341E) AASs. See Table 1 for detailed information regarding each isolate.
Abbreviations: UPGMA, unweighted pair group method with arithmetic averages; DST, diploid sequence type; MLST, multi-locus sequence typing; AAS, amino acid substitution; FNS, fluconazole non-susceptible.

The clinical features of all 14 patients are summarized in Table 2. All 14 patients were adults with various underlying diseases, but no patient was infected with HIV. Previous amphotericin B (two patients) or fluconazole (one patient) exposure was identified in only three patients, indicating that almost all (92.9%) FNS C. albicans BSI isolates were from patients not previously exposed to azole. Among the 14 patients with FNS BSIs, eight had a fatal outcome within 30 days, three (R5, R7, and D3) died without receiving antifungal therapy, two (R4 and D1) died despite fluconazole therapy, and three (R1, R6, and R8) with a hematological malignancy died despite >3 days of echinocandin or amphotericin B therapy. The overall 30-day mortality rate of the patients was 57.1% (8/14), which was higher than the mean 30-day mortality rate (36.4%, 123/338) of patients with C. albicans BSIs reported at 11 Korean hospitals from 2017 to 2018, although the difference was not significant [15].

Table 2 . Clinical features of 14 patients with fluconazole non-susceptible bloodstream isolates of Candida albicans

Isolate No.	Age (yr)/sex	Diagnosis	Prior antifungal exposure	Immuno-suppression	CVC	Duration of fungemia (days)	Antifungal treatment	Patient outcome (days)*
R1	30/F	Acute myeloid leukemia	Yes (AMB)	Yes	Yes	5	AMB, ANI	Death (6)
R2	63/F	Pancreatic cancer	No	Yes	Yes	1	CAS	Death (52)
R3	52/M	Diabetes mellitus	No	No	No	10	AMB	Improved
R4	75/M	COPD	No	No	No	3	FLC	Death (19)
R5	49/F	Breast cancer	No	No	Yes	1	None	Death (2)
R6	52/M	T/NK-cell lymphoma	No	No	Yes	5	CAS	Death (7)
R7	74/M	Diabetes mellitus	No	No	Yes	1	None	Death (1)
R8	74/F	Chronic myeloid leukemia	Yes (FLC)	Yes	No	2	CAS	Death (4)
R9	62/M	Rheumatoid arthritis	No	No	Yes	4	MICA	Improved
R10	79/F	Traumatic subdural hemorrhage	No	No	Yes	1	FLC	Improved
R11	80/M	Spinal abscess	No	No	Yes	2	CAS	Improved
D1	79/M	Diabetes mellitus	Yes (AMB)	No	Yes	8	FLC	Death (15)
D2	43/M	Down syndrome	No	Yes	Yes	6	AMB	Improved
D3	67/F	Fulminant myocarditis, COPD	No	No	Yes	1	None	Death (3)

*Time to death after the first positive culture.

Abbreviations: F, female; M, male; COPD, chronic obstructive pulmonary disease; AMB, amphotericin B; FLC, fluconazole; CVC, central venous catheter; ANI, anidulafungin; CAS, caspofungin; MICA, micafungin.

Given the marked genetic diversity among Korean C. albicans BSI isolates in our previous MLST study [14], it is interesting that in the present study, five FNS isolates shared two DSTs (1179 and 1539). Additionally, the dendrogram obtained through MLST testing revealed that three isolates of DST 1179 (isolates R3–R5) were more closely related to four FNS isolates (R1, R6, R10, and R11) (Fig. 1). However, there was no time- or location-based clustering of these isolates, which excludes the potential of cross-transmission in the hospitals. Alternatively, some patients could accidentally acquire clonal FNS isolates of C. albicans already present in the environment of healthcare settings in Korea, leading to the development of healthcare-associated BSIs; further studies are needed to confirm this possibility.

In summary, our results showed that most FNS C. albicans BSI isolates from Korean hospitals harbor mutations in ERG11 or TAC1 and that fungemia can develop without azole exposure. This is the first study to describe both the molecular and clinical features of FNS BSI isolates of C. albicans obtained from candidemia surveillance studies.

None.

Shin JH designed the study; Choi MJ and Byun SA performed the laboratory measurements and molecular studies; Kim MN, Lee WG, Lee J, Yong D, Chang CL, Won EJ, and Kim SH collected the clinical isolates and data; Shin JH, Kwon YJ, and Lee SY wrote the preliminary manuscript; Shin JH, Kwon YJ, and Lee SY analyzed the data; Shin JH revised the manuscript; Kim MN, Lee WG, Lee J, Chang CL, Won EJ, and Kim SH provided valuable comments and recommendations. All authors revised and accepted the final version of the manuscript.

No potential conflicts of interest relevant to this article are reported.

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (grant no. NRF-2022R1A2B5B0100322).