Evolution of Fluconazole Resistance Mechanisms and Clonal Types of Candida parapsilosis Isolates from a Tertiary Care Hospital in South Korea

ABSTRACT

We investigated the evolution of fluconazole resistance mechanisms and clonal types of Candida parapsilosis isolates from a tertiary care hospital in South Korea. A total of 45 clinical isolates, including 42 collected between 2017 and 2021 and 3 collected between 2012 and 2013, were subjected to antifungal susceptibility testing, sequencing of fluconazole resistance genes (ERG11, CDR1, TAC1, and MRR1), and microsatellite typing. Twenty-two isolates carried Y132F (n = 21; fluconazole MIC = 2 to >256 mg/L) or Y132F+R398I (n = 1; fluconazole MIC = 64 mg/L) in ERG11 and four isolates harbored N1132D in CDR1 (fluconazole MIC = 16 to 64 mg/L). All 21 Y132F isolates exhibited similar microsatellite profiles and formed a distinct group in the dendrogram. All four N1132D isolates displayed identical microsatellite profiles. Fluconazole MIC values of the Y132F isolates varied depending on their MRR1 mutation status (number of isolates, year of isolation, and MIC): K177N (n = 8, 2012 to 2020, 2 to 8 mg/L); K177N + heterozygous G982R (n = 1, 2017, 64 mg/L); K177N + heterozygous S614P (n = 2, 2019 to 2020, 16 mg/L); and K177N + homozygous S614P (n = 10, 2020 to 2021, 64 to > 256 mg/L). Our study revealed that Y132F in ERG11 and N1132D in CDR1 were the major mechanisms of fluconazole resistance in C. parapsilosis isolates. Furthermore, our results suggested that the clonal evolution of Y132F isolates persisting and spreading in hospital settings for several years occurred with the acquisition of heterozygous or homozygous MRR1 mutations associated with a gradual increase in fluconazole resistance.

INTRODUCTION

Candida species are a major cause of nosocomial bloodstream infections, resulting in substantial morbidity and mortality (1). Candida albicans remains the most common species causing candidemia; however, the proportion of candidemia caused by non-albicans Candida species is increasing worldwide (2). Candida parapsilosis is the most common non-albicans Candida species causing candidemia in many countries, including South Korea (3–6). Candida parapsilosis has the ability to survive on inanimate surfaces and colonize the hands of health care workers, facilitating the spread of nosocomial infections (7–12). Fluconazole has been widely used as a first-line agent for the treatment of C. parapsilosis infections (13, 14), and fluconazole resistance among C. parapsilosis isolates has been considered to be uncommon (2, 15–17). However, in recent years, the clonal spread of fluconazole-resistant C. parapsilosis isolates in hospital settings, particularly in intensive care units (ICUs), has been reported in many parts of the world, including South Korea (12, 18–29). Furthermore, multidrug-resistant (MDR) C. parapsilosis isolates exhibiting resistance to both azoles and echinocandins have recently been identified in Iran and Turkey (30, 31).

Fluconazole inhibits lanosterol 14-α demethylase (Erg11p) encoded by ERG11, blocking the synthesis of ergosterol, an essential component of fungal cell membranes. One of the major mechanisms of fluconazole resistance in Candida species is a decrease in the fluconazole affinity of Erg11p caused by ERG11 mutations (32–34). In particular, the Y132F mutation in ERG11 is the predominant mechanism for fluconazole resistance in C. parapsilosis (12, 18, 19, 22, 23, 25–29, 35, 36). Previous studies from our group and others have shown that C. parapsilosis isolates carrying Y132F have the capacity to persist and cause clonal spread in hospital settings (18, 22, 29). In addition to ERG11 mutations, the overexpression of genes encoding drug efflux pumps (CDR1, CDR2, and MDR1) is another important mechanism of fluconazole resistance in Candida species, primarily caused by gain-of-function mutations in the transcription factor genes TAC1 and MRR1 (TAC1: regulator of CDR1 and CDR2 expression; MRR1: regulator of MDR1 expression) (32–34). However, little is known regarding the precise roles of these genes in the fluconazole resistance of C. parapsilosis.

In a previous study, we analyzed 47 fluconazole-resistant C. parapsilosis isolates obtained from eight tertiary care hospitals in South Korea, including Samsung Medical Center (SMC), between 2005 and 2016 (22). The study included three isolates obtained from the neonatal ICU of SMC between 2012 and 2013, and these isolates were found to carry Y132F in ERG11 and an identical microsatellite genotype (22). Recently, we witnessed a dramatic increase in fluconazole resistance in C. parapsilosis blood isolates collected from SMC between January 2017 and April 2021 (Fig. 1), and we hypothesized that this increase was due to the long-term persistence and clonal transmission of fluconazole-resistant C. parapsilosis isolates carrying Y132F within the hospital. To test this hypothesis, we investigated the fluconazole resistance mechanisms and microsatellite genotypes of 42 C. parapsilosis isolates obtained from different wards of SMC between January 2017 and April 2021 and compared them with those of three isolates obtained from the neonatal ICU between 2012 and 2013.

FIG 1.

Frequency of fluconazole resistance in Candida parapsilosis blood isolates from Samsung Medical Center (SMC) between January 2017 and April 2021. C. parapsilosis accounted for 6% to 17% of candidemia cases. The frequency of fluconazole resistance among C. parapsilosis blood isolates increased from 21% to 75% between January 2017 and April 2021.

RESULTS

Azole susceptibility testing and ERG11, TAC1, and MRR1 sequencing.

Of the 45 isolates tested in this study, 24 (53.3%) were nonsusceptible to fluconazole, including 21 resistant (MIC ≥ 8 mg/L) and 3 susceptible dose-dependent (SDD) (MIC = 4 mg/L) isolates (Table 1). Of the 24 fluconazole-nonsusceptible isolates, 16 (66.7%) were nonsusceptible to voriconazole, including 11 resistant (MIC ≥ 1 mg/L) and 5 intermediate (MIC = 0.25 to 0.5 mg/L) isolates (Table 2). Among the 24 fluconazole-nonsusceptible isolates, 20 (83.3%) carried Y132F (n = 19) or Y132F+R398I (n = 1) in ERG11, whereas the remaining 4 (16.7%) had no mutations in ERG11, TAC1, or MRR1 and were submitted for whole-genome sequencing (WGS) (Table 1). Y132F in ERG11 was also found in two fluconazole-susceptible isolates. Fluconazole MIC values of the 21 Y132F isolates varied depending on their MRR1 mutation status (number of isolates and fluconazole MIC): K177N (n = 8, MIC = 2 to 8 mg/L); K177N + heterozygous G982R (n = 1, 64 mg/L); K177N + heterozygous S614P (n = 2, 16 mg/L); and K177N + homozygous S614P (n = 10, 64 to > 256 mg/L). TAC1 mutations were only identified in 10 fluconazole-susceptible isolates.

TABLE 1.

Fluconazole MIC distribution of 45 Candida parapsilosis isolates according to the mutation status of fluconazole resistance genes (ERG11, CDR1, MRR1, and TAC1)

Mutation status				No. of isolates with fluconazole MICs (mg/L)												Categories based on breakpoints
ERG11	CDR1	MRR1	TAC1	0.25	0.5	1	2	4	8	16	32	64	128	256	>256	Susceptible	SDDa	Resistant
Y132F	None	K177N+S614P	None									1		6	3			10
Y132F	None	K177N+S614Pb	None							2								2
Y132F	None	K177N+G982Rb	None									1						1
None	N1132D	None	None							1	2	1						4
Y132F+R398I	None	None	None									1						1
Y132F	None	K177N	None				2	3	3							2	3	3
R398I	None	None	L877P		1	3										4
R398I	None	None	E615D+L877P			1										1
R398I	None	None	None		1											1
None	None	None	L877P			1										1
None	None	None	D774H			1										1
None	None	None	R208G	2	1											3
None	None	None	None	1	2	4	1									8
Total				3	5	10	3	3	3	3	2	4	0	6	3	21 (46.7%)	3 (6.7%)	21 (46.7%)

^aSDD, susceptible-dose dependent.

^bHeterozygous mutations.

TABLE 2.

Antifungal susceptibility testing results, mutation status of fluconazole resistance genes (ERG11, CDR1, MRR1 and TAC1), and microsatellite genotypes/clusters for each isolate

Yr	Isolate	Source	Antifungal agent MIC (mg/L)a							Mutation status				Microsatellite genotype/cluster
			FLC	VRC	ITR	POS	AND	CAS	MCF	ERG11	CDR1	MRR1	TAC1
2012	Y9-2012b	Blood	8 (R)	0.12 (S)	0.03	0.03	1 (S)	0.25 (S)	1 (S)	Y132F	None	K177N	None	M1/cluster 1
	Y10-2012b	Blood	4 (SDD)	0.06 (S)	0.03	0.015	2 (S)	0.25 (S)	2 (S)	Y132F	None	K177N	None	M1/cluster 1
2013	Y11-2013b	Blood	4 (SDD)	0.06 (S)	0.03	0.015	1 (S)	0.25 (S)	1 (S)	Y132F	None	K177N	None	M1/cluster 1
2017	SM01	Blood	64 (R)	0.25 (I)	0.03	0.015	0.25 (S)	0.25 (S)	0.25 (S)	Y132F	None	K177N+G982Rc	None	M1/cluster 1
2019	SM02	Blood	1 (S)	0.03 (S)	0.06	0.06	1 (S)	0.5 (S)	1 (S)	R398I	None	None	E615D+L877P	M7/non-cluster
	SM03	Blood	1 (S)	0.015 (S)	0.06	0.03	0.5 (S)	0.25 (S)	1 (S)	None	None	None	None	M17/non-cluster
	SM04	Blood	1 (S)	0.03 (S)	0.12	0.06	0.5 (S)	0.25 (S)	0.5 (S)	None	None	None	None	M24/non-cluster
	SM05	Blood	64 (R)	2 (R)	0.25	0.12	0.5 (S)	0.25 (S)	0.5 (S)	Y132F+R398I	None	None	None	M14/non-cluster
	SM06	Blood	16 (R)	0.25 (I)	0.06	0.06	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614Pc	None	M2/cluster 2
	SM07	Blood	8 (R)	0.12 (S)	0.06	0.015	1 (S)	0.25 (S)	2 (S)	Y132F	None	K177N	None	M1/cluster 1
2020	SM08	Blood	256 (R)	1 (R)	0.015	0.008	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614P	None	M2/cluster 2
	SM09	Blood	1 (S)	0.015 (S)	0.06	0.03	2 (S)	1 (S)	2 (S)	None	None	None	None	M12/non-cluster
	SM10	Blood	16 (R)	0.12 (S)	0.12	0.03	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614Pc	None	M2/cluster 2
	SM11	Blood	64 (R)	1 (R)	0.5	0.5	1 (S)	0.5 (S)	1 (S)	None	N1132D	None	None	M13/cluster 5
	SM12	Blood	1 (S)	0.03 (S)	0.06	0.03	1 (S)	0.5 (S)	1 (S)	R398I	None	None	L877P	M9/non-cluster
	SM13	Blood	1 (S)	0.015 (S)	0.06	0.03	2 (S)	0.5 (S)	1 (S)	R398I	None	None	L877P	M10/non-cluster
	SM14	Blood	1 (S)	0.03 (S)	0.12	0.06	1 (S)	0.25 (S)	1 (S)	None	None	None	L877P	M6/non-cluster
	SM15	Blood	1 (S)	0.015 (S)	0.06	0.03	0.5 (S)	0.25 (S)	0.5 (S)	None	None	None	None	M15/non-cluster
	SM16	Blood	4 (SDD)	0.06 (S)	0.06	0.015	1 (S)	0.5 (S)	1 (S)	Y132F	None	K177N	None	M1/cluster 1
	SM17	Blood	0.5 (S)	0.008 (S)	0.03	0.03	1 (S)	1 (S)	1 (S)	None	None	None	None	M22/non-cluster
	SM18	Blood	8 (R)	0.06 (S)	0.03	0.015	1 (S)	0.25 (S)	1 (S)	Y132F	None	K177N	None	M1/cluster 1
	SM19	Blood	0.5 (S)	0.015 (S)	0.06	0.03	1 (S)	0.5 (S)	1 (S)	None	None	None	None	M23/non-cluster
	SM20	Blood	0.25 (S)	0.008 (S)	0.03	0.03	1 (S)	0.5 (S)	1 (S)	None	None	None	R208G	M21/cluster 6
	SM21	Blood	2 (S)	0.06 (S)	0.015	0.008	1 (S)	1 (S)	1 (S)	Y132F	None	K177N	None	M5/cluster 4
	SM22	Blood	2 (S)	0.06 (S)	0.015	0.008	1 (S)	0.5 (S)	1 (S)	Y132F	None	K177N	None	M5/cluster 4
	SM23	Blood	256 (R)	2 (R)	0.12	0.06	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614P	None	M2/cluster 2
	SM24	Blood	256 (R)	2 (R)	0.12	0.06	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614P	None	M2/cluster 2
2021	SM25	Blood	256 (R)	4 (R)	0.06	0.06	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614P	None	M3/cluster 3
	SM26	Skin	256 (R)	2 (R)	0.06	0.06	2 (S)	0.5 (S)	4 (I)	Y132F	None	K177N+S614P	None	M4/non-cluster
	SM27	Blood	256 (R)	4 (R)	0.06	0.12	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614P	None	M2/cluster 2
	SM28	Skin	>256 (R)	4 (R)	0.12	0.06	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614P	None	M3/cluster 3
	SM29	Skin	>256 (R)	4 (R)	0.12	0.12	1 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614P	None	M2/cluster 2
	SM30	Urine	16 (R)	0.12 (S)	0.25	0.25	0.5 (S)	0.5 (S)	0.5 (S)	None	N1132D	None	None	M13/cluster 5
	SM31	Ear discharge	0.5 (S)	0.03 (S)	0.12	0.06	1 (S)	0.5 (S)	1 (S)	R398I	None	None	None	M18/non-cluster
	SM32	Skin	>256 (R)	4 (R)	0.06	0.06	2 (S)	0.5 (S)	2 (S)	Y132F	None	K177N+S614P	None	M2/cluster 2
	SM33	Peritoneal fluid	32 (R)	0.5 (I)	0.25	0.25	1 (S)	0.5 (S)	1 (S)	None	N1132D	None	None	M13/cluster 5
	SM34	Ear discharge	2 (S)	0.03 (S)	0.06	0.06	1 (S)	0.25 (S)	1 (S)	None	None	None	None	M25/non-cluster
	SM35	Skin	1 (S)	0.03 (S)	0.06	0.06	0.5 (S)	0.25 (S)	1 (S)	None	None	None	D774H	M16/non-cluster
	SM36	Skin	64 (R)	0.5 (I)	0.06	0.03	2 (S)	0.25 (S)	2 (S)	Y132F	None	K177N+S614P	None	M2/cluster 2
	SM37	Bile	32 (R)	0.25 (I)	0.25	0.25	1 (S)	0.5 (S)	1 (S)	None	N1132D	None	None	M13/cluster 5
	SM38	Urine	0.5 (S)	0.015 (S)	0.06	0.06	0.5 (S)	0.25 (S)	1 (S)	None	None	None	R208G	M21/cluster 6
	SM39	Pus	0.25 (S)	0.008 (S)	0.03	0.015	0.5 (S)	0.12 (S)	1 (S)	None	None	None	R208G	M20/non-cluster
	SM40	Skin	1 (S)	0.03 (S)	0.03	0.06	2 (S)	0.25 (S)	2 (S)	R398I	None	None	L877P	M8/non-cluster
	SM41	Blood	0.5 (S)	0.015 (S)	0.06	0.03	0.25 (S)	0.25 (S)	0.25 (S)	R398I	None	None	L877P	M11/non-cluster
	SM42	Pus	0.25 (S)	0.008 (S)	0.03	0.015	0.25 (S)	0.25 (S)	0.5 (S)	None	None	None	None	M19/non-cluster

^aFLC, fluconazole; VRC, voriconazole; ITR, itraconazole; POS, posaconazole; AND, anidulafungin; CAS, caspofungin; MCF, micafungin; R, resistant; S, susceptible; I, intermediate; SDD, susceptible-dose dependent.

^bIsolates included in our previous multicenter study (22).

^cHeterozygous mutations.

Whole-genome sequencing and CDR1 sequencing.

WGS analysis revealed that all four fluconazole-resistant isolates without mutations in ERG11, TAC1, and MRR1 (SM11, SM30, SM33, and SM37) carried the N1132D mutation in CDR1 but had no mutations in other genes associated with fluconazole resistance, such as CDR2 and MDR1 (Table 2). These isolates exhibited relatively higher MIC values for itraconazole and posaconazole than those carrying Y132F. The remaining 41 isolates were screened for CDR1 mutation status, and all were confirmed to contain no mutations in this gene.

Echinocandin susceptibility testing and FKS1 and FKS2 sequencing.

Among the 45 isolates tested by the Sensititre YeastOne panel, none exhibited nonsusceptibility to anidulafungin or caspofungin; only 1 isolate (2.2%) was intermediate to micafungin (MIC = 4 mg/L) (Table 2). The isolate (SM26) was resistant to both fluconazole and voriconazole and carried Y132F in ERG11. This isolate was screened for FKS1 and FKS2; however, no mutations other than a naturally occurring polymorphism (P660A) in the hot spot 1 region of FKS1 were detected. Using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) method, the isolate showed a MIC value of 2 mg/L and was interpreted as micafungin-susceptible.

Microsatellite genotypes.

Microsatellite typing of 45 isolates produced 25 distinct genotypes, among which 7 (M1, M2, M3, M4, M5, M13, and M14) were exclusively found in 26 isolates with fluconazole resistance-conferring mutations (Y132F or Y132F+R398I in ERG11 or N1132D in CDR1) (Table 2). The remaining 18 genotypes were exclusively observed in 19 isolates without fluconazole resistance-conferring mutations. The unweighted pair group method with arithmetic mean (UPGMA) tree based on the allelic data obtained from microsatellite typing is shown in Fig. 2. A total of six clusters were noted, five of which were found in isolates with fluconazole resistance-conferring mutations. Specifically, 20 out of 21 Y132F isolates formed four clusters (clusters 1 to 4) and all 4 N1132D isolates formed one cluster (cluster 5). In contrast, only 2 out of 19 isolates without fluconazole resistance-conferring mutations formed one cluster (cluster 6). Of interesting note, all 21 Y132F isolates exhibited similar microsatellite profiles and formed a distinct group in the UPGMA tree that was clearly divergent from the other isolates. In particular, the microsatellite profiles of 3 Y132F isolates collected from the neonatal ICU between 2012 and 2013 (Y9-2012, Y10-2012, and Y11-2013) were identical to those of 4 Y132F isolates (SM01, SM07, SM16, and SM18) collected from other ICUs between 2017 and 2020, corresponding to the microsatellite genotype M1.

FIG 2.

Unweighted pair group method with arithmetic mean (UPGMA) tree constructed based on the microsatellite profiles of 45 C. parapsilosis isolates.

Evolution of fluconazole resistance mechanisms in 21 clonal Y132F isolates.

Figure 3 depicts the evolutionary change of fluconazole resistance mechanisms in 21 clonal Y132F isolates which formed a distinct group in the UPGMA tree. Eight Y132F isolates with K177N in MRR1 showed a fluconazole MIC of ≤ 8 mg/L and were recovered from 2012 to 2020. Two Y132F isolates with K177N + heterozygous S614P in MRR1 had a fluconazole MIC of 16 mg/L and were recovered in 2019 and 2020. Ten Y132F isolates with K177N + homozygous S614P in MRR1 had a fluconazole MIC of 64 to >256 mg/L and were recovered in 2020 and 2021. Notably, all 7 Y132F isolates recovered in 2021 harbored K177N + homozygous S614P in MRR1, and their fluconazole MIC values were consistently high (64 to >256 mg/L). Of the 21 clonal Y132F isolates, 7 were recovered from patients with prior exposure to fluconazole, of which 3 were associated with breakthrough candidemia. The first isolate harboring K177N + heterozygous S614P in MRR1 (SM06) was obtained from a patient with prior exposure to fluconazole, and the first isolate harboring K177N + homozygous S614P in MRR1 (SM08) was obtained from a patient who developed breakthrough candidemia while receiving fluconazole therapy.

FIG 3.

Evolution of MRR1 mutations and fluconazole MICs in 21 clonal Y132F isolates recovered during the period of 2012 to 2021. Numbers in colored boxes denote the number of clonal Y132F isolates with particular MRR1 mutations (yellow, K177N; dark yellow, K177N + heterozygous S614P; red, K177N + homozygous S614P; green, K177N + heterozygous G982R). Numbers in parentheses denote the number of clonal Y132F isolates obtained from patients with prior exposure to fluconazole. Superscript asterisks indicate the occurrence of breakthrough candidemia.

DISCUSSION

The emergence and clonal spread of fluconazole-resistant C. parapsilosis isolates, particularly those with Y132F in ERG11, in hospital settings poses a serious public health threat in many countries, including South Korea (18, 22, 29). In this study, we showed that the recent increase in fluconazole resistance in C. parapsilosis blood isolates from SMC was due to the long-term persistence and clonal transmission of Y132F isolates. Furthermore, our study demonstrated for the first time that the acquisition of heterozygous or homozygous MRR1 mutations in Y132F isolates contributed to a gradual increase in fluconazole resistance by assessing 21 clonal Y132F isolates collected during the period of 2012 to 2021. These findings suggested that clonal Y132F isolates evolved toward higher levels of fluconazole resistance in SMC.

To date, the Y132F mutation in ERG11 has been identified exclusively in fluconazole-nonsusceptible C. parapsilosis isolates (18, 22, 24, 27, 35, 37). In contrast to these findings, we detected Y132F in two fluconazole-susceptible C. parapsilosis isolates (SM21 and SM22) and noted substantial variability in the fluconazole MIC values of C. parapsilosis isolates with Y132F (2 to >256 mg/L). Sequencing analysis of MRR1 and TAC1 showed that all 8 clonal Y132F isolates with fluconazole MICs of ≤8 mg/L contained K177N in MRR1, whereas all 13 clonal Y132F isolates with fluconazole MICs of ≥16 mg/L harbored K177N+S614P (n = 12) or K177N+G982R in MRR1 (n = 1). These findings suggested that increased levels of fluconazole resistance in clonal Y132F isolates were associated with the acquisition of mutations such as S614P or G982R in MRR1. The acquisition of these mutations in clonal Y132F isolates may have been induced by the repeated exposure to fluconazole, considering that the Y132F isolates in which S614P in MRR1 was first identified (heterozygous S614P: SM06; homozygous S614P: SM08) were obtained from patients with prior exposure to fluconazole.

Several MRR1 mutations have been found to be associated with fluconazole resistance in C. parapsilosis (18, 38, 39); however, to our knowledge, S614P and G982R have not been previously reported. Although we did not examine the expression of MRR1 and MDR1, S614P in MRR1 was highly likely to be a gain-of-function mutation because the MIC values of Y132F isolates with S614P in homozygous form (64 to >256 mg/L) were higher than those of Y132F isolates with S614P in heterozygous form (16 mg/L). G982R in MRR1 was also likely to be a gain-of-function mutation because a Y132F isolate with K177N+G982R in MRR1 belonging to the microsatellite genotype M1 showed a higher MIC value (64 mg/L) compared to Y132F isolates with K177N in MRR1 belonging to the same genotype (4 to 8 mg/L). Unlike S614P and G982R, K177N in MRR1 was unlikely to be a gain-of-function mutation, as our previous study and another by Grossman et al. (18) showed that this mutation was identified not only in fluconazole-resistant isolates but also in highly fluconazole-susceptible isolates (MIC ≤ 1 mg/L) (22). As shown in this study, Y132F isolates without gain-of-function mutations in MRR1 have low levels of fluconazole resistance and thus carry the risk of being classified as susceptible or SDD by in vitro susceptibility testing.

Recently, Asadzadeh et al. (37) performed WGS or amplicon sequencing of C. parapsilosis isolates from Kuwait and identified a novel missense mutation (N1132D) in CDR1 in the majority of fluconazole-resistant isolates. Similarly, we performed WGS for four fluconazole-resistant isolates without mutations in ERG11, TAC1, and MRR1 (SM11, SM30, SM33, and SM37) and identified N1132D in all of them. We and Asadzadeh et al. showed that N1132D was present exclusively in fluconazole-resistant isolates (37), indicating that it could be a fluconazole resistance-conferring mutation in C. parapsilosis. In addition, our N1132D isolates displayed relatively high MIC values for itraconazole and posaconazole, suggesting that N1132D may mediate cross-resistance to itraconazole and posaconazole in C. parapsilosis. However, the precise role of N1132D in the azole resistance of C. parapsilosis needs to be further investigated.

Similar to the Y132F isolates, four N1132D isolates displayed identical microsatellite profiles and thus formed a clonal cluster. Our findings provide the first evidence that C. parapsilosis isolates with N1132D have the capacity to persist and cause clonal spread in hospital settings. Notably, these N1132D isolates were obtained from the general surgery ICU and ward. Patients and health care workers frequently move between the general surgery ICU and ward, and we speculate that this frequent movement facilitated the cross-transmission of N1132D isolates from the general surgery ICU to the general surgery ward.

In this study, all isolates with Y132F were collected from ICUs and nearly all of them (20/21, 95.2%) formed clonal clusters, while only 2 out of the 19 isolates without fluconazole resistance-conferring mutations (10.5%) formed a clonal cluster. Furthermore, all 21 Y132F isolates exhibited similar microsatellite profiles and formed a distinct group in the UPGMA tree that was clearly divergent from the other isolates, indicating that they were of the same clonal origin. By analyzing the 21 clonal Y132F isolates, we demonstrated that the clonal evolution of Y132F isolates occurred with the acquisition of heterozygous or homozygous mutations in MRR1 which may confer higher levels of fluconazole resistance. We speculate that Y132F isolates with K177N in MRR1 persisted and evolved in the ICU environment and that during evolution, these isolates increased the level of fluconazole resistance by acquiring S614P or G982R in one MRR1 allele and undergoing loss of heterozygosity in the other. Due to increased fluconazole resistance, Y132F isolates homozygous for K177N+S614P in MRR1 had a selective advantage in the presence of antifungal therapy and finally emerged as the dominant group among fluconazole-resistant C. parapsilosis isolates from SMC.

Unlike fluconazole resistance, echinocandin resistance among C. parapsilosis isolates is rarely found (2). However, MDR C. parapsilosis isolates exhibiting resistance to both fluconazole and micafungin have recently been reported, limiting therapeutic options against C. parapsilosis infections (30, 31). Thus, the emergence and clonal spread of MDR C. parapsilosis isolates in hospital settings should be closely monitored. In the present study, one fluconazole-resistant isolate with Y132F (SM26) was intermediate to micafungin (MIC = 4 mg/L). Because the isolate harbored no causative mutations in FKS1 and FKS2 and was classified as susceptible to micafungin by the CLSI BMD method (MIC = 2 mg/L), we concluded that it was micafungin-susceptible and therefore not MDR.

One major limitation of this study is that we did not perform functional studies on the mutations identified in our isolates. Therefore, the exact roles of these mutations in the fluconazole resistance of C. parapsilosis remain unclear. Another limitation is that the retrospective nature of our study did not allow us to obtain environmental samples or identify the environmental reservoir of C. parapsilosis.

In conclusion, we revealed that Y132F in ERG11 and N1132D in CDR1 were the major mechanisms of fluconazole resistance in C. parapsilosis clinical isolates from SMC. Furthermore, our results indicated that Y132F and N1132D isolates can persist and cause clonal spread in hospital settings and therefore should be closely monitored. Given that increased levels of fluconazole resistance in clonal Y132F isolates were associated with evolutionary changes such as the acquisition of MRR1 mutations, continuous surveillance of fluconazole resistance rates, resistance mechanisms, and clonal relatedness of C. parapsilosis isolates collected from hospitals is warranted.

MATERIALS AND METHODS

Clinical isolates.

This retrospective study analyzed 45 C. parapsilosis clinical isolates stored in the culture collection of SMC. This included 23 blood isolates obtained from January 2019 to December 2020 and 3 blood and 15 nonblood isolates obtained from January to April 2021. In addition, 1 blood isolate collected in 2017 and 3 collected from the neonatal ICU between 2012 and 2013 and described in our previous study (22) were included. All isolates were from different patients, and no serial isolates were included. Of the 45 isolates analyzed, 30 (66.7%) were collected from ICUs (neonatal ICU, n = 15 [33.3%]; medical ICU, n = 8 [17.8%]; general surgery ICU, n = 5 [11.1%]; cardiac surgery ICU, n = 2 [4.4%]). The remaining 15 isolates (33.3%) were collected from general wards or outpatient clinics (general surgery ward, n = 3 [6.7%]; hematology/oncology ward, n = 3 [6.7%]; emergency ward, n = 2 [4.4%]; infectious disease ward, n = 1 [2.2%]; pulmonology ward, n = 1 [2.2%]; neurosurgery ward, n = 1 [2.2%]; pediatric ward, n = 1 [2.2%]; outpatient clinics, n = 3 [6.7%]). The majority of the isolates tested were obtained from blood (n = 30, 66.7%). The remaining isolates were obtained from skin (n = 7, 15.6%), urine (n = 2, 4.4%), ear discharge (n = 2, 4.4%), pus (n = 2, 4.4%), peritoneal fluid (n = 1, 2.2%), and bile (n = 1, 2.2%). All isolates were confirmed as C. parapsilosis sensu stricto via matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (Vitek MS; bioMérieux, Marcy l’Etoile, France) and sequencing analysis of the internal transcribed spacer (ITS) region using the universal primers ITS4 and ITS5 (40). Genomic DNA of the isolates was extracted as described by Tavanti et al. (41) and the isolates and extracted genomic DNA were stored frozen at −70°C until being used in this study.

Antifungal susceptibility testing.

In vitro susceptibility to fluconazole, voriconazole, itraconazole, posaconazole, anidulafungin, caspofungin, and micafungin was assessed using the Sensititre YeastOne panel (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. In addition, isolates which showed nonsusceptibility to anidulafungin, caspofungin, or micafungin but contained no mutations in FKS1 and FKS2 were retested using the broth microdilution (BMD) method according to CLSI M27-Ed4 guidelines (42). MIC values were interpreted based on breakpoints described in the CLSI M60-Ed2 document (43). Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as quality control strains.

ERG11, TAC1, and MRR1 sequencing.

ERG11 mutation status was determined using the primers described by Souza et al. (19). Amplification was carried out using the primer pairs ERG11_CP_F1/ERG11_CP_R1 and ERG11_CP_F2/ERG11_CP_R2. The amplified products were sequenced using the same primer pairs. The mutation status of TAC1 and MRR1 was determined using the primers described by Berkow et al. (44) and the primers designed in this study. Amplification of TAC1 was performed using the primer pair TAC1-A/TAC1-E, and the amplified products were sequenced using the primers TAC1-A, TAC1-B, TAC1-C, TAC1-D, TAC1-E, and TAC1-382seq. Amplification of MRR1 was conducted using the primer pairs MRR1-F/MRR1-1887R and MRR1-C/MRR1-R. The amplified products were sequenced using the primers MRR1-F, MRR1-1887R, MRR1-B, MRR1-C, MRR1-D, MRR1-seqE, and MRR1-2357R.

Whole-genome sequencing.

Four fluconazole-resistant isolates without mutations in ERG11, TAC1, and MRR1 were analyzed via WGS. Genomic DNA was extracted using the MG Genomic DNA purification kit (Macrogen, Seoul, South Korea) and sequenced using HiSeq X 10 (Illumina, San Diego, CA, USA). Raw paired-end reads were quality trimmed using BBDuk. Trimmed reads were mapped to the genome of C. parapsilosis CDC317 (GenBank accession no. ASM18276v2) and annotated using Geneious Prime v2021.1.1 (https://www.geneious.com). The mutation status of the genes encoding or regulating efflux pumps was assessed.

CDR1 sequencing.

Four fluconazole-resistant isolates without mutations in ERG11, TAC1, and MRR1 were found to harbor the N1132D mutation in CDR1 via WGS analysis, and the CDR1 mutation status of the remaining 41 isolates was evaluated to determine whether N1132D was a fluconazole resistance-conferring mutation or a benign polymorphism. Amplification and sequencing of CDR1 was performed using the primer pair CDR1_F and CDR1_R.

FKS1 and FKS2 sequencing.

Isolates showing nonsusceptibility to anidulafungin, caspofungin, or micafungin were subjected to FKS1 and FKS2 sequencing. The hot spot 1 and 2 regions of FKS1 were amplified using the primer pairs designed in this study (FKS1-1437F/FKS1-2887R and FKS1-3546F/FKS1-3546R). The hot spot 1 and 2 regions of FKS2 were amplified using the primer pairs described by Martí-Carrizosa et al. (CpF2H1F/CpF2H1R and CpF2H2F/CpF2H2R) (45).The amplified products were sequenced using the same primers used for PCR amplification. The primers used in this study are listed in Table S1 in the supplemental material.

Microsatellite typing.

Isolates were genotyped using four microsatellite loci (CP1, CP4, CP6, and B5) as described by Sabino et al. (46). Allelic data obtained from microsatellite typing were imported into GenAlEx 6.5 for calculating a genetic distance matrix (47, 48). MEGA-X was used to construct the UPGMA tree from the genetic distance matrix (49). A cluster was defined as a group of ≥2 isolates showing the same allelic profiles at all loci (identical genotype).

Clinical investigation.

Information on previous exposure to antifungal therapy and breakthrough candidemia was obtained from retrospective chart review.

Ethical approval.

This study was approved by the Institutional Review Board of SMC (IRB no. SMC 2021-08-079).

Data availability.

Whole-genome sequencing data are available in the NCBI Sequence Read Archive (BioProject accession no. PRJNA872778).