FKS2 Mutations Associated with Decreased Echinocandin Susceptibility of Candida glabrata following Anidulafungin Therapy

Abstract

This is the first case report of Candida glabrata-disseminated candidiasis describing the acquisition of echinocandin resistance following anidulafungin treatment. The initial isolates recovered were susceptible to echinocandins. However, during 27 days of anidulafungin treatment, two resistant strains were isolated (from the blood and peritoneal fluid). The resistant peritoneal fluid isolate exhibited a Ser663Pro mutation in position 1987 of FKS2 HS1 (hot spot 1), whereas the resistant blood isolate displayed a phenylalanine deletion (Phe659).

Candida glabrata infections represent a serious clinical problem due to patient comorbidities, the inherent high mortality rate, and the predisposition to rapidly develop azole resistance (6, 11, 20). Therefore, efforts were made to develop new therapeutic alternatives. A new class of antifungal agents has arisen, including caspofungin (CSF), micafungin (MCF), and more recently, anidulafungin (ANF), which inhibit β (1,3)-glucan synthase activity. This enzyme, including the regulatory subunit Rho-1p, is responsible for producing β (1,3)-glucan, a key component of the fungal cell wall, and is encoded by FKS genes.

Due to their excellent clinical effectiveness and safety profile, these antifungals became the first-line therapy in many hospitals for the treatment of invasive candidiasis (17, 18, 26). Despite the wide use of CSF, reports describing primary or secondary echinocandin resistance in candidiasis are rare. Reduced susceptibility to echinocandins has been linked to mutations in hot spot regions of FKS genes (19). We report for the first time the in vivo acquisition of echinocandin resistance following ANF therapy in a patient with C. glabrata invasive candidiasis.

A 71-year-old female patient was admitted to the intensive care unit (ICU) with a diagnosis of acute pancreatitis that developed after a laparoscopic cholecystectomy. The patient's medical history included hypertension and dyslipidemia. An exploratory laparoscopy was performed on the second day after admission to the ICU. The surgical findings were peritoneal necrosis, necroses of the ileum and jejunum, and pancreatic necrosis. During the hospital stay, several antibacterial drugs were administered to the patient, namely tazobactam, piperacillin, meropenem, tigecycline, and vancomycin. She had no previous antifungal exposure. Her medical condition was further complicated by multiple Candida infections, including disseminated candidiasis and central venous catheter-related infections. Starting on day 6, she received a 200-mg load of ANF, followed by 100 mg ANF/day for 27 days. Cultures of multiple sites, including blood, exudates, central venous catheter (CVC), and urine, were positive for C. albicans and C. glabrata. While C. albicans fungemia cleared following 24 h of ANF therapy, C. glabrata persisted. On day 17, the patient received noradrenaline support. Subsequently, she was submitted to a second exploratory laparoscopic surgery on day 30. The procedure revealed extensive cytosteatonecrosis. Amphotericin B was then initiated, and 2 days later, the patient developed multiorgan failure. The patient died 34 days after admission. No autopsy was performed.

Twenty-one isolates were recovered from the patient's blood and other biological samples throughout ANF treatment (Fig. 1). The assessment of the MICs of azoles, amphotericin B, and echinocandins for all clinical isolates was performed in RPMI 1640 (Sigma), according to the CLSI M27-A3 protocol (4, 5).

FIG. 1.

Time line of antifungal therapy administered to the patient. The number of days after admission and fungal strains isolated from biological samples are also shown. ANF, anidulafungin; AMB lipo, amphotericin B lipid complex.

From the set of C. glabrata isolates, two isolates were resistant to echinocandins, one recovered from the blood (isolate 18-1) and the other from the peritoneal fluid (isolate 30-2) (isolated 18 and 30 days after ICU admission, respectively) (Fig. 1 and Table 1). The acquisition of ANF resistance was associated with an increase in the CSF and MCF MIC values, suggesting the development of cross-resistance among these three compounds. Although MIC values are usually considered predictors of clinical response to antimicrobial therapy in invasive fungal infections, no strong correlation has yet been found between in vitro susceptibility results and clinical outcome (6, 12, 13, 15, 16, 21, 22).

TABLE 1.

In vitro antifungal susceptibility of C. glabrata isolates to CSF, ANF, and MCF^a

Strain	Isolation (no. of days after admission)	Sample type	MIC50 (μg/ml)			FKS2 HS1 SNP	AA profile
			CSF	ANF	MCF
10-1	10	Blood	0.125	≤0.06	≤0.06	Positions 1987-1989, TCT	Ser
18-1	18	Blood	>32	4	4	Positions 1977-1979, CTT deletion	ΔPhe659
19-1	19	Peritoneal fluid	≤0.06	≤0.06	≤0.06	Positions 1987-1989, TCT	Ser
30-2	30	Peritoneal fluid	32	4	8	T1987C	Ser663Pro

^aDetermined according to the CLSI M27-A3 protocol (4). MICs were determined by considering a prominent inhibition, corresponding to 50%, as the endpoint. SNP, single nucleotide polymorphism; AA, amino acid.

In the clinical case described herein, a correlation between the in vitro results and the in vivo lack of efficacy of the drug was found. The MIC values obtained (4 μg/ml) from the blood (strain 18-1) and peritoneal fluid (strain 30-2) isolates were above the susceptibility breakpoint.

Random amplification of polymorphic DNA (RAPD) was carried out using isolates obtained from different biological samples to determine their isogenicity. Using the primers OPE-18 (5′ GGACTGCAGA 3′) and OPA-18 (5′ AGCTGACCGT 3′) (2), identical band profiles were displayed by all isolates, with the exception of strain 30-2, which was recovered from the peritoneal fluid prior to patient death (Fig. 2). Considering the surgical interventions, the evidence of CVC infection, and the long stay in the ICU (34 days), where nosocomial infections are likely to occur, it is plausible that this strain had an exogenous source.

FIG. 2.

Random amplification of polymorphic DNA gel patterns of C. glabrata isolates 18-1, 30-2, 10-1, and 19-1, resistant and susceptible to echinocandins, obtained with primers OPE-18 and OPA-18.

Mutations in FKS genes have been shown to be responsible for echinocandin resistance during caspofungin treatment, namely of C. glabrata (3, 9, 10, 14, 24). To determine whether ANF resistance was associated with mutations in the target genes, genomic DNA of C. glabrata isolates was extracted and, using the hot spot 1 (HS1) regions of the FKS1 and FKS2 genes, were amplified with specific primers (FKS1 HS1F2 [5′-CTTATGTTTGATTTTTGCA-3′]) (8, 24). DNA products were sequenced in an ABI Prism 3130 genetic analyzer (Applied Biosystems). The coding sequences of the Candida glabrata FKS1 and FKS2 genes (GenBank accession numbers XM_446406 and XM_448401, respectively) were aligned with those obtained from the clinical isolates. No sequence alterations were observed in HS1 of the FKS1 gene; however, several point mutations were found in HS1 of the FKS2 gene, with most of them corresponding to synonymous substitutions that did not result in nucleotide changes. Furthermore, the isolate 30-2, which was recovered following a long period of ANF exposure, displayed a C-T mutation at position 1987. This mutation leads to a replacement of serine 663 by proline in HS1 of FKS2 (Table 1). In the same HS, a deletion of 3 nucleotides was found in the blood isolate 18-1, which results in the deletion of one of the two consecutive phenylalanines at positions 658 and 659 (Table 1). These findings are consistent with previous reports describing increased resistance to echinocandins associated with mutations, namely, the S663P substitution and Phe659 deletion in the FKS2 gene (9, 10). The highest frequency of resistance-associated mutations is found within HS1 (1). HS1 is a highly conserved region among the Fks family; hence, the amino acid changes in this region implicate a modification of the echinocandin target and a reduced susceptibility phenotype.

The significant increase in the chitin content following in vitro echinocandin exposure has been suggested as an escape or salvage mechanism for echinocandins (7, 23, 25). In order to unveil the role of chitin in the resistance displayed by isolates 18-1 and 30-2, the cell wall chitin content was measured initially and after 30 days of subculturing in drug-free medium. While the resistant phenotype remained, the chitin content decreased (data not shown). Attending to such results, we were compelled to conclude that the deletion and the mutation detected in the FKS2 gene confer echinocandin resistance to the isolates 18-1 and 30-2, respectively.

Overall, our findings suggest that structural alterations in the HS1 of the FKS2 molecule due to the Ser663Pro substitution and Phe659 deletion lead to a dramatic decrease in echinocandin efficacy. Although just a few cases describing the development of echinocandin resistance have been reported so far, our case report emphasizes the crucial need for antifungal susceptibility surveillance in patients under extended echinocandin therapy.