Differential in Vitro Activity of Anidulafungin, Caspofungin and Micafungin Against Candida Parapsilosis Isolates Recovered from a Burn Unit

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ORIGINAL ARTICLE 10.1111/j.1469-0691.2008.02660.x

Differential in vitro activity of anidulafungin, caspofungin and micafungin against Candida parapsilosis isolates recovered from a burn unit

M. A. Ghannoum1, A. Chen2,3, M. Buhari3, J. Chandra1, P. K. Mukherjee1, D. Baxa2, A. Golembieski2 and J. A. Vazquez2,3 1) Center for Medical Mycology, University Hospitals of Cleveland/Case Western Reserve University, Cleveland, OH, 2) Division of Infectious Diseases, Henry Ford Hospital, Detroit, MI and 3) Division of Infectious Diseases, Wayne State University School of Medicine, Detroit, MI, USA

Abstract

Recent studies suggest that differences in antifungal activity among echinocandins may exist. In this study, the activities of three echinocandins (anidulafungin, caspofungin, and micafungin) against Candida parapsilosis isolates from burn unit patients, healthcare workers and the hospital environment were determined. Additionally, the effect of these echinocandins on the cell morphology of caspofungin-susceptible and caspofungin-non-susceptible isolates was assessed using scanning electron microscopy (SEM). The C. parapsilosis isolates obtained from patients were susceptible to anidulafungin, but were less so to caspofungin and micafungin. Isolates obtained from healthcare workers or environmental sources were susceptible to all antifungals. SEM data demonstrated that although anidulafungin and caspofungin were equally active against a caspofungin-susceptible C. parapsilosis strain, they differed in their ability to damage a caspofungin-non-susceptible strain, for which lower concentrations of anidulafungin (1 mg/L) than of caspofungin (16 mg/L) were needed to induce cellular damage and distortion of the cellular morphology. To determine whether the difference in the antifungal susceptibility of C. parapsilosis isolates to anidulafungin as compared to the other two echinocandins could be due to different mutations in the FKS1 gene, the sequences of the 493-bp region of this gene associated with echinocandin resistance were compared. No differences in the corresponding amino acid sequences were observed, indicating that differences in activity between anidulafungin and the other echinocandins are not related to mutations in this region. The results of this study provide evidence that differences exist between the activities of anidulafungin and the other echinocandins.

Keywords: Antifungal, Candida, cross-resistance, echinocandins, fungi, hot spot mutation Original Submission: 17 March 2008; Revised Submission: 8 July 2008; Accepted: 8 July 2008 Editor: E. Roilides Clin Microbiol Infect 2009; 15: 274–279

have become the mainstay of antifungal therapy for the treat- Corresponding author and reprint requests: M. A. Ghannoum, ment of mucosal and systemic fungal infections [16–18]. Over- Center for Medical Mycology, University Hospitals of Cleveland and Case Western Reserve University, 11100 Euclid Avenue, Cleveland, all, echinocandins demonstrate reduced in vitro activity against OH 44106, USA C. parapsilosis when compared to other Candida spp., and E-mail: [email protected] recent studies have reported the rare development of resistance to caspofungin and micafungin [19–23]. Moudgal et al. [19] reported the isolation of multi-echinocandin-resistant Introduction and multi-azole-resistant strains of C. parapsilosis that were serially recovered from a patient with prosthetic valve endocarditis after being treated with caspofungin and ﬂuconazole. Candida species represent the fourth most common cause of Of importance was the reduced activity of caspofungin and nosocomial bloodstream infections [1]. Approximately half of micafungin in sequential isolates, while anidulafungin activity the cases of candidaemia are due to non-albicans species, such remained unchanged. Although the mechanism of action of as Candida glabrata, Candida parapsilosis and Candida tropicalis the echinocandins is similar, it is likely that differences exist in [1–3]. Among the non-albicans spp., C. parapsilosis is associated antifungal activity among the three echinocandins and also with bloodstream infections, particularly in patients with among the fungal resistance mechanisms. indwelling catheters and prosthetic devices, including critically To evaluate this possibility, C. parapsilosis isolates obtained ill patients in neonatal units and intensive-care units (ICUs) [4–7], from burn unit patients, healthcare workers (HCWs) and the patients undergoing peritoneal dialysis, and patients with pros- environment of the burn unit in a large tertiary-care teaching thetic heart valves [8–15]. The echinocandins and the triazoles

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hospital in Detroit, MI were characterized. These studies Scanning electron microscopy (SEM) showed anidulafungin to be different from caspofungin and The effects of anidulafungin and caspofungin on the ultrastruc- micafungin in its antifungal activity against C. parapsilosis. ture and cell morphology of two C. parapsilosis isolates (one caspofungin-susceptible and the other caspofungin-non-susceptible) were evaluated using SEM. Yeast cells were grown in Materials and Methods the presence of different concentrations of either anidulafungin or caspofungin, and processed for SEM as described previ- Candida strains ously [29]. Samples were sputter-coated with Au/Pd (60 : 40) The C. parapsilosis isolates evaluated in this study were and viewed with an ESEM (model XL3C Philips) scanning elec- recovered from patients, HCWs, and environmental sources tron microscope (Philips Research, Eindhoven, The Nether- in a tertiary-care burn ICU. Patients were prospectively fol- lands), using a voltage of 15 kV and a spot size of 4¢¢. lowed during hospitalization in the burn unit from June to November 2004. Samples were collected from each patient DNA sequence analysis of FKS1 from non-sterile sites. Candida species recovered from the To determine whether differences in echinocandin susceptibil- bloodstream during the study period were also collected. ities were due to mutations in the hot spot region of FKS1 Samples from the hands of HCWs in the burn unit were (which has been associated with development of echinocandin obtained using the ‘hand-washing’ technique as described resistance) [30–32], DNA sequence analysis of this region was previously [24]. Environmental samples from various loca- performed. A 493-bp portion of FKS1 was amplified, using tions in the unit were collected using replicate organism PCR and Candida albicans primers F1719 and R2212, and detection and counting plates (RODAC; BD Diagnostics Sys- sequenced using the same primers as previously described tems, Franklin Lakes, NJ, USA) as previously described [25]. [22,23] (Park S, Paderu P, Perlin DS. 46th InterScience Con- During the study period, both caspofungin and fluconazole ference on Antimicrobial Agents and Chemotherapy, Abstract were commonly used as empirical therapy or for the treat- M-1755. San Francisco, CA: American Society for Microbiol- ment of documented fungal infections. All yeasts were identi- ogy, 2006). Sequences of the amplified segments were aligned fied to the species level using the germ-tube test and and compared using the Blast program. chlamydospore formation, and were confirmed using the API 32C kit (bioMerieux Inc., Durham, NC, USA) [26]. Only Results strains that were found to differ, according to pulsed-field gel electrophoresis, from isolates from either the same patient or from different patients were included in the study. Isolation of Candida strains In addition, reference strains (obtained from the American During the 6-month study period, 22 patients were Type Culture Collection (ATCC)) of C. parapsilosis, Candida prospectively followed during their stay in the burn unit. Can- orthopsilosis and Candida metapsilosis were included as control dida isolates were recovered from 18 of the 22 patients and strains (ATCC 96139, ATCC 96143 and ATCC 22019, from multiple non-sterile sites (oropharynx, female genital respectively). tract, perineum and wound). Among the 18 patients, 11 (61%) were colonized with C. parapsilosis, and seven (39%) Antifungal susceptibility were colonized with C. albicans. In total, 59 C. parapsilosis iso- The in vitro susceptibilities of C. parapsilosis isolates obtained lates were recovered from these 11 patients. Samples were from patients, HCWs and the burn unit environment were obtained from 24 HCWs, of whom ten (42%) had Candida determined according to the guidelines of the CLSI as spp. From these ten HCWs, C. parapsilosis was recovered in described in the M27-A2 microdilution method for yeasts seven cases (70%), whereas the rest were colonized with [27]. Recently, the CLSI Antifungal Subcommittee has recom- C. albicans. Environmental sampling resulted in the isolation of mended a susceptibility breakpoint of £2 mg/L for anidulafun- three C. parapsilosis strains. Overall, 69 C. parapsilosis isolates gin, caspofungin and micafungin, while designating all isolates (59 from burn unit patients, seven from HCWs, and three with an MIC >2 mg/L as non-susceptible [28]. from the environment) and 11 C. albicans isolates (seven from patients and four from HCWs) were obtained. However, 33 Genotyping C. parapsilosis isolates were identified as different strain types Genotyping of C. parapsilosis strains was performed by karyo- after CHEF electrophoresis, and were used in the final analy- typing using CHEF elctrophoresis as described previously sis. These organisms consisted of 23 isolates from patients, [19]. seven isolates from HCWs, and three isolates from the

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environment. In this study, analysis of the C. parapsilosis isolates was the focus.

Antifungal susceptibility testing Table 1 summarizes the susceptibility patterns of the 23 C. parapsilosis isolates obtained from burn patients. When caspofungin and micafungin were tested against these C. parapsilosis isolates, elevated MIC90 values were found (8 and 16 mg/L, respectively), whereas the MIC90 value for anidulafungin was four-fold to eight-fold lower

(MIC90 2 mg/L, Table 1). These results indicated that anidulafungin had more potent antifungal activity than the other two candins against the C. parapsilosis isolates tested. The antifungal susceptibilities of the C. parapsilosis isolates obtained from the hands of HCWs and the burn unit envi- FIG. 1. Electrophoretic karyotype of Candida parapsilosis isolates ronment are shown in Table 2. Whereas C. parapsilosis iso- recovered from eight different patients. Lanes A and B: controls. lates were susceptible to anidulafungin and caspofungin (with Lanes C–J: patient isolates. MIC50 and MIC90 values of 1 mg/L for both), they tended to be non-susceptible to micafungin (MIC50 and MIC90 of 8 mg/ L for both). (data not shown). Moreover, two of the three environmental C. parapsilosis isolates were also similar to those recovered Strain differentiation from HCWs and patients. These results provide evidence to Strain differentiation of all the 59 C. parapsilosis isolates conﬁrm the transmission of clinical C. parapsilosis isolates recovered from 11 burn patients revealed 23 different strain between the environment, HCWs and patients. types (Fig. 1). Eighteen per cent of the patients had the same strain throughout their entire stay in the burn unit. In addi- SEM analysis tion, 71% of C. parapsilosis strains obtained from HCWs The ability of caspofungin and anidulafungin to affect the were similar to the strains recovered from several patients ultrastructure of C. parapsilosis was evaluated in two isolates (one caspofungin-susceptible and one caspofungin-non-susceptible) by SEM. As shown in Fig. 2, untreated C. parapsilosis Candida parapsi- TABLE 1. Antifungal susceptibilities of 23 cells showed normal yeast ultrastructure with smooth cell losis isolates recovered from burn unit patients morphology (Fig. 2a,d). In contrast, treatment of the caspo- Caspofungin Micafungin Anidulafungin fungin-susceptible strain with anidulafungin or caspofungin affected the cell morphology dramatically, resulting in cell MIC50 181collapse and deformity, and leakage of cytoplasmic material MIC90 816 2 MIC range 0.125–8 0.03–16 0.03–2 that ultimately led to cell damage and lysis (Fig. 2b,c, respec-

MIC50 and MIC90 (mg/L) represent the minimum drug concentration that tively). inhibits 50% and 90% of isolates tested, respectively. This uniformity of activity between anidulafungin and caspofungin did not extend to the caspofungin-non-susceptible C. parapsilosis strain, where differences in their effects TABLE 2. Antifungal susceptibilities of ten Candida parapsi- were noted. In this regard, exposure of the caspofungin-non- losis isolates recovered from the hands of healthcare susceptible C. parapsilosis strain to 2 mg/L anidulafungin led workers and burn unit environmental surfaces to cell swelling, lysis and collapse (Fig. 2e), whereas caspofun- Caspofungin Micafungin Anidulafungin gin at the same concentration (2 mg/L) had no effect on the morphology of this strain (data not shown). For caspofungin MIC50 18 1 MIC90 18 1to exert a signiﬁcant effect on the morphology of the C. par- MIC range 0.5–2 0.5–8 0.5–2 apsilosis caspofungin-non-susceptible strain, it was necessary MIC50 and MIC90 (mg/L) represent the minimum drug concentration that inhibits 50% and 90% of isolates tested, respectively. to increase the concentration to 16 mg/L (Fig. 2f). These SEM results demonstrated that anidulafungin and caspofungin

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(a) (b) (c)

(d) (e) (f)

FIG. 2. Effect of anidulafungin (ANID) and caspofungin (CASPO) on surface structure and morphology of (a–c) caspofungin-susceptible or (d–f) caspofungin-non-susceptible C. parapsilosis isolates. The susceptible C. parapsilosis isolate was exposed to: (a) no drug (control); (b) anidulafungin, 0.25 mg/L; or (c) caspofungin, 0.25 mg/L. The non-susceptible C. parapsilosis isolate was exposed to: (d) no drug; (e) anidulafungin, 2 mg/L; or (f) caspofungin, 16 mg/L. Bars represent 5 lm in (a)–(e) and 10 lm in (f). Magnification: ·8000 (a), and ·5000 (b–f). possess equivalent activity against the caspofungin-susceptible The data confirm and extend the findings of Moudgal et al. C. parapsilosis strain. However, they differ in their ability to [19], who reported isolation of sequential multi-echinocan- damage the C. parapsilosis caspofungin-non-susceptible strain: din-resistant C. parapsilosis isolates from a patient during lower concentrations of anidulafungin than of caspofungin therapy for prosthetic valve endocarditis. The C. parapsilosis were sufficient to induce cellular damage and to distort the isolates studied here and those of Moudgal et al. demon- cellular morphology of this strain. strated that although they had reduced susceptibility to ca-

spofungin and micafungin (elevated MIC90 of both agents),

FKS1 sequence analysis they remained susceptible to anidulafungin (low MIC90). To determine whether the difference in the antifungal sus- SEM was performed to determine whether the observed ceptibility of C. parapsilosis isolates to anidulafungin as com- differences in antifungal susceptibility of the C. parapsilosis pared to the other two echinocandins could be due to isolates to anidulafungin and caspofungin are reflected in the different mutations in FKS1, the sequence of the 493 bp effect of these echinocandins at the morphological level. This region of this gene was determined in eight isolates. analysis showed that a much lower concentration of anidula- Blast search analysis of the sequenced FKS1 region fungin (1 mg/L) than of caspofungin (16 mg/L) was needed to revealed 94% identity with that of the C. albicans 1,3-b-D- cause a dramatic effect on cell morphology in the caspofun- glucan synthase gene. Alignment of the deduced amino acid gin-non-susceptible C. parapsilosis isolates. These findings pro- sequences from two susceptible C. parapsilosis isolates and vide further evidence that differences exist among the three six non-susceptible isolates did not reveal any differences echinocandins; that is, caspofungin and micafungin behave in the amino acid patterns within the previously reported similarly, but differently from anidulafungin. ‘‘hot spot’’ regions [30–32]. The difference between anidulafungin and the other two echinocandins regarding antifungal susceptibility has also been demonstrated recently in C. tropicalis by Pasquale et al. [33]. Discussion These authors reported the case of a 28-year-old female with acute myelogenous leukaemia, fever, otitis media and In this study, differential echinocandin activity against C. par- invasive candidal oesophagitis. Treatment with caspofungin apsilosis isolates recovered from patients in a burn ICU was was initiated, but therapy failed after 5 days. In vitro echino- investigated for the first time. These unique isolates exhib- candin susceptibility testing of the C. tropicalis isolate showed ited non-susceptibility to caspofungin and micafungin, while MICs of anidulafungin, caspofungin and micafungin of 1.0, 4.0 remaining susceptible to anidulafungin. and 8.0 mg/L, respectively. Similar to the present findings,

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these investigators found cross-resistance between caspofun- evaluate mutations in the corresponding genes. Alternatively, gin and micafungin in the isolated C. tropicalis species. it is possible that although all echinocandins target the same Resistance of C. albicans isolates to caspofungin was enzyme (1,3-b-D-glucan synthase), differences in the affinity recently reported by Laverdiere et al. [34], who showed a of the enzyme for these agents may exist, possibly due to progressive increase in MICs of different echinocandins for variations in the drugs’ molecular structures. This scenario is four sequential C. albicans isolates obtained from a human not unique to the echinocandins, as differential activity pat- immunodeficiency virus-infected patient with chronic oesoph- terns have been shown among the different members of the agitis. These isolates exhibited progressive resistance to azole class of antifungals [36,37]. micafungin following prolonged treatment, and showed In conclusion, these results provide evidence of differential increased caspofungin and anidulafungin MICs, indicating anti-C. parapsilosis activities of anidulafungin and of caspofun- reduced susceptibility to all three echinocandins. Thus, unlike gin and micafungin. Moreover, the data show that the differ- the present results with C. parapsilosis, where cross-resis- ences in the activity of the three echinocandins against the tance between micafungin and caspofungin does not extend C. parapsilosis isolates tested are not related to mutations in to anidulafungin, in C. albicans, cross-resistance may span FKS1. The clinical implications of these findings are yet to be all three echinocandins, suggesting that cross-resistance to determined. echinocandins may be species-dependent. Additionally, an interesting result was that non-susceptibil- Transparency Declaration ity was only seen in the C. parapsilosis isolates obtained from patients in the burn unit, and not in those obtained from the hands of HCWs or the environmental samples. Although the This study was partially supported by funds from Pfizer Phar- reason for this reduced susceptibility to caspofungin and mi- maceutical Company. M.A. Ghannoum has acted as a consul- cafungin in C. parapsilosis isolates obtained from burn unit tant, speaker or received grants and contracts from the patients is unknown, possible explanations include the following companies: Pfizer, Merck, Novartis, Schering following: (i) when C. parapsilosis is exposed to the host Plough, and Stiefel Pharmaceuticals. P.K. Mukherjee has acted environment, it may adapt by expressing genes that aid in its as a consultant, speaker or received grants and contracts survival; and (ii) all of these isolates were recovered from a from the following companies: Great Lakes Pharmaceuticals, busy tertiary-care burn unit with high rates of candidaemia Staval Pharmaceuticals, Gebauer Pharmaceuticals, and Astella. and candidiasis, and with usage rates for both fluconazole The other authors have no conflict of interest with the stu- and caspofungin that are extremely high when compared to dies performed. other parts of the hospital. These factors exert significant selective pressure on Candida. References The mechanism(s) underlying the reduced susceptibility of C. parapsilosis to caspofungin and micafungin, but not to anidulafungin, is unknown. Several studies suggest that amino 1. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. 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