Caspofungin at Catheter Lock Concentrations Eradicates Mature Biofilms of Candida lusitaniae and Candida guilliermondii

Abstract

The antibiofilm activities of caspofungin, anidulafungin, micafungin, and liposomal amphotericin B were studied against Candida lusitaniae, Candida guilliermondii, and a Candida albicans control strain. While anidulafungin and micafungin (0.007 to 2,048 mg/liter) showed reduced activity against biofilms of both test species, caspofungin displayed concentration-dependent antibiofilm activity, reaching complete and persistent eradication at concentrations achievable during lock therapy (512 to 2,048 mg/liter, P < 0.05). Although liposomal amphotericin B strongly inhibited mature biofilms, it possessed lower antibiofilm activity than caspofungin (P < 0.05).

TEXT

Although Candida albicans remains the most frequent yeast species isolated from blood, an increasing proportion of non-albicans Candida species has emerged (1–3). The biofilm-forming capacity of Candida spp. has been implicated as a potential virulence factor in the development of candidemia for patients with implanted vascular catheters (4–6). While removal and replacement of the infected vascular catheter is recommended when feasible in nonneutropenic patients diagnosed with candidemia, this process may not be feasible due to coagulopathies, limited venous access, or life-threatening complications (7, 8). Under such circumstances, antifungal lock therapy may present an alternative strategy for the management of catheter-related infections as an adjunct to systemic antifungal therapy. This method involves the use of very high antifungal concentrations instilled into vascular catheters for several hours to days in an attempt to eradicate microbial biofilms without imposing systemic toxicity (9).

While Candida biofilms are generally recalcitrant to conventional antifungal agents, liposomal amphotericin B and echinocandins have demonstrated good antibiofilm activities which are both species and drug specific (10–14). Candida lusitaniae and Candida guilliermondii are known to have relatively high MICs to echinocandins (10, 13, 14) that may create greater challenges in eradication of their biofilms from vascular catheters (15). We have previously shown that caspofungin at high concentrations (≥64 mg/liter) was able to cause damage to the highly recalcitrant biofilm structures of C. lusitaniae and C. guilliermondii by 70% to 96% (14). These findings indicated that caspofungin could be an effective agent for antifungal lock therapy, especially against C. lusitaniae and C. guilliermondii. In this study, we aimed to evaluate the antibiofilm activities of anidulafungin (ANID), caspofungin (CAS), micafungin (MFG), and liposomal amphotericin B (LAMB) used at systemically achievable but also at lock concentrations against C. lusitaniae and C. guilliermondii bloodstream isolates.

(This study was presented in part at the 53rd Annual Meeting of Interscience Conference of Antimicrobial Agents and Chemotherapy [ICAAC], Denver, CO, 10 to 13 September 2013.)

Six isolates of C. lusitaniae, five of C. guilliermondii, and the control strain of C. albicans M61were studied. C. guilliermondii and C. lusitaniae strains were isolated from catheter-associated bloodstream infections (14) and C. albicans M61 from an infected intravascular catheter (12). Anidulafungin (Pfizer), caspofungin (Merck), micafungin (Astellas), and liposomal amphotericin B (Gilead) were obtained from their manufacturers. Antifungal agents were used at 2-fold dilutions as follows: ANID and LAMB, 0.007 to 2,048 mg/liter; CAS, 0.03 to 2,048 mg/liter; MFG, 0.06 to 2,048 mg/liter. For the formation of biofilms, from each Candida isolate 10⁶ blastoconidia/ml was incubated at 37°C for 48 h in RPMI 1640 medium, pH 7.2, using 96-well polystyrene plates as previously described (16). Mature biofilms or planktonic cells were incubated in RPMI 1640, containing each antifungal at 2-fold dilutions at 37°C for 24 h (preexposure phase). Drug-free wells with growth media containing each clinical isolate were used as controls.

The activity of each drug against biofilms or planktonic cells was assessed by using an XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay as described previously with minor modifications (17). MICs for biofilms (sMICs) and planktonic cells (pMICs) were determined as the minimum antifungal concentrations that caused ≥50% fungal damage compared to drug-free controls. Biofilm eradication, evidenced spectrophotometrically by XTT assay, was confirmed by plating aliquots of drug-treated or untreated biofilms from each condition onto Sabouraud dextrose agar (SDA) after the drug treatment period.

In order to evaluate persistence of drug antibiofilm activity after the biofilm exposure to high drug concentrations for 24 h, the drug-containing media were removed and the drug-treated biofilms were incubated with fresh drug-free RPMI 1640 at 37°C for a “postlock” period of 72 h. Specifically, the extent of escaping fungal growth was determined by inoculating aliquots of scraped biofilms on SDA plates at 0, 24, 48, and 72 h after drug removal. The plates were incubated at 37°C for 48 h prior to reading.

All experiments were performed twice with five replicates per condition, except for the control M61 strain, which was repeated five times using five replicates per condition. Postlock persistence testing was repeated twice for all strains. Results from all different Candida species and strains tested were compared by analysis of variance (ANOVA) with Bonferroni posttest using the Instat 3 biostatistics software.

With the exception of the planktonic MIC of ANID for C. guilliermondii that was six 2-fold dilutions (64-fold) higher than the corresponding MIC for C. albicans M61, the MICs of all four antifungal agents against planktonic cells of the Candida species studied displayed good in vitro activity with comparable susceptibility profiles (Table 1, 0.06 to 1 mg/liter), as also previously documented by us and others (13, 14, 18, 19). However, mature biofilms of C. lusitaniae and C. guilliermondii showed higher MICs to the three echinocandins than their corresponding planktonic cells (Table 1; P < 0.01). ANID and MFG demonstrated reduced activity against mature biofilms of C. lusitaniae and C. guilliermondii with sMICs of 32 to >2,048 mg/liter (Table 1). Biofilms of C. lusitaniae were significantly more resistant than those of C. guilliermondii to ANID and MFG at concentrations of ≥1,024 mg/liter (Fig. 1a and b; P < 0.05). In contrast, CAS displayed concentration-dependent antibiofilm activity against C. lusitaniae and C. guilliermondii, reaching complete eradication at lock concentrations ranging from 512 to 2,048 mg/liter (Fig. 1c). Fungal damage results obtained by XTT were confirmed by plating biofilm aliquots (data not shown). Additionally, persistence of CAS antibiofilm activity at 512 to 2,048 mg/liter was evidenced by “postlock” time points of 0, 24, 48, and 72 h, which persisted at 99% to 100% for all Candida species.

TABLE 1.

Planktonic and biofilm MIC₅₀s of echinocandins and liposomal amphotericin B for C. lusitaniae, C. guilliermondii, and C. albicans^b

Antifungal agent	C. lusitaniae (n = 6)		C. guilliermondii (n = 5)		C. albicans M61a (n = 1)
	pMIC	sMIC	pMIC	sMIC	pMIC	sMIC
ANID	0.125 (0.06–0.125)	>2,048c	1 (0.5–1)	32 (8–128)c	0.015	0.25c
CAS	1 (0.5–1)	64 (32–64)c	1 (0.5–1)	64 (32–64)c	0.125	0.5
MFG	≤0.06	>2,048c	0.125 (0.06–0.125)	>2,048c	0.06	0.25
LAMB	0.06 (0.03–0.06)	0.125 (0.03–0.125)	0.125 (0.06–0.125)	2 (1–4)c	0.125	0.25

^aC. albicans M61 is the control strain.

^bValues are medians (ranges) in mg/liter.

^cSignificant differences between MICs of echinocandins and liposomal amphotericin B against planktonic cells (pMIC) versus biofilms (sMIC) of Candida spp. (P < 0.01).

FIG 1.

Fungal damage of biofilms of C. albicans M61, C. lusitaniae, and C. guilliermondii bloodstream isolates caused by different lock concentrations of ANID, MFG, CAS, and LAMB. Fungal damage was assessed by XTT assay. Results are means ± standard errors (SE) of percentages of biofilm damage of C. albicans M61 (open bars), C. lusitaniae (black bars), and C. guilliermondii (gray bars) isolates for each drug. Asterisks show significant differences between biofilms of organisms for the concentrations indicated by horizontal lines (P < 0.05). The discontinuous line denotes 50% MIC.

sMICs of LAMB for C. lusitaniae, C. guilliermondii, and C. albicans were 0.125, 2, and 0.25 mg/liter (Table). At 256 to 2,048 mg/liter, LAMB demonstrated similar biofilm damage against C. lusitaniae (74% ± 5 to 78% ± 4) and C. guilliermondii (67% ± 3.5 to 73% ± 4.3; Fig. 1d), but the antibiofilm activity observed was significantly less than that achieved by CAS against both Candida spp. (P < 0.05). Similar to our results, Toulet et al. (20) showed strong inhibition of C. albicans and Candida glabrata biofilms by LAMB without leading to complete eradication of biofilms.

In conclusion, our study showed that CAS as lock therapy was significantly more active against biofilms formed by C. lusitaniae and C. guilliermondii than ANID and MFG. While antibiofilm activity of LAMB against C. lusitaniae and C. guilliermondii was considerable, it was significantly lower than that achieved by CAS. Our findings suggest that CAS, if used at higher concentrations and longer periods of time, could potentially be useful in the management of catheter-related candidemia as lock therapy against C. lusitaniae and C. guilliermondii if first verified by laboratory animal studies.