In Vitro Activities of Anidulafungin and Other Antifungal Agents against ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2011, p. 3031–3035 0066-4804/11/$12.00 doi:10.1128/AAC.01569-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 55, No. 6

In Vitro Activities of Anidulafungin and Other Antifungal Agents against Biofilms Formed by Clinical Isolates of Different Candida and Aspergillus Species䌤† Barbara Fiori,1‡ Brunella Posteraro,1‡ Riccardo Torelli,1 Mario Tumbarello,2 David S. Perlin,3 Giovanni Fadda,1 and Maurizio Sanguinetti1* Institutes of Microbiology1 and Infectious Diseases,2 Universita ` Cattolica del Sacro Cuore, Largo F. Vito, 1, 00168 Rome, Italy, and Public Health Research Institute, New Jersey Medical School, UMDNJ, Newark, New Jersey3 Received 14 November 2010/Returned for modification 27 December 2010/Accepted 13 March 2011

We tested the activities of anidulafungin and other antifungal agents against clinical isolates of different fungal species. For Candida species, high sessile MIC90s (SMIC90s) were obtained for fluconazole, voriconazole, and amphotericin B, whereas the anidulafungin SMIC90s were very low, as were those for caspofungin. Comparatively, for Aspergillus species, higher SMIC90 values were obtained not only for amphotericin B and voriconazole but also for the echinocandins. showed a time-dependent decrease in efficacy for amphotericin B, voriconazole, and caspofungin as the complexity of the A. fumigatus hyphal structure increased, suggesting that the matrix material may play a role in impeding drug action. The aim of the present study was to further evaluate the in vitro activities of anidulafungin and caspofungin against biofilms formed by clinical isolates of different Candida and Aspergillus species and compare them with those of the older antifungals amphotericin B, fluconazole (only for Candida isolates), and voriconazole. (This work was partly presented at the 4th Trends in Medical Mycology [Athens, Greece, 18 to 21 October 2009].) We tested 120 Candida species (20 Candida albicans, 40 Candida parapsilosis, 20 Candida tropicalis, 20 Candida glabrata, and 20 Candida krusei) isolates and 40 Aspergillus species (10 Aspergillus fumigatus, 10 Aspergillus flavus, 10 Aspergillus terreus, and 10 Aspergillus niger) isolates that were obtained from patients hospitalized at our institution from January 2000 through July 2010. All of the Candida isolates were from blood cultures, including 42 from patients with central venous catheters (26), whereas the Aspergillus isolates were from cultures of bronchoalveolar lavage fluid, sputum, skin, nasal, and tissue specimens. C. albicans SC5314 was used as a positive control (21). Anidulafungin and voriconazole (Pfizer, Inc., New York, NY), amphotericin B and fluconazole (Sigma Aldrich, Milan, Italy), and caspofungin (Merck Research Laboratories, Rahway, NJ) were obtained as standard powders and prepared according to Clinical and Laboratory Standards Institute (CLSI) guidelines (5, 6). The MICs for planktonic cells were determined by broth microdilution using the standard CLSI M27-A3 and M38-A2 methods and defined as the lowest drug concentrations that caused either an approximately 50% (for azoles and [only for Candida isolates] echinocandins) or a 100% (for amphotericin B) growth inhibition compared with that of the drug-free growth control (5, 6). For Aspergillus isolates, the minimum effective concentrations (MECs) for echinocandins were determined according to the CLSI M38-A2 document and defined as the lowest drug concentrations that led to the growth

Treatment of invasive Candida infections is often complicated by the ability of Candida species to form biofilms that exhibit elevated intrinsic resistance to various antifungal agents, in particular azoles and polyenes. This antifungal resistance may contribute to the increased pathogenicity of Candida species (2, 7), as observed in catheter-associated bloodstream infections due to biofilm-forming isolates (26). Thus, removal of intravascular devices is currently recommended for management of device-related Candida infections whenever practicable (13). A recent study exploring the early transcriptional responses of mature Candida albicans biofilm exposed to various antifungal agents suggests that enhanced extracellularmatrix or beta-glucan synthesis during biofilm growth (18) might prevent fluconazole and amphotericin B from reaching biofilm cells, thus impairing their cell toxicity and the associated transcriptional responses (27). As echinocandins are noncompetitive inhibitors of beta-glucan synthesis (20), this may explain why the drugs have been shown to be effective against Candida biofilms (4, 8, 9, 11, 28). In another study, liposomal formulation of amphotericin B was shown to eradicate C. albicans biofilm in a continuous catheter flow model, whereas fluconazole and caspofungin were less effective (24). Similar to Candida, filamentous molds such as Aspergillus fumigatus have been shown to be implicated in biofilm-associated infections (17, 22). In vitro, A. fumigatus has the ability to form adherent multicellular communities that are resistant to the effects of antifungal drugs (15, 23). Typical biofilm structures, consisting of fungal mycelium surrounded by an extracellular matrix composed of galactomannan, ␣-1,3-glucans, and melanin (3), were also observed in aspergilloma or during invasive aspergillosis (12). Interestingly, Mowat et al. (16) * Corresponding author. Mailing address: Istituto di Microbiologia, Universita` Cattolica del Sacro Cuore, Largo F. Vito, 1, 00168 Rome, Italy. Phone: 39 06 30154964. Fax: 39 06 3051152. E-mail: msanguinetti @rm.unicatt.it. † Supplemental material for this article may be found at http://aac .asm.org/. ‡ The authors equally contributed to this work. 䌤 Published ahead of print on 21 March 2011. 3031

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TABLE 1. Distributions of echinocandins MICs for different Candida species isolates under planktonic or biofilm growth conditions Drug

Anidulafungin

Species (no. of isolates)

C. albicans (20) C. parapsilosis (40) C. tropicalis (20) C. glabrata (20) C. krusei (20)

Caspofungin


a

Type of MICa

Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC

No. of isolates at MIC (␮g/ml) of: ⱕ0.03

0.06

7 2

13 14

8 1 20 14 2 6

0.25

0.5

1

2

4 9 2

22 9

9 11

14

4

9

2

5 8

3 30 1

2 9

15

5

11

4

9 3 4

5

12 17

2

4 14 2

2 4 7

7 2

7 10

13

5

19

1 2 3

3

0.125

4

8

16

8

4

3

⬎16

2 4 11

7

7

2

Planktonic MICs were determined by the CLSI broth microdilution method, whereas biofilm SMICs were measured by the XTT reduction assay.

of small, rounded, compact hyphal forms compared to the hyphal growth seen in the drug-free growth control well (6). The MICs and MECs were recorded after incubation for 24 to 48 h. The MICs for sessile (biofilm) cells (SMICs) were determined by a 96-well microtiter-based method as previously developed (21). This technique is based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) by metabolically active sessile cells to yield a water-soluble colored formazan product that can then be measured spectrophotometrically in a microtiter plate reader. Briefly, biofilm-containing wells were filled with RPMI 1640 containing doubling concentrations of the antifungal drugs, whereas untreated biofilm wells were filled with RPMI 1640 without drugs for 24 h. Negative-control wells (containing XTT only) were also included. From the resulting colorimetric readings, and after subtraction of the corresponding values for negative controls, the SMICs were calculated and expressed as the lowest drug concentrations at which a 50% decrease in absorbance was detected in comparison with the level for the biofilms formed in the absence of drug (21). For Candida isolates, echinocandin MICs and SMICs were also determined in the presence of 50% serum, as described previously (19). The distributions of antifungal MICs and SMICs for the different Candida species are shown in Tables 1 to 3 and Tables S1 and S2 in the supplemental material. The anidulafungin SMIC90s (␮g/ml) for the isolates of C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei were 0.125, 1, 0.06, 0.06, and 0.25, respectively. The caspofungin SMIC90s (␮g/ml) for the isolates of C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei were 0.5, 8, 1, 0.5, and 1, respectively. As shown in Table 2, serum increased anidulafungin SMIC90s (␮g/ml) to 1, 16, 4, 2, and 8 and caspofungin SMIC90s (␮g/ml) to 8, ⬎16, 8, 8, and ⬎16 for the isolates of C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei, respectively. The amphotericin B SMIC90s (␮g/ml) for the isolates of

C. albicans, C. parapsilosis, and C. krusei were ⬎32, whereas those for the isolates of C. tropicalis and C. glabrata were 32 and 16, respectively. The fluconazole and voriconazole SMIC90s (␮g/ml) for isolates of all the species studied were ⬎1,024 and ⬎128, respectively. The distributions of antifungal MICs (or MECs for echinocandins) and SMICs for the different Aspergillus species are shown in Tables 4, 5, and 6. The anidulafungin and caspofungin SMIC90s (␮g/ml) for isolates of all the species studied (A. fumigatus, A. flavus, A. terreus, and A. niger) were both ⬎16. The amphotericin B SMIC90s (␮g/ml) for the isolates of A. fumigatus, A. flavus, A. terreus, and A. niger were 16, 16, ⬎32, and 8, respectively. The voriconazole SMIC90s (␮g/ml) for the isolates of A. fumigatus, A. flavus, A. terreus, and A. niger were ⬎128, 128, 128, and 64, respectively. As expected, fluconazole and voriconazole were ineffective against biofilms of all five Candida species, with none of the isolates showing an azole SMIC of ⬍128 ␮g/ml. Despite SMIC90 values nearing those of the azoles, the SMICs (␮g/ml) of amphotericin B ranged from 0.5 to ⬎32 for isolates of C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata and from 1 to ⬎32 for isolates of C. krusei. As detailed in Table 3, isolates from all Candida species were captured almost equally at the different SMIC values, except for 40 C. parapsilosis isolates, for which SMICs of 0.5 to 2 ␮g/ml comprised the majority of them (28 isolates). Consistent with recent data reported by Choi et al. (4), we showed that amphotericin B was weakly effective against the biofilms of all five species. This was also in agreement with the previous observation by Kuhn et al. (11) of unique efficacy by lipid-formulated amphotericin B against biofilms of C. albicans and C. parapsilosis isolates (SMICs of 0.25 and 1 ␮g/ml, respectively), while conventional amphotericin B failed to inhibit the same Candida strains when grown as biofilms (SMICs of 4 and 8 ␮g/ml, respectively). In addition, the use of microtiter plates as the surface for biofilm

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TABLE 2. Distributions of echinocandins MICs for different Candida species isolates under planktonic or biofilm growth conditions in the presence of 50% serum Species (no. of isolates)

Drug

Anidulafungin


Caspofungin


a

No. of isolates at MIC (␮g/ml) of:

Type of MICa

ⱕ0.03

0.06

0.125

0.25

0.5

1

2

Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC

12

8 4

14

2

Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC

5

14 2 12 6

8

13

6 12

4

8

16

⬎16

10 4

18 19

12 15

2

8 8

4 10

2

9 22

5 3 8

7 19

13

2 4 6

5

6

14 8

2 6 16

2 9

7

3

12

4 4 5 3 3

4

12

6 6

9 7 9


Planktonic MIC90 shifts were consistently observed for both anidulafungin (8 ␮g/ml) and caspofungin (16 ␮g/ml), reflecting their greater antifungal potencies in the absence of serum. By this test condition, 30 of 40 C. parapsilosis isolates were found to exhibit caspofungin SMICs of ⱖ2 ␮g/ml. Consistent with these findings, studies by Khun et al. (11) on two biofilmassociated C. parapsilosis isolates, one (PA/71) from a sputum culture and the other (P92) from a blood culture, demonstrated that caspofungin inhibited only PA/71 (SMIC of 0.125 ␮g/ml) while exhibiting a high SMIC (4 ␮g/ml) for strain P92. On the other hand, sessile cells from 12 C. parapsilosis isolates studied by Choi et al. (4) were all less susceptible to caspofungin (SMIC range of 1 to ⬎16 ␮g/ml). Whether the behavior of C. parapsilosis biofilm (10) or the rate of drug diffusion through the biofilm (1), also linked to the echinocandin relative hydro-

development, instead of disks made from catheter materials used elsewhere, could explain our high amphotericin B SMIC90 value differing from that reported by some investigators (25). As shown, anidulafungin was the agent most active against sessile cells of isolates from all five Candida species. Caspofungin, the other echinocandin antifungal tested, was slightly less effective in this study. Interestingly, a prominent difference was noted between the SMIC90 values for anidulafungin (1 ␮g/ml) and caspofungin (8 ␮g/ml) for C. parapsilosis isolates, whereas the planktonic MIC90s were 0.5 ␮g/ml for both drugs. However, this difference disappeared in the presence of serum, where both drugs showed higher SMIC90 (16 and ⬎16 ␮g/ml, respectively), since serum was shown to differentially reduce the antifungal efficacies of echinocandin drugs due to their extensive binding to serum proteins (19).

TABLE 3. Distributions of amphotericin B MICs for different Candida species isolates under planktonic or biofilm growth conditions Species (no. of isolates)

C. albicans (20) C. parapsilosis (40) C. tropicalis (20) C. glabrata (20) C. krusei (20) a

Type of MICa


0.125

0.25

Planktonic MIC Biofilm SMIC

12

8


15


0.06

6

10


6


4

12

8

16

32

⬎32

2

2

4

1

2

6

5

2

0.5

1

2

4

2

4

4

2

13 12

6

10

2

1

1

7

1

3

1

9 1

5

1

1

6

4

1

1

6

2

2

4 5 12

2

4


3

5

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TABLE 4. Distributions of echinocandins MECs or MICs for different Aspergillus species isolates under planktonic or biofilm growth conditions Drug

Anidulafungin

Type of MEC or MICa

A. fumigatus (10)

Planktonic MEC Biofilm SMIC Planktonic MEC Biofilm SMIC Planktonic MEC Biofilm SMIC Planktonic MEC Biofilm SMIC

A. flavus (10) A. terreus (10) A. niger (10) Caspofungin

A. fumigatus (10) A. flavus (10) A. terreus (10) A. niger (10)

a

No. of isolates at MEC or MIC (␮g/ml) of:

Species (no. of isolates)

Planktonic MEC Biofilm SMIC Planktonic MEC Biofilm SMIC Planktonic MEC Biofilm SMIC Planktonic MEC Biofilm SMIC

ⱕ0.03

0.06

9

1

0.125

0.25

0.5

1

2

4

8

16

⬎16

2

4

4

3

5

2

10 10 10 10 4

4

2

3

7

2

8

10 10 10 10 10 3

7

Planktonic MECs were determined by the CLSI broth microdilution method, whereas biofilm SMICs were measured by the XTT reduction assay.

phobicity, would account for the different activity displayed by both echinocandins in our study is not known. Also, novel biochemical and genetic mechanisms in biofilm formation might be responsible for the observed differences in echinocandin susceptibilities among different Candida species, as suggested by Choi et al. (4). In contrast to previous biofilm studies mainly focusing on C. albicans and C. parapsilosis isolates (8, 9, 11, 25), our analysis addressing clinical isolates of C. krusei confirms and extends data on the potency of anidulafungin and, to a lesser extent, caspofungin (8, 9) against Candida biofilms. As almost all of the bloodstream isolates in our study had been conclusively associated with a device infection (26), this strengthens the importance of our findings in the context of clinical treatment of biofilm-related candidiasis and warrants investigation on the use of anidulafungin as an antifungal catheter lock solution to prevent invasive Candida infection in high-risk patients. With regard to the Aspergillus species, our SMIC results paralleled but expanded those of the previous few investigations involving A. fumigatus, which until now was the only Aspergillus species studied concerning antifungal drug suscep-

tibility under biofilm conditions (3, 15, 16, 23). Despite a highlevel activity profile for anidulafungin and caspofungin (MEC range of 0.03 to 0.06 ␮g/ml) against planktonic Aspergillus species isolates, which confirmed earlier data (14), we noticed that A. fumigatus (and three other species) isolates exhibited elevated SMICs for anidulafungin (ranging from 8 to ⬎16 ␮g/ml, except for A. terreus isolates, whose SMICs were ⬎16 ␮g/ml) and caspofungin (ranging from 16 to ⬎16 ␮g/ml). Accordingly, Seidler et al. (23) found that the echinocandin (caspofungin and micafungin) MICs for the A. fumigatus strain ATCC 9197 embedded in biofilm were ⬎8 ␮g/ml, whereas higher caspofungin SMIC values (64 to 128 ␮g/ml) were obtained for the A. fumigatus strain Af293 when grown at 24 h (as dense intertwined hyphae) (16) as well as for the five A. fumigatus strains, including four clinical isolates studied by Mowat et al. (15). In contrast (and in comparison with voriconazole [SMICs ranging from 32 to ⬎128 ␮g/ml]), amphotericin B was the most effective antifungal agent, as SMICs (␮g/ml) of 8 were found for eight isolates of A. fumigatus and A. flavus and SMICs of ⱕ8 were found for nine isolates of A. niger. Although expected, our SMIC data suggest a poor utility of these agents

TABLE 5. Distributions of amphotericin B MICs for different Aspergillus species isolates under planktonic or biofilm growth conditions Species (no. of isolates)

Type of MICa

A. fumigatus (10) A. flavus (10) A. terreus (10) A. niger (10) a


0.06

0.125

0.25

0.5

1


2

7

1


3

5

4

8

16

32

4

5

1

4

6

2

Planktonic MIC Biofilm SMIC Planktonic MIC Biofilm SMIC

2

4

6 10

3

7 2

7

1


⬎32

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TABLE 6. Distributions of voriconazole MICs for different Aspergillus species isolates under planktonic or biofilm growth conditions Species (no. of isolates)

Type of MICa

A. fumigatus (10) A. flavus (10) A. terreus (10) A. niger (10) a


0.125

0.25

0.5


3

6

1


1


4


6 4 8

1

2

4

8

16

64

128

⬎128

1

4

5

8

2

1

7

2

8

2

32

3 2 1

1


against the most common Aspergillus species identified in biofilm-associated infections. However, as echinocandins, caspofungin in particular, were shown in vitro to be active against germinated conidia but not against complex filamentous Aspergillus forms (16), these drugs have potential for prophylaxis or early antifungal therapy for patients with suspected aspergillosis. This work was supported by a grant from Pfizer Pharmaceutical Group, Italy. REFERENCES 1. Al-Fattani, M. A., and L. J. Douglas. 2004. Penetration of Candida biofilms by antifungal agents. Antimicrob. Agents Chemother. 48:3291–3297. 2. Angiolella, L., et al. 2008. Increase of virulence and its phenotypic traits in drug-resistant strains of Candida albicans. Antimicrob. Agents Chemother. 52:927–936. 3. Beauvais, A., et al. 2007. An extracellular matrix glues together the aerialgrown hyphae of Aspergillus fumigatus. Cell. Microbiol. 9:1588–1600. 4. Choi, H. W., et al. 2007. Species-specific differences in the susceptibilities of biofilms formed by Candida bloodstream isolates to echinocandin antifungals. Antimicrob. Agents Chemother. 51:1520–1523. 5. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A3. Clinical and Laboratory Standards Institute, Wayne, PA. 6. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard, 2nd ed., M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA. 7. Hasan, F., I. Xess, X. Wang, N. Jain, and B. C. Fries. 2009. Biofilm formation in clinical Candida isolates and its association with virulence. Microbes Infect. 11:753–761. 8. Jacobson, M. J., K. E. Piper, G. Nguyen, J. M. Steckelberg, and R. Patel. 2008. In vitro activity of anidulafungin against Candida albicans biofilms. Antimicrob. Agents Chemother. 52:2242–2243. 9. Katragkou, A., et al. 2008. Differential activities of newer antifungal agents against Candida albicans and Candida parapsilosis biofilms. Antimicrob. Agents Chemother. 52:357–360. 10. Kuhn, D. M., J. Chandra, P. K. Mukherjee, and M. A. Ghannoum. 2002. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect. Immun. 70:878–888. 11. Kuhn, D. M., T. George, J. Chandra, P. K. Mukherjee, and M. A. Ghannoum. 2002. Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob. Agents Chemother. 46:1773–1780.

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