J Infect Chemother xxx (2015) 1e7
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms Akira Kawai, Yuka Yamagishi, Hiroshige Mikamo* Department of Clinical Infectious Diseases, Aichi Medical University Graduate School of Medicine, Aichi, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2015 Received in revised form 23 May 2015 Accepted 25 May 2015 Available online xxx
Non-albicans Candida species are being isolated with increasing frequency. In this study, biofilm formation by Candida tropicalis, Candida parapsilosis and Candida glabrata was evaluated and the activities of liposomal amphotericin B (LAB), micafungin (MFG) and fluconazole (FLC) against these biofilms were assessed using a clinically relevant in vitro model system. LAB exhibited strong activities against the three non-albicans Candida species and showed dose-dependent efficacy. MFG displayed a paradoxical growth effect against the C. tropicalis biofilm. FLC was ineffective for non-albicans biofilms. This study shows that Candida biofilms have unique susceptibility to LAB. The dose-dependent effects of LAB indicate that this drug may be a useful treatment for biofilm formation by non-albicans Candida species in cases in which the catheter cannot be removed for clinical reasons. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Antifungals Biofilms Candida tropicalis Candida parapsilosis Candida glabrata Paradoxical growth effect (PGE)
1. Introduction Candidemia is increasing gradually in recent clinical practices and is an important cause of mortality [1,2]. Candida albicans is still the most frequently isolated Candida species, but non-albicans Candida species are increasingly common. The most recent national survey in Japan found that C. albicans comprised less than 50% of isolates [3]. Candida parapsilosis (23%), Candida glabrata (18%), and Candida tropicalis (12%) as a group represented the majority of infections [3], and C. tropicalis fungemia is an independent risk factor associated with increased mortality [4]. Candida species have a propensity to grow as biofilms on implanted medical devices such as central venous and urinary catheters [5]. Thus, Candida species cause biomaterial-related infections and give rise to infective pathologies typically associated with biofilm formation. These species form biofilms with comparable levels of antifungal resistance to C. albicans [6,7]. Candidiasis associated with intravenous lines and bioprosthetic devices is especially problematic, since these devices can act as substrates for biofilm growth. Antifungal therapy is effective, but the affected devices generally need to be removed [4]. Removal of these devices
* Corresponding author. Zip code 480-1195, 1-1 Yazakokarimata, Nagakute, Aichi, Japan. Tel.: þ81 561 62 3311x77668; fax: þ81 561 61 1842. E-mail address: [email protected] (H. Mikamo).
has serious implications in cases with infected heart disease valves, joint prostheses, and central nervous system shunts. In fact, failure of antifungal therapy can lead to chronic infections that can only be treated by surgery and/or removal of the medical devices. This study aims to investigate and compare the susceptibility of key antifungal agents classes (polyenes, echinocandins, azoles) against three major species of non-albicans Candida biofilms. We recently determined the in vitro antifungal activities of liposomal amphotericin B (LAB), micafungin (MFG) and fluconazole (FLC) against Candida strains in their planktonic states. In the current study, we used these drugs as examples of the three major classes of antifungal agents and evaluated their efficacy against C. tropicalis, C. parapsilosis and C. glabrata bloodstream isolates growing as biofilms. 2. Materials and methods 2.1. Organisms Formed biofilm was measured the ability and antifungal susceptibility of the clinical isolates, then was chosen the most suitable clinical strains which have strong biofilm formation ability and high antifungal susceptibility for an experiment (data not shown). Three clinical non-albicans Candida spp. isolates were evaluated: C. tropicalis AMTC001414, C. parapsilosis AMTC45867, and C. glabrata
http://dx.doi.org/10.1016/j.jiac.2015.05.007 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
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A. Kawai et al. / J Infect Chemother xxx (2015) 1e7
AMTC5382. All the isolates were from patients admitted to Aichi Medical University Hospital, Japan and were recovered between 2008 and 2013. 2.2. Antifungal agents Liposomal amphotericin B (LAB), micafungin (MFG), and fluconazole (FLC) were supplied by Sumitomo Dainippon Pharma (Osaka, Japan), Astellas (Tokyo, Japan), and Pfizer (Tokyo, Japan), respectively. The agents were obtained in powder form and dissolved in sterile water. 2.3. MICs against planktonic cells The susceptibility of planktonic cells was determined by the Clinical Laboratory Standard Institute M27- A3 method [8] using RPMI-1640 medium with addition of 50% FBS. The planktonic MICs of LAB and FLC were visually determined as the minimum antifungal concentration that caused >50% fungal damage compared to that of the drug-free growth control. The MIC values against planktonic cells of MCF was determined as antifungal concentration that can not be visually observed fungal growth. 2.4. Biofilm formation and testing The susceptibility of biofilms was determined using the 2,3bis(2-methoxy-4-nitro-5sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay [9,10]. FBS was added to 24-well tissue culture plates and incubated for 24 h at 37 C. After washing three times with PBS, the 24-well plates containing 200 ml of a suspension of 1 107/ml Candida cells were incubated for 1.5 h at 37 C. The cells were grown overnight in Sabouraud dextrose broth as preculture condition. After washing with sterile PBS to remove unattached cells, 500 ml of fresh RPMI1640 plus 50% FBS was added and the plates were incubated for 18 h at 37 C to allow biofilm formation. Formed biofilm was visually confirmed the fungal morphology in a bottom of the well, then the biofilm formation was visualized by using confocal laser scanning microscopy with Concanavalin A and Fun-1 dyes [11]. The bottom of the well were washed with sterile PBS prior to testing with each antifungal agent from 0.5 to 256 mg/ml. After exposure to the doubling dilution of different concentrations of each antifungal agent (600 ml) for 24 h at 37 C, the plates were reacted for 4 h at 37 C with XTT-menadione solution. The medium was removed and centrifuged for 10 min at 6000 g to pellet suspended cells and debris from the plates. The supernatant was measured at 492 nm using a spectrophotometer (Hitachi U-1500). The antifungal concentration causing 50% reduction of the biofilm compared with the metabolic activity of a drug-free control was determined as the sessile MIC (SMIC). Antifungal susceptibility tests of biofilms were performed at least in duplicate.
3. Results 3.1. Antifungal susceptibilities against non-albicans Candida planktonic cells Antifungal susceptibilities against C. tropicalis, C. parapsilosis, C. glabrata planktonic cells to LAB, MFG and FLC are shown in Table 1. The MICs of LAB were 16, 8 and 8 mg/ml for C. tropicalis, C. parapsilosis and C. glabrata, respectively. The planktonic cells of C. tropicalis and C. glabrata were highly susceptible to MFG, with MICs of 1 mg/ml, whereas the MIC of MFG for C. parapsilosis was 32 mg/ml. The MICs of FLC were 4, 4 and 256 mg/ml for planktonic cells of C. tropicalis, C. parapsilosis and C. glabrata, respectively. FLC showed high MIC that displayed resistance for C. glabrata. 3.2. Antifungal susceptibilities of Candida biofilms In the XTT reduction assay, SMICs of the key antifungal agents tested against biofilms formed by three major non-albicans Candida isolates are shown in Table 1. A clear difference in antifungal susceptibilities was seen against C. tropicalis biofilm. LAB showed high antifungal efficacy against biofilm of C. tropicalis, with SMIC50/80 values of 2e64. For MFG and FLC, the respective SMIC50/80 values were >256 mg/ml against C. tropicalis biofilm. All antifungal agents were characterized by high SMIC50 against biofilms of C. parapsilosis (8e256 mg/ml), with the lowest SMIC50 characterizing LAB (Table 1; 8 mg/ml). LAB and MFG were activity than FLC for C. glabrata biofilm, MFG had the lowest value among them (1 mg/ml). In contrast, FLC was ineffective, with no notable sessile activity against any strains tested (SMIC50/80 >256 mg/ml). Representative curves for inhibition of growth of C. tropicalis, C. parapsilosis and C. glabrata biofilms in the presence of different concentrations of LAB, MFG and FLC are shown in Figs. 1e3. LAB dose-dependently reduced the metabolic activity against C. tropicalis biofilm (Fig. 1). In comparison, C. tropicalis biofilm were less susceptible to MFG at concentrations above SMIC (8e64 mg/ml) than at 0.5e4 mg/ml. This observation is referred to as a paradoxical growth effect. This was not observed for LAB with C. tropicalis. FLC had little effect on the C. tropicalis biofilm (MIC50 >256 mg/ml). LAB also indicated dose-dependent reduction of metabolic activity against C. parapsilosis biofilm (Fig. 2). In comparison, MFG and FLC had little effect on C. parapsilosis biofilm. However, growth inhibition at low FLC concentrations, paradoxical growth above MIC, and inhibition at high FLC concentrations occurred for C. parapsilosis biofilm. The dose-dependent effects of LAB also showed in the C. glabrata biofilm (Fig. 3). MFG had a rapid effect at all concentrations. In comparison, FLC was ineffective against the C. glabrata biofilm. Overall, these results suggest that LAB was more effective and was not observed the paradoxical growth effect against all biofilms.
Table 1 Antifungal susceptibility testing of non-albicans Candida isolates under planktonic (MIC) and biofilm (SMIC) growing conditions. MIC(mg/mL), minimal inhibitory concentration; SMIC(mg/mL), sessile minimal inhibitory concentration (SMIC50/SMIC80). Antifungal agents
LAB MCF FLC
C. tropicalis AMTC001414
C. parapsilosis AMTC45867
Planktonic cell
Biofilm
Planktonic cell
Biofilm
C. glabrata AMTC5382 Planktonic cell
Biofilm
MIC
SMIC50
SMIC80
MIC
SMIC50
SMIC80
MIC
SMIC50
SMIC80
16 1 4
2 >256 >256
64 >256 >256
8 32 4
8 16 >256
256 64 >256
8 1 256
4 1 >256
128 1 >256
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
A. Kawai et al. / J Infect Chemother xxx (2015) 1e7
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Fig. 1. Activities of different concentrations of LAB, MFG and FLC against C. tropicalis biofilms. Graphs shows the XTT activity of C. tropicalis normalized to control (untreated), which was taken as 100%. Each result is representative of at least two experiments.
4. Discussion Using a biofilm model system, we measured SMIC50 values for isolates from three clinically relevant and morphologically different
Candida species: C. tropicalis, C. parapsilosis and C. glabrata [12]. Measurements based on XTT metabolic activity can be used to monitor biofilm formation indirectly [9,10]. Most studies have focused on C. albicans biofilm [13e17]; however, the aim of this
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
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Fig. 2. Activities of different concentrations of LAB, MFG and FLC against C. parapsilosis biofilms. Graphs shows the XTT activity of C. parapsilosis normalized to control (untreated), which was taken as 100%. Each result is representative of at least two experiments.
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
A. Kawai et al. / J Infect Chemother xxx (2015) 1e7
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Fig. 3. Activities of different concentrations of LAB, MFG and FLC against C. glabrata biofilms. Graphs shows the XTT activity of C. glabrata normalized to control (untreated), which was taken as 100%. Each result is representative of at least two experiments.
study was to investigate and compare the susceptibility of three major species of non-albicans Candida biofilms to key antifungal drugs. LAB has dose-dependent activities against all non-albicans Candida species and does not exhibit a paradoxical growth effect
with any biofilms, while the greater mortality associated with C. tropicalis [4] suggests that the biofilm formed by C. tropicalis is more resistant to antifungal agents. Thus, it is particularly important to investigate the susceptibility of biofilms to antifungal agents.
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
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Our results showed that C. tropicalis biofilm was more susceptible to LAB than to MFG and FLC. This suggests that LAB has an extended spectrum of activity against various Candida species and exhibits fungicidal activity, perhaps because it can relatively easily penetrate the biofilm and reach the inner candida membrane. These findings suggest that high dose LAB may have a good outcome, but this agent may also exhibit dose-dependent nephrotoxicity in clinical practice. In contrast, the MIC of LAB for planktonic cells of C. tropicalis was relatively higher than that found by Bizerra et al. [18]. This suggests that addition of FBS to a eutrophic planktonic cell medium increases fungal proliferation. Paradoxical growth effects occurred for MFG on C. tropicalis and FLC on C. parapsilosis, but LAB had no paradoxical effects and good activity against all Candida biofilms. MFG reduced the metabolic activity of C. tropicalis biofilm at low concentrations, but paradoxical growth above MIC prevented MFG from completely eliminating the activity, even at the highest concentration tested. Similar paradoxical effects on growth of C. tropicalis biofilm have been found for other echinocandins [19,20]. There are several proposed explanations for these effects at high concentrations of echinocandins [20,21]. Caspofungin, a member of the echinocandin family used worldwide, inhibits synthesis of 1,3-beta-glucan, a fundamental component of fungal cell walls, by inhibition of 1,3-beta-glucan synthase [22e24]. The paradoxical growth effect has been investigated by measuring the levels of 1,3-beta-glucan, 1,6-beta-glucan and chitin in a C. albicans strain after exposure to high caspofungin [25,26]. In clinical practice in Japan, high-dose MFG may be used for invasive candidiasis, but there is no evidence for dose-dependent antifungal activity of MFG. Factors such as protein binding in human serum might also influence the MIC for MFG, since serum shifts the activity of MFG in in vitro susceptibility tests on Candida spp [27,28]. Further studies in vivo and in vitro are thus needed to examine the effects of high dose MFG and the impact of human serum on MFG activity. FLC has a limited spectrum against non-albicans Candida species, but may be more effective than echinocandins against C. parapsilosis [29]. The paradoxical growth effect of FLC on C. parapsilosis may be due to biofilm formation by C. parapsilosis being strongly strain-dependent [30]. Biofilm formation enhances the capacity of C. parapsilosis to colonize catheters and intravascular central lines, which produces a drug-resistant phenotype [31,32]. Biofilms also show significantly enhanced resistance to some antifungal agents and altered phenotypes, making eradication difficult [30,33]. Recent clinical data have shown that patients with defined biofilm-forming bloodstream non-albicans Candida species have a shorter hospital stay and lower mortality when treated with echinocandins such as caspofungin and liposomal amphotericin B [34]. Our findings are generally consistent with these observations, but not all patients respond equally to antifungal treatment. This may be partly due to the level of biofilm formation by individual strains and/or differential biofilm responses to different antifungal classes. Interestingly, no antifungal agents completely eliminate biofilms, suggesting that adjunctive therapy is required [35]. Further studies are needed to maximize antifungal efficacy against biofilms formed by non-albicans Candida species and to improve clinical management of patients with these infections. Conflict of interest Sumitomo Dainippon Pharma is acknowledged for financial support of this study.
Acknowledgements We thank ICAM Co., Ltd. (Japan) for technical assistance with the susceptibility tests of planktonic cells and biofilms. References [1] Tumbarello M, Posteraro B, Trecarichi EM, Fiori B, Rossi M, Porta R, et al. Biofilm production by Candida species and inadequate antifungal therapy as predictors of mortality for patients with candidemia. J Clin Microbiol 2007;45: 1843e50. [2] Nagao M. A multicentre analysis of epidemiology of the nosocomial bloodstream infections in Japanese university hospitals. Clin Microbiol Infect 2013;19:852e8. [3] Takakura S, Fujihara N, Saito T, Kudo T, Iinuma Y, Ichiyama S. National surveillance of species distribution in blood isolates of Candida species in Japan and their susceptibility to six antifungal agents including voriconazole and micafungin. J Antimicrob Chemother 2004;53:283e9. [4] Andes DR, Safdar N, Baddley JW, Playford G, Reboli AC, Rex JH, et al. Mycoses study Group. Impact of treatment strategy on outcomes in patients with candidemia and other forms of invasive candidiasis: a patient-level quantitative review of randomized trials. Clin Infect Dis 2012;54:1110e22. pez-Ribot JL. Candida biofilms on implanted bio[5] Ramage G, Martínez JP, Lo materials: a clinically significant problem. FEMS Yeast Res 2006;6:979e86. [6] Pfaller M, Neofytos D, Diekema D, Azie N, Meier-Kriesche HU, Quan SP, et al. Epidemiology and outcomes of candidemia in 3648 patients: data from the prospective antifungal therapy (PATH Alliance®) registry, 2004-2008. Diagn Microbiol Infect Dis 2012;74:323e31. mez-García de la Pedrosa E, Pintado V, Cobo J, [7] Fortún J, Martín-D avila P, Go Fresco G, et al. Emerging trends in candidemia: a higher incidence but a similar outcome. J Infect 2012;65:64e70. [8] Wayne PA. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard. CLSI document M27eA3. 3rd ed. Clinical and Laboratory Standards Institute; 2008. [9] Hawser SP, Norris H, Jessup CJ, Ghannoum MA. Comparison of a 2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric method with the standardized national committee for clinical laboratory standards method of testing clinical yeast isolates for susceptibility to antifungal agents. J Clin Microbiol 1998;36:1450e2. pez-Ribot JL. Standardized method [10] Ramage G, Vande Walle K, Wickes BL, Lo for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 2001;45:2475e9. [11] Kuhn DM, Chandra J, Mukherjee PK, Ghannoum MA. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect Immun 2002;70:878e88. [12] Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Adherence and biofilm formation of non-Candida albicans Candida species. Trends Microbiol 2011;19:241e7. [13] Kuhn DM, George T, Chandra J, Mukherjee PK, Ghannoum MA. Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother 2002;46: 1773e80. pez-Ribot JL. In vitro [14] Ramage G, VandeWalle K, Bachmann SP, Wickes BL, Lo pharmacodynamic properties of three antifungal agents against preformed Candida albicans biofilms determined by time-kill studies. Antimicrob Agents Chemother 2002;46:3634e6. [15] Schinabeck MK, Long LA, Hossain MA, Chandra J, Mukherjee PK, Mohamed S, et al. Rabbit model of Candida albicans biofilm infection: liposomal amphotericin B antifungal lock therapy. Antimicrob Agents Chemother 2004;48: 1727e32. [16] Shuford JA, Rouse MS, Piper KE, Steckelberg JM, Patel R. Evaluation of caspofungin and amphotericin B deoxycholate against Candida albicans biofilms in an experimental intravascular catheter infection model. J Infect Dis 2006;194:710e3. [17] Uppuluri P, Srinivasan A, Ramasubramanian A, Lopez-Ribot JL. Effects of fluconazole, amphotericin B, and caspofungin on Candida albicans biofilms under conditions of flow and on biofilm dispersion. Antimicrob Agents Chemother 2011;55:3591e3. [18] Bizerra FC, Nakamura CV, de Poersch C, Estivalet Svidzinski TI, Borsato Quesada RM, Goldenberg S, et al. Characteristics of biofilm formation by Candida tropicalis and antifungal resistance. FEMS Yeast Res 2008;8:442e50. [19] Ferreira JA, Carr JH, Starling CE, de Resende MA, Donlan RM. Biofilm formation and effect of caspofungin on biofilm structure of Candida species bloodstream isolates. Antimicrob Agents Chemother 2009;53:4377e84. [20] Chamilos G, Lewis RE, Albert N, Kontoyiannis DP. Paradoxical effect of echinocandins across Candida species in vitro: evidence for echinocandin-specific and candida species-related differences. Antimicrob Agents Chemother 2007;51:2257e9. [21] Melo AS, Colombo AL, Arthington-Skaggs BA. Paradoxical growth effect of caspofungin observed on biofilms and planktonic cells of five different Candida species. Antimicrob Agents Chemother 2007;51:3081e8.
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[29] Pfaller MA, Andes D, Diekema DJ, Espinel-Ingroff A, Sheehan D. CLSI subcommittee for antifungal susceptibility Testing. Wild-type MIC distributions, epidemiological cutoff values and species-specific clinical breakpoints for fluconazole and Candida: time for harmonization of CLSI and EUCAST broth microdilution methods. Drug Resist Updat 2010;13:180e95. [30] Kuhn DM, Ghannoum MA. Candida biofilms: antifungal resistance and emerging therapeutic options. Curr Opin Investig Drugs 2004;5:186e97. cser A, Nosanchuk JD. Candida parapsilosis, an emerging fungal [31] Trofa D, Ga pathogen. Clin Microbiol Rev 2008;21:606e25. [32] Douglas LJ. Candida biofilms and their role in infection. Trends Microbiol 2003;11:30e6. [33] Kumamoto CA. Candida biofilms. Curr Opin Microbiol 2002;5:608e11. [34] Tumbarello M, Fiori B, Trecarichi EM, Posteraro P, Losito AR, De Luca A, et al. Risk factors and outcomes of candidemia caused by biofilm-forming isolates in a tertiary care hospital. PLoS One 2012;7:e33705. [35] Katragkou A, McCarthy M, Alexander EL, Antachopoulos C, Meletiadis J, JabraRizk MA, et al. In vitro interactions between farnesol and fluconazole, amphotericin B or micafungin against Candida albicans biofilms. J Antimicrob Chemother 2015 Feb;70:470e8.
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Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms Akira Kawai, Yuka Yamagishi, Hiroshige Mikamo* Department of Clinical Infectious Diseases, Aichi Medical University Graduate School of Medicine, Aichi, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2015 Received in revised form 23 May 2015 Accepted 25 May 2015 Available online xxx
Non-albicans Candida species are being isolated with increasing frequency. In this study, biofilm formation by Candida tropicalis, Candida parapsilosis and Candida glabrata was evaluated and the activities of liposomal amphotericin B (LAB), micafungin (MFG) and fluconazole (FLC) against these biofilms were assessed using a clinically relevant in vitro model system. LAB exhibited strong activities against the three non-albicans Candida species and showed dose-dependent efficacy. MFG displayed a paradoxical growth effect against the C. tropicalis biofilm. FLC was ineffective for non-albicans biofilms. This study shows that Candida biofilms have unique susceptibility to LAB. The dose-dependent effects of LAB indicate that this drug may be a useful treatment for biofilm formation by non-albicans Candida species in cases in which the catheter cannot be removed for clinical reasons. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Antifungals Biofilms Candida tropicalis Candida parapsilosis Candida glabrata Paradoxical growth effect (PGE)
1. Introduction Candidemia is increasing gradually in recent clinical practices and is an important cause of mortality [1,2]. Candida albicans is still the most frequently isolated Candida species, but non-albicans Candida species are increasingly common. The most recent national survey in Japan found that C. albicans comprised less than 50% of isolates [3]. Candida parapsilosis (23%), Candida glabrata (18%), and Candida tropicalis (12%) as a group represented the majority of infections [3], and C. tropicalis fungemia is an independent risk factor associated with increased mortality [4]. Candida species have a propensity to grow as biofilms on implanted medical devices such as central venous and urinary catheters [5]. Thus, Candida species cause biomaterial-related infections and give rise to infective pathologies typically associated with biofilm formation. These species form biofilms with comparable levels of antifungal resistance to C. albicans [6,7]. Candidiasis associated with intravenous lines and bioprosthetic devices is especially problematic, since these devices can act as substrates for biofilm growth. Antifungal therapy is effective, but the affected devices generally need to be removed [4]. Removal of these devices
* Corresponding author. Zip code 480-1195, 1-1 Yazakokarimata, Nagakute, Aichi, Japan. Tel.: þ81 561 62 3311x77668; fax: þ81 561 61 1842. E-mail address: [email protected] (H. Mikamo).
has serious implications in cases with infected heart disease valves, joint prostheses, and central nervous system shunts. In fact, failure of antifungal therapy can lead to chronic infections that can only be treated by surgery and/or removal of the medical devices. This study aims to investigate and compare the susceptibility of key antifungal agents classes (polyenes, echinocandins, azoles) against three major species of non-albicans Candida biofilms. We recently determined the in vitro antifungal activities of liposomal amphotericin B (LAB), micafungin (MFG) and fluconazole (FLC) against Candida strains in their planktonic states. In the current study, we used these drugs as examples of the three major classes of antifungal agents and evaluated their efficacy against C. tropicalis, C. parapsilosis and C. glabrata bloodstream isolates growing as biofilms. 2. Materials and methods 2.1. Organisms Formed biofilm was measured the ability and antifungal susceptibility of the clinical isolates, then was chosen the most suitable clinical strains which have strong biofilm formation ability and high antifungal susceptibility for an experiment (data not shown). Three clinical non-albicans Candida spp. isolates were evaluated: C. tropicalis AMTC001414, C. parapsilosis AMTC45867, and C. glabrata
http://dx.doi.org/10.1016/j.jiac.2015.05.007 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
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A. Kawai et al. / J Infect Chemother xxx (2015) 1e7
AMTC5382. All the isolates were from patients admitted to Aichi Medical University Hospital, Japan and were recovered between 2008 and 2013. 2.2. Antifungal agents Liposomal amphotericin B (LAB), micafungin (MFG), and fluconazole (FLC) were supplied by Sumitomo Dainippon Pharma (Osaka, Japan), Astellas (Tokyo, Japan), and Pfizer (Tokyo, Japan), respectively. The agents were obtained in powder form and dissolved in sterile water. 2.3. MICs against planktonic cells The susceptibility of planktonic cells was determined by the Clinical Laboratory Standard Institute M27- A3 method [8] using RPMI-1640 medium with addition of 50% FBS. The planktonic MICs of LAB and FLC were visually determined as the minimum antifungal concentration that caused >50% fungal damage compared to that of the drug-free growth control. The MIC values against planktonic cells of MCF was determined as antifungal concentration that can not be visually observed fungal growth. 2.4. Biofilm formation and testing The susceptibility of biofilms was determined using the 2,3bis(2-methoxy-4-nitro-5sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay [9,10]. FBS was added to 24-well tissue culture plates and incubated for 24 h at 37 C. After washing three times with PBS, the 24-well plates containing 200 ml of a suspension of 1 107/ml Candida cells were incubated for 1.5 h at 37 C. The cells were grown overnight in Sabouraud dextrose broth as preculture condition. After washing with sterile PBS to remove unattached cells, 500 ml of fresh RPMI1640 plus 50% FBS was added and the plates were incubated for 18 h at 37 C to allow biofilm formation. Formed biofilm was visually confirmed the fungal morphology in a bottom of the well, then the biofilm formation was visualized by using confocal laser scanning microscopy with Concanavalin A and Fun-1 dyes [11]. The bottom of the well were washed with sterile PBS prior to testing with each antifungal agent from 0.5 to 256 mg/ml. After exposure to the doubling dilution of different concentrations of each antifungal agent (600 ml) for 24 h at 37 C, the plates were reacted for 4 h at 37 C with XTT-menadione solution. The medium was removed and centrifuged for 10 min at 6000 g to pellet suspended cells and debris from the plates. The supernatant was measured at 492 nm using a spectrophotometer (Hitachi U-1500). The antifungal concentration causing 50% reduction of the biofilm compared with the metabolic activity of a drug-free control was determined as the sessile MIC (SMIC). Antifungal susceptibility tests of biofilms were performed at least in duplicate.
3. Results 3.1. Antifungal susceptibilities against non-albicans Candida planktonic cells Antifungal susceptibilities against C. tropicalis, C. parapsilosis, C. glabrata planktonic cells to LAB, MFG and FLC are shown in Table 1. The MICs of LAB were 16, 8 and 8 mg/ml for C. tropicalis, C. parapsilosis and C. glabrata, respectively. The planktonic cells of C. tropicalis and C. glabrata were highly susceptible to MFG, with MICs of 1 mg/ml, whereas the MIC of MFG for C. parapsilosis was 32 mg/ml. The MICs of FLC were 4, 4 and 256 mg/ml for planktonic cells of C. tropicalis, C. parapsilosis and C. glabrata, respectively. FLC showed high MIC that displayed resistance for C. glabrata. 3.2. Antifungal susceptibilities of Candida biofilms In the XTT reduction assay, SMICs of the key antifungal agents tested against biofilms formed by three major non-albicans Candida isolates are shown in Table 1. A clear difference in antifungal susceptibilities was seen against C. tropicalis biofilm. LAB showed high antifungal efficacy against biofilm of C. tropicalis, with SMIC50/80 values of 2e64. For MFG and FLC, the respective SMIC50/80 values were >256 mg/ml against C. tropicalis biofilm. All antifungal agents were characterized by high SMIC50 against biofilms of C. parapsilosis (8e256 mg/ml), with the lowest SMIC50 characterizing LAB (Table 1; 8 mg/ml). LAB and MFG were activity than FLC for C. glabrata biofilm, MFG had the lowest value among them (1 mg/ml). In contrast, FLC was ineffective, with no notable sessile activity against any strains tested (SMIC50/80 >256 mg/ml). Representative curves for inhibition of growth of C. tropicalis, C. parapsilosis and C. glabrata biofilms in the presence of different concentrations of LAB, MFG and FLC are shown in Figs. 1e3. LAB dose-dependently reduced the metabolic activity against C. tropicalis biofilm (Fig. 1). In comparison, C. tropicalis biofilm were less susceptible to MFG at concentrations above SMIC (8e64 mg/ml) than at 0.5e4 mg/ml. This observation is referred to as a paradoxical growth effect. This was not observed for LAB with C. tropicalis. FLC had little effect on the C. tropicalis biofilm (MIC50 >256 mg/ml). LAB also indicated dose-dependent reduction of metabolic activity against C. parapsilosis biofilm (Fig. 2). In comparison, MFG and FLC had little effect on C. parapsilosis biofilm. However, growth inhibition at low FLC concentrations, paradoxical growth above MIC, and inhibition at high FLC concentrations occurred for C. parapsilosis biofilm. The dose-dependent effects of LAB also showed in the C. glabrata biofilm (Fig. 3). MFG had a rapid effect at all concentrations. In comparison, FLC was ineffective against the C. glabrata biofilm. Overall, these results suggest that LAB was more effective and was not observed the paradoxical growth effect against all biofilms.
Table 1 Antifungal susceptibility testing of non-albicans Candida isolates under planktonic (MIC) and biofilm (SMIC) growing conditions. MIC(mg/mL), minimal inhibitory concentration; SMIC(mg/mL), sessile minimal inhibitory concentration (SMIC50/SMIC80). Antifungal agents
LAB MCF FLC
C. tropicalis AMTC001414
C. parapsilosis AMTC45867
Planktonic cell
Biofilm
Planktonic cell
Biofilm
C. glabrata AMTC5382 Planktonic cell
Biofilm
MIC
SMIC50
SMIC80
MIC
SMIC50
SMIC80
MIC
SMIC50
SMIC80
16 1 4
2 >256 >256
64 >256 >256
8 32 4
8 16 >256
256 64 >256
8 1 256
4 1 >256
128 1 >256
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
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Fig. 1. Activities of different concentrations of LAB, MFG and FLC against C. tropicalis biofilms. Graphs shows the XTT activity of C. tropicalis normalized to control (untreated), which was taken as 100%. Each result is representative of at least two experiments.
4. Discussion Using a biofilm model system, we measured SMIC50 values for isolates from three clinically relevant and morphologically different
Candida species: C. tropicalis, C. parapsilosis and C. glabrata [12]. Measurements based on XTT metabolic activity can be used to monitor biofilm formation indirectly [9,10]. Most studies have focused on C. albicans biofilm [13e17]; however, the aim of this
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
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Fig. 2. Activities of different concentrations of LAB, MFG and FLC against C. parapsilosis biofilms. Graphs shows the XTT activity of C. parapsilosis normalized to control (untreated), which was taken as 100%. Each result is representative of at least two experiments.
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
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Fig. 3. Activities of different concentrations of LAB, MFG and FLC against C. glabrata biofilms. Graphs shows the XTT activity of C. glabrata normalized to control (untreated), which was taken as 100%. Each result is representative of at least two experiments.
study was to investigate and compare the susceptibility of three major species of non-albicans Candida biofilms to key antifungal drugs. LAB has dose-dependent activities against all non-albicans Candida species and does not exhibit a paradoxical growth effect
with any biofilms, while the greater mortality associated with C. tropicalis [4] suggests that the biofilm formed by C. tropicalis is more resistant to antifungal agents. Thus, it is particularly important to investigate the susceptibility of biofilms to antifungal agents.
Please cite this article in press as: Kawai A, et al., In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.05.007
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Our results showed that C. tropicalis biofilm was more susceptible to LAB than to MFG and FLC. This suggests that LAB has an extended spectrum of activity against various Candida species and exhibits fungicidal activity, perhaps because it can relatively easily penetrate the biofilm and reach the inner candida membrane. These findings suggest that high dose LAB may have a good outcome, but this agent may also exhibit dose-dependent nephrotoxicity in clinical practice. In contrast, the MIC of LAB for planktonic cells of C. tropicalis was relatively higher than that found by Bizerra et al. [18]. This suggests that addition of FBS to a eutrophic planktonic cell medium increases fungal proliferation. Paradoxical growth effects occurred for MFG on C. tropicalis and FLC on C. parapsilosis, but LAB had no paradoxical effects and good activity against all Candida biofilms. MFG reduced the metabolic activity of C. tropicalis biofilm at low concentrations, but paradoxical growth above MIC prevented MFG from completely eliminating the activity, even at the highest concentration tested. Similar paradoxical effects on growth of C. tropicalis biofilm have been found for other echinocandins [19,20]. There are several proposed explanations for these effects at high concentrations of echinocandins [20,21]. Caspofungin, a member of the echinocandin family used worldwide, inhibits synthesis of 1,3-beta-glucan, a fundamental component of fungal cell walls, by inhibition of 1,3-beta-glucan synthase [22e24]. The paradoxical growth effect has been investigated by measuring the levels of 1,3-beta-glucan, 1,6-beta-glucan and chitin in a C. albicans strain after exposure to high caspofungin [25,26]. In clinical practice in Japan, high-dose MFG may be used for invasive candidiasis, but there is no evidence for dose-dependent antifungal activity of MFG. Factors such as protein binding in human serum might also influence the MIC for MFG, since serum shifts the activity of MFG in in vitro susceptibility tests on Candida spp [27,28]. Further studies in vivo and in vitro are thus needed to examine the effects of high dose MFG and the impact of human serum on MFG activity. FLC has a limited spectrum against non-albicans Candida species, but may be more effective than echinocandins against C. parapsilosis [29]. The paradoxical growth effect of FLC on C. parapsilosis may be due to biofilm formation by C. parapsilosis being strongly strain-dependent [30]. Biofilm formation enhances the capacity of C. parapsilosis to colonize catheters and intravascular central lines, which produces a drug-resistant phenotype [31,32]. Biofilms also show significantly enhanced resistance to some antifungal agents and altered phenotypes, making eradication difficult [30,33]. Recent clinical data have shown that patients with defined biofilm-forming bloodstream non-albicans Candida species have a shorter hospital stay and lower mortality when treated with echinocandins such as caspofungin and liposomal amphotericin B [34]. Our findings are generally consistent with these observations, but not all patients respond equally to antifungal treatment. This may be partly due to the level of biofilm formation by individual strains and/or differential biofilm responses to different antifungal classes. Interestingly, no antifungal agents completely eliminate biofilms, suggesting that adjunctive therapy is required [35]. Further studies are needed to maximize antifungal efficacy against biofilms formed by non-albicans Candida species and to improve clinical management of patients with these infections. Conflict of interest Sumitomo Dainippon Pharma is acknowledged for financial support of this study.
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