Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Imidazolium salts as antifungal agents: strong antibiofilm activity against multidrug-resistant Candida tropicalis isolates V.Z. Bergamo1,2, R.K. Donato3, D.F. Dalla Lana2, K.J.Z. Donato3, G.G. Ortega4, H.S. Schrekker3 and A.M. Fuentefria1,2 1 Institute of Basic Health Sciences, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, RS, Brazil 2 Laboratory of Applied Mycology, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, RS, Brazil 3 Laboratory of Technological Processes and Catalysis, Institute of Chemistry, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, RS, Brazil 4 Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, RS, Brazil
Significance and Impact of the Study: The imidazolium salt 1-n-hexadecyl-3-methylimidazolium chloride (C16MImCl) strongly prevents, in concentrations as low as 0028 lg ml 1, the biofilm formation of multidrug-resistant Candida tropicalis isolates, either in solution or applied on the surface of commercial catheters. This presents an effective antimicotic candidate and alternative for invasive clinical procedure toolset asepsis.
Keywords antibiofilm, antibiotic lock therapy, antifungal agent, Candida tropicalis, candidiasis, imidazolium salt, ionic liquid. Correspondence Alexandre M. Fuentefria, Laboratory of Applied Mycology, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, Porto Alegre – RS, Brazil. E-mail: [email protected] 2014/1105: received 28 May 2014, revised 26 September 2014 and accepted 26 September 2014
Abstract The in vitro activity of the imidazolium salt C16MImCl against planktonic and biofilm cells of multidrug-resistant isolates of Candida tropicalis was evaluated, both in solution and applied on a commercial catheter surface. This was determined by inhibition and susceptibility assays of biofilm and planktonic cells. In both cases, C16MImCl prevented in vitro biofilm formation of C. tropicalis strains, including multidrug-resistant ones. Outstanding performances were observed, even at extremely low concentrations. Furthermore, this is the first report of the antifungal lock property of C16MImCl, using a tracheal catheter as the test specimen to mimic a clinical in vivo condition. As such, C16MImCl has been identified as a promising antimicotic pharmaceutical candidate for the treatment of candidiasis infections.
doi:10.1111/lam.12338
Introduction Biofilms are highly structured microbial communities, constituted by sessile cells incorporated in an extracellular polymer matrix (composed of, e.g. DNA, polysaccharides, proteins and lipids). In comparison with planktonic cells, sessile cells generally present higher resistance, which has a considerable impact on the treatment of biofilm related fungal infections (Donlan and Costerton 2002). Previous works showed that Candida biofilm cells are 30–2000 times more resistant than planktonic cells against several antifungal agents, including amphotericin B and the azolics like fluconazole (Hawser and Douglas 1995). 66
The antifungal drug resistance is a key characteristic of Candida biofilms, which is associated with increased cell density and decreased metabolic activity. This causes a slow diffusion of the antimicrobial agents into the extracellular matrix water channels, hindering their access to the target fungal cells inside the biofilm biomass (Gordon et al. 1988; Kumamoto 2002; Nett et al. 2007). The Candida species are important infection causing pathogens, mainly in immunosuppressed and seriously ill patients, making them elements of high clinical relevance (Edmond et al. 1999). Although Candida albicans is the main etiologic agent in candidiasis, C. tropicalis infections in immunosuppressed patients have increased intensely on a
Letters in Applied Microbiology 60, 66--71 © 2014 The Society for Applied Microbiology
V.Z. Bergamo et al.
global scale, thus characterizing this organism as emerging pathogenic yeast (Silva et al. 2012). Various materials used in clinical practices, for example catheters, implants and prostheses, usually represent a risk of developing Candida nosocomial infections (Crump and Collignon 2000). Candida tropicalis is an important biofilm producer among the nonalbicans Candida species, especially in tropical and subtropical areas (Chai et al. 2010; Nucci et al. 2010), which comport more than half of the world’s human population (Mellinger et al. 2000; Bizerra et al. 2008). A frequently used strategy to avoid biofilm formation at the surface of clinical instrumentation is the antibiotic lock therapy (ALT). It was initially idealized to treat catheter-associated bacterial infections, avoiding the need of catheter exchange. Until now, the efficacy of ALT against Candida infections has been only vaguely explored and the high drug concentration involved in the standard procedures makes it a chemically invasive strategy (Walraven and Lee 2013). Furthermore, a broad range of commercial antifungal agents, frequently used as therapeutics, present inefficacy and side effects, explaining the urge for prospecting new effective antifungal substances with low toxicity. Recently, our group identified imidazolium salts as excellent candidates for this end use (Schrekker et al. 2013). Differently from their neutral analogue drugs, for example azolic antifungals, the imidazolium salt’s ionicity provides properties that are unusual and highly interesting for pharmaceutical formulations. Furthermore, the potential efficacy of imidazolium salts against some species of bacteria and fungi, for example C. albicans, Aspergillus niger and Aspergillus flavus, has been reported (Cornellas et al. 2011). Considering these and our previous screening results, in combination with the low toxicity to humans from some of these imidazolium salts at MIC (no genotoxic effect to cell membrane structure or DNA damage in human leukocyte cells) (Schrekker et al. 2013), we decided to explore in further detail the potential activity of C16MImCl against planktonic and biofilm cells of multiresistant and sensitive isolates of C. tropicalis, either in solution or applied on the surface of discs of commercial catheters. This approach could provide a cheaper, more sustainable and less chemically aggressive lock therapy alternative for invasive clinical instrumentation, as well as a treatment for persistent biofilm originated candidiasis. Results and discussion The biofilm forming capacity of different C. tropicalis strains was confirmed in our study with the following procedures: (i) the crystal violet screening assay using microplates and (ii) the counts of cells grown on a catheter
Imidazolium salt against biofilm
surface. In the crystal violet assay, all the strains showed being biofilm formers. The individual absorbance (A) values were 0082, 0281, 0086, 0101, 0099 and 0150, respectively, for 17A, 57A, 72A, 72P, 94P and 102A. Strain 57A was identified as the strongest and strain 72A as the weakest biofilm former. The ability of forming biofilms was also confirmed for all isolates of C. tropicalis by the assay on the catheter surface. This was verified by determining log (CFU cm 2), which ranged from 50 to 61. Among the C. tropicalis isolates, 72A was the one with the highest value, corresponding to the strongest biofilm formation, followed in decreasing order by the isolates 102A, 72P, 94P, 17A and 57A. The two applied assays presented an almost perfectly inverted correlation between the strongest and the weakest biofilm producers, suggesting that fungi with slower forming biofilms have a higher adhesion capacity to the catheter surface. An effective method for evaluating the drug efficacy in such treatment is the in vitro susceptibility assay of planktonic and biofilm cells (Ramage et al. 2001). In the susceptibility test, the PMIC of C16MImCl was 0014 lg ml 1 for all the strains tested. On the other hand, fluconazole presented higher and very nonconstant PMIC values (0125–128 lg ml 1). This improved profile of C16MImCl was also observed with biofilm cells. It also presented low BMIC values, which depended on the strain tested (0028–0225 lg ml 1). As determined for the planktonic cells, the antibiofilm activity of fluconazole is also more strain dependent (BMIC = 40– 128 lg ml 1). Importantly, the best result obtained for the commercial antifungal is 100 times less efficient than the worst result for C16MImCl (Table 1). The MIC values observed for both biofilm and planktonic cells using this technique, applying C16MImCl, were extremely low when compared to the MIC found for the commercial antifungal fluconazole (Table 1). These results are also far more impacting than the ones reported for other antifungal agents, for example Peman et al. (2008) showed that MIC for biofilms regarding anidulafungin, caspofungin and micafungin are 112, 897 and 16 lg ml 1, respectively. C16MImCl shows MIC values between 0014 and 0225 lg ml 1, and, as a consequence, 100 times lower concentrations could already exert an exceptional antibiofilm effect against C. tropicalis. In contrast, the majority of the isolates in this study were resistant to fluconazole, with the exception of strain 17A that was sensitive when subjected to the biofilm formation test. Considering the inhibition of biofilm formation, fluconazole (80 lg ml 1) showed a variation in the log (CFU cm 2) values from 47 to 54, where 102A presented the lowest and 72A the highest value. In comparison, the antibiofilm activity presented by C16MImCl was extremely
Letters in Applied Microbiology 60, 66--71 © 2014 The Society for Applied Microbiology
67
Imidazolium salt against biofilm
V.Z. Bergamo et al.
Table 1 PMIC, BMIC and antibiofilm activity against planktonic and biofilm cells of multidrug-resistant isolates of Candida tropicalis C16MImCl
Planktonic
Biofilm
Fluconazole
Strains
MIC (lg ml 1)
Antibiofilm activity (log CFU cm 2)
MIC (lg ml 1)
Antibiofilm activity (log CFU cm 2)
17A 57A 72A 72P 94P 102A 17A 57A 72A 72P 94P 102A
0014 0014 0014 0014 0014 0014 0028 0056 0056 0056 0225 0056
n.a. n.a. n.a. n.a. n.a. n.a. 44 44 47 0 44 47
0125 0125 128 128 64 0125 4 64 8 128 32 128
n.a. n.a. n.a. n.a. n.a. n.a. 51 51 54 50 51 47
n.a., not applicable.
strong as the values varied from 0 (total inhibition, for strain 72P) to 47 (for strain 72A). Several azole compounds, for example fluconazole, are fungistatic drugs that inhibit the ergosterol biosynthesis and consequently disturb the fungal plasmatic membrane integrity. They target 14-a-lanosterol demethylase (Erg11 or Cyp51), a cytochrome P-450 enzyme that catalyses a key step in the ergosterol biosynthetic pathway (Nucci and Colombo 2007). Apparently, C16MImCl also has an action profile over the fungal plasmatic membrane, interfering in the ergosterol ability of regenerating the membrane by decreasing the amount of available sterol in the fungal cell. This leads to a process of depletion of intracellular constituents that are essential for the fungal growth and survival. The amphipathicity of the fungal target perfectly matches the physicochemical properties of the imidazolium salt, which explains its affinity with the fungal components (Schrekker et al. 2013). The use of chelating and coordinating agents such as ethylenediaminetetraacetic acid (EDTA) is reported as effective to inhibit fungal biofilm formation, but only when applied to catheter lock therapy using extremely high concentrations (40 000 lg ml 1) (Percival et al. 2005). This evidences also the structural effectiveness of C16MImCl. Its conjugated surfactant, chelating and antifungal characteristics appear to allow an easier penetration and mobility into the hydrogel from C. tropicalis biofilms, traversing the rigid structure formed by the layers of sessile cells and thereby exerting its antifungal effect at lower doses. As fluconazole is an antifungal agent with neutral character, it does not adapt to the environment with variable hydrophilic–hydrophobic conditions, becoming trapped into the biofilm grid, thus not exerting an effective antifungal activity (Fig. 1) (Nweze et al. 2012). This could explain the strong antibiofilm activity of C16MImCl (0–47) in 68
comparison with fluconazole (47–54) (Table 1), even in drastically lower concentrations. Additionally, this class of salt presents low phytotoxicity (Biczak et al. 2014), as well as low toxicity to human cells (Schrekker et al. 2013). This fact coupled with their high efficacy at low concentrations makes them a possible choice for a stronger and safer antibiofilm action, especially in recurrent cases of biofilm associated with multidrug resistance. Importantly, despite the structural similarity of these salts with traditional azole antifungals, the fact that they carry a positively charged imidazolium ring appears to be a key factor for the enhanced antifungal activity. Furthermore, the long aliphatic chain attached to the strong electron withdrawing positive imidazolium ring seems essential for the proper amphiphilicity of this compound, providing a surfactant property that facilitates the antibiofilm action against pathogens that do not fully respond to conventional treatments. The catheter antifungal lock therapy is a useful technique for reducing the need of catheter exchange/relocation when one deals with long-term hospitalized patients (Walraven and Lee 2013). The advanced physicochemical characteristics together with the pronounced antifungal and antibiofilm properties of C16MImCl suggest a possibility for a milder alternative to the catheter lock therapy. This would avoid the drawbacks of a high drug concentration on the catheter surface, reducing both the exposition of patients to chemicals and treatment costs. Materials and methods Microbial strains Six clinical isolates of C. tropicalis (72A, 72P, 94P, 102A, 17A, 57A) were used in this study. The isolates 72A, 72P
Letters in Applied Microbiology 60, 66--71 © 2014 The Society for Applied Microbiology
V.Z. Bergamo et al.
Imidazolium salt against biofilm
(a) B
C
I
A T
N
O
H
OH
F
E T
I
E
N
N
F N
F
N
L
N
R
M
(b)
S U R F A C E
Figure 1 Schematic representation of fluconazole (a) and C16MImCl (b) antibiofilm effect on a catheter surface.
and 94P are resistant to fluconazole, amphotericin B, voriconazole and anidulafungin. All microbial strains are deposited in the Mycology Collection of the Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, Brazil. Synthesis of antifungal agents The imidazolium salt 1-n-hexadecyl-3-methylimidazolium chloride (C16MImCl) was synthesized as described in the literature, and the spectral data were in agreement with those reported previously (Gordon et al. 2008). Fluconazole was prepared in agreement with the CLSI 2012; (M27-S4) guidelines.
transferred to microtitre plates and supplemented with 180 ll of tryptone soy broth (TSB). After 24 h of incubation at 35°C, the culture was carefully removed and to each well was added sterile distilled water to wash and remove weakly adherent cells. Methanol (150 ll) was added after 20 min and removed. The microtitre plates were stained for 15 min with 150 ll of 05% crystal violet at room temperature, followed by removing the liquid and washing the plates with running water. Finally, the biofilm-adsorbed dye was re-suspended in 150 ll of ethanol, and after 30 min the absorption was measured. The following classification has been applied: strong biofilm producer >0280 A; average biofilm producer 0. 170– 0279 A; weak biofilm producer 0. 070–0170 A; and not a biofilm producer
ORIGINAL ARTICLE
Imidazolium salts as antifungal agents: strong antibiofilm activity against multidrug-resistant Candida tropicalis isolates V.Z. Bergamo1,2, R.K. Donato3, D.F. Dalla Lana2, K.J.Z. Donato3, G.G. Ortega4, H.S. Schrekker3 and A.M. Fuentefria1,2 1 Institute of Basic Health Sciences, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, RS, Brazil 2 Laboratory of Applied Mycology, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, RS, Brazil 3 Laboratory of Technological Processes and Catalysis, Institute of Chemistry, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, RS, Brazil 4 Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, RS, Brazil
Significance and Impact of the Study: The imidazolium salt 1-n-hexadecyl-3-methylimidazolium chloride (C16MImCl) strongly prevents, in concentrations as low as 0028 lg ml 1, the biofilm formation of multidrug-resistant Candida tropicalis isolates, either in solution or applied on the surface of commercial catheters. This presents an effective antimicotic candidate and alternative for invasive clinical procedure toolset asepsis.
Keywords antibiofilm, antibiotic lock therapy, antifungal agent, Candida tropicalis, candidiasis, imidazolium salt, ionic liquid. Correspondence Alexandre M. Fuentefria, Laboratory of Applied Mycology, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, Porto Alegre – RS, Brazil. E-mail: [email protected] 2014/1105: received 28 May 2014, revised 26 September 2014 and accepted 26 September 2014
Abstract The in vitro activity of the imidazolium salt C16MImCl against planktonic and biofilm cells of multidrug-resistant isolates of Candida tropicalis was evaluated, both in solution and applied on a commercial catheter surface. This was determined by inhibition and susceptibility assays of biofilm and planktonic cells. In both cases, C16MImCl prevented in vitro biofilm formation of C. tropicalis strains, including multidrug-resistant ones. Outstanding performances were observed, even at extremely low concentrations. Furthermore, this is the first report of the antifungal lock property of C16MImCl, using a tracheal catheter as the test specimen to mimic a clinical in vivo condition. As such, C16MImCl has been identified as a promising antimicotic pharmaceutical candidate for the treatment of candidiasis infections.
doi:10.1111/lam.12338
Introduction Biofilms are highly structured microbial communities, constituted by sessile cells incorporated in an extracellular polymer matrix (composed of, e.g. DNA, polysaccharides, proteins and lipids). In comparison with planktonic cells, sessile cells generally present higher resistance, which has a considerable impact on the treatment of biofilm related fungal infections (Donlan and Costerton 2002). Previous works showed that Candida biofilm cells are 30–2000 times more resistant than planktonic cells against several antifungal agents, including amphotericin B and the azolics like fluconazole (Hawser and Douglas 1995). 66
The antifungal drug resistance is a key characteristic of Candida biofilms, which is associated with increased cell density and decreased metabolic activity. This causes a slow diffusion of the antimicrobial agents into the extracellular matrix water channels, hindering their access to the target fungal cells inside the biofilm biomass (Gordon et al. 1988; Kumamoto 2002; Nett et al. 2007). The Candida species are important infection causing pathogens, mainly in immunosuppressed and seriously ill patients, making them elements of high clinical relevance (Edmond et al. 1999). Although Candida albicans is the main etiologic agent in candidiasis, C. tropicalis infections in immunosuppressed patients have increased intensely on a
Letters in Applied Microbiology 60, 66--71 © 2014 The Society for Applied Microbiology
V.Z. Bergamo et al.
global scale, thus characterizing this organism as emerging pathogenic yeast (Silva et al. 2012). Various materials used in clinical practices, for example catheters, implants and prostheses, usually represent a risk of developing Candida nosocomial infections (Crump and Collignon 2000). Candida tropicalis is an important biofilm producer among the nonalbicans Candida species, especially in tropical and subtropical areas (Chai et al. 2010; Nucci et al. 2010), which comport more than half of the world’s human population (Mellinger et al. 2000; Bizerra et al. 2008). A frequently used strategy to avoid biofilm formation at the surface of clinical instrumentation is the antibiotic lock therapy (ALT). It was initially idealized to treat catheter-associated bacterial infections, avoiding the need of catheter exchange. Until now, the efficacy of ALT against Candida infections has been only vaguely explored and the high drug concentration involved in the standard procedures makes it a chemically invasive strategy (Walraven and Lee 2013). Furthermore, a broad range of commercial antifungal agents, frequently used as therapeutics, present inefficacy and side effects, explaining the urge for prospecting new effective antifungal substances with low toxicity. Recently, our group identified imidazolium salts as excellent candidates for this end use (Schrekker et al. 2013). Differently from their neutral analogue drugs, for example azolic antifungals, the imidazolium salt’s ionicity provides properties that are unusual and highly interesting for pharmaceutical formulations. Furthermore, the potential efficacy of imidazolium salts against some species of bacteria and fungi, for example C. albicans, Aspergillus niger and Aspergillus flavus, has been reported (Cornellas et al. 2011). Considering these and our previous screening results, in combination with the low toxicity to humans from some of these imidazolium salts at MIC (no genotoxic effect to cell membrane structure or DNA damage in human leukocyte cells) (Schrekker et al. 2013), we decided to explore in further detail the potential activity of C16MImCl against planktonic and biofilm cells of multiresistant and sensitive isolates of C. tropicalis, either in solution or applied on the surface of discs of commercial catheters. This approach could provide a cheaper, more sustainable and less chemically aggressive lock therapy alternative for invasive clinical instrumentation, as well as a treatment for persistent biofilm originated candidiasis. Results and discussion The biofilm forming capacity of different C. tropicalis strains was confirmed in our study with the following procedures: (i) the crystal violet screening assay using microplates and (ii) the counts of cells grown on a catheter
Imidazolium salt against biofilm
surface. In the crystal violet assay, all the strains showed being biofilm formers. The individual absorbance (A) values were 0082, 0281, 0086, 0101, 0099 and 0150, respectively, for 17A, 57A, 72A, 72P, 94P and 102A. Strain 57A was identified as the strongest and strain 72A as the weakest biofilm former. The ability of forming biofilms was also confirmed for all isolates of C. tropicalis by the assay on the catheter surface. This was verified by determining log (CFU cm 2), which ranged from 50 to 61. Among the C. tropicalis isolates, 72A was the one with the highest value, corresponding to the strongest biofilm formation, followed in decreasing order by the isolates 102A, 72P, 94P, 17A and 57A. The two applied assays presented an almost perfectly inverted correlation between the strongest and the weakest biofilm producers, suggesting that fungi with slower forming biofilms have a higher adhesion capacity to the catheter surface. An effective method for evaluating the drug efficacy in such treatment is the in vitro susceptibility assay of planktonic and biofilm cells (Ramage et al. 2001). In the susceptibility test, the PMIC of C16MImCl was 0014 lg ml 1 for all the strains tested. On the other hand, fluconazole presented higher and very nonconstant PMIC values (0125–128 lg ml 1). This improved profile of C16MImCl was also observed with biofilm cells. It also presented low BMIC values, which depended on the strain tested (0028–0225 lg ml 1). As determined for the planktonic cells, the antibiofilm activity of fluconazole is also more strain dependent (BMIC = 40– 128 lg ml 1). Importantly, the best result obtained for the commercial antifungal is 100 times less efficient than the worst result for C16MImCl (Table 1). The MIC values observed for both biofilm and planktonic cells using this technique, applying C16MImCl, were extremely low when compared to the MIC found for the commercial antifungal fluconazole (Table 1). These results are also far more impacting than the ones reported for other antifungal agents, for example Peman et al. (2008) showed that MIC for biofilms regarding anidulafungin, caspofungin and micafungin are 112, 897 and 16 lg ml 1, respectively. C16MImCl shows MIC values between 0014 and 0225 lg ml 1, and, as a consequence, 100 times lower concentrations could already exert an exceptional antibiofilm effect against C. tropicalis. In contrast, the majority of the isolates in this study were resistant to fluconazole, with the exception of strain 17A that was sensitive when subjected to the biofilm formation test. Considering the inhibition of biofilm formation, fluconazole (80 lg ml 1) showed a variation in the log (CFU cm 2) values from 47 to 54, where 102A presented the lowest and 72A the highest value. In comparison, the antibiofilm activity presented by C16MImCl was extremely
Letters in Applied Microbiology 60, 66--71 © 2014 The Society for Applied Microbiology
67
Imidazolium salt against biofilm
V.Z. Bergamo et al.
Table 1 PMIC, BMIC and antibiofilm activity against planktonic and biofilm cells of multidrug-resistant isolates of Candida tropicalis C16MImCl
Planktonic
Biofilm
Fluconazole
Strains
MIC (lg ml 1)
Antibiofilm activity (log CFU cm 2)
MIC (lg ml 1)
Antibiofilm activity (log CFU cm 2)
17A 57A 72A 72P 94P 102A 17A 57A 72A 72P 94P 102A
0014 0014 0014 0014 0014 0014 0028 0056 0056 0056 0225 0056
n.a. n.a. n.a. n.a. n.a. n.a. 44 44 47 0 44 47
0125 0125 128 128 64 0125 4 64 8 128 32 128
n.a. n.a. n.a. n.a. n.a. n.a. 51 51 54 50 51 47
n.a., not applicable.
strong as the values varied from 0 (total inhibition, for strain 72P) to 47 (for strain 72A). Several azole compounds, for example fluconazole, are fungistatic drugs that inhibit the ergosterol biosynthesis and consequently disturb the fungal plasmatic membrane integrity. They target 14-a-lanosterol demethylase (Erg11 or Cyp51), a cytochrome P-450 enzyme that catalyses a key step in the ergosterol biosynthetic pathway (Nucci and Colombo 2007). Apparently, C16MImCl also has an action profile over the fungal plasmatic membrane, interfering in the ergosterol ability of regenerating the membrane by decreasing the amount of available sterol in the fungal cell. This leads to a process of depletion of intracellular constituents that are essential for the fungal growth and survival. The amphipathicity of the fungal target perfectly matches the physicochemical properties of the imidazolium salt, which explains its affinity with the fungal components (Schrekker et al. 2013). The use of chelating and coordinating agents such as ethylenediaminetetraacetic acid (EDTA) is reported as effective to inhibit fungal biofilm formation, but only when applied to catheter lock therapy using extremely high concentrations (40 000 lg ml 1) (Percival et al. 2005). This evidences also the structural effectiveness of C16MImCl. Its conjugated surfactant, chelating and antifungal characteristics appear to allow an easier penetration and mobility into the hydrogel from C. tropicalis biofilms, traversing the rigid structure formed by the layers of sessile cells and thereby exerting its antifungal effect at lower doses. As fluconazole is an antifungal agent with neutral character, it does not adapt to the environment with variable hydrophilic–hydrophobic conditions, becoming trapped into the biofilm grid, thus not exerting an effective antifungal activity (Fig. 1) (Nweze et al. 2012). This could explain the strong antibiofilm activity of C16MImCl (0–47) in 68
comparison with fluconazole (47–54) (Table 1), even in drastically lower concentrations. Additionally, this class of salt presents low phytotoxicity (Biczak et al. 2014), as well as low toxicity to human cells (Schrekker et al. 2013). This fact coupled with their high efficacy at low concentrations makes them a possible choice for a stronger and safer antibiofilm action, especially in recurrent cases of biofilm associated with multidrug resistance. Importantly, despite the structural similarity of these salts with traditional azole antifungals, the fact that they carry a positively charged imidazolium ring appears to be a key factor for the enhanced antifungal activity. Furthermore, the long aliphatic chain attached to the strong electron withdrawing positive imidazolium ring seems essential for the proper amphiphilicity of this compound, providing a surfactant property that facilitates the antibiofilm action against pathogens that do not fully respond to conventional treatments. The catheter antifungal lock therapy is a useful technique for reducing the need of catheter exchange/relocation when one deals with long-term hospitalized patients (Walraven and Lee 2013). The advanced physicochemical characteristics together with the pronounced antifungal and antibiofilm properties of C16MImCl suggest a possibility for a milder alternative to the catheter lock therapy. This would avoid the drawbacks of a high drug concentration on the catheter surface, reducing both the exposition of patients to chemicals and treatment costs. Materials and methods Microbial strains Six clinical isolates of C. tropicalis (72A, 72P, 94P, 102A, 17A, 57A) were used in this study. The isolates 72A, 72P
Letters in Applied Microbiology 60, 66--71 © 2014 The Society for Applied Microbiology
V.Z. Bergamo et al.
Imidazolium salt against biofilm
(a) B
C
I
A T
N
O
H
OH
F
E T
I
E
N
N
F N
F
N
L
N
R
M
(b)
S U R F A C E
Figure 1 Schematic representation of fluconazole (a) and C16MImCl (b) antibiofilm effect on a catheter surface.
and 94P are resistant to fluconazole, amphotericin B, voriconazole and anidulafungin. All microbial strains are deposited in the Mycology Collection of the Universidade Federal do Rio Grande do Sul-UFRGS, Porto Alegre, Brazil. Synthesis of antifungal agents The imidazolium salt 1-n-hexadecyl-3-methylimidazolium chloride (C16MImCl) was synthesized as described in the literature, and the spectral data were in agreement with those reported previously (Gordon et al. 2008). Fluconazole was prepared in agreement with the CLSI 2012; (M27-S4) guidelines.
transferred to microtitre plates and supplemented with 180 ll of tryptone soy broth (TSB). After 24 h of incubation at 35°C, the culture was carefully removed and to each well was added sterile distilled water to wash and remove weakly adherent cells. Methanol (150 ll) was added after 20 min and removed. The microtitre plates were stained for 15 min with 150 ll of 05% crystal violet at room temperature, followed by removing the liquid and washing the plates with running water. Finally, the biofilm-adsorbed dye was re-suspended in 150 ll of ethanol, and after 30 min the absorption was measured. The following classification has been applied: strong biofilm producer >0280 A; average biofilm producer 0. 170– 0279 A; weak biofilm producer 0. 070–0170 A; and not a biofilm producer
