Mycopathologia (2014) 177:19–27 DOI 10.1007/s11046-014-9727-7
In Vitro Effect of Amphotericin B on Candida albicans, Candida glabrata and Candida parapsilosis Biofilm Formation Małgorzata Pra_zyn´ska • Eugenia Gospodarek
Received: 2 August 2013 / Accepted: 6 January 2014 / Published online: 17 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Candida spp. biofilm is considered highly resistant to conventional antifungals. The aim of this study was to investigate the in vitro effect of amphotericin B on Candida spp. biofilms at different stages of maturation. We investigated the activity of amphotericin B against 78 clinical isolates of Candida spp., representing three species, growing as planktonic and sessile cells, by a widely accepted broth microdilution method. The in vitro effect on sessile cell viability was evaluated by MTT reduction assay. All examined strains were susceptible to amphotericin B when grown as free-living cells. At the early stages of biofilm maturation 96.7–100.0 % strains, depending on species, displayed amphotericin B sessile minimal inhibitory concentration (SMIC) B1 lg/mL. Mature Candida spp. biofilm of 32.1–90.0 % strains displayed amphotericin B SMIC B1 lg/mL. Based on these results, amphotericin B displays species- and straindepending activity against Candida spp. biofilms.
Electronic supplementary material The online version of this article (doi:10.1007/s11046-014-9727-7) contains supplementary material, which is available to authorized users. M. Pra_zyn´ska (&) E. Gospodarek Department of Microbiology, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun´, 9 Maria Skłodowska-Curie Street, 85-094 Bydgoszcz, Poland e-mail: [email protected]
Keywords Antifungals Candidosis Polyenes Sessile cells
Introduction A biofilm is defined as a spatially organized hydrated community of microorganisms surrounded by a selfproduced extracellular polymeric matrix and adherent to a biotic or abiotic surface. This is considered the most prevalent growth form of microorganisms [1]. Fungi Candida spp., especially C. albicans, C. parapsilosis, C. glabrata, and C. tropicalis, possess a well-documented ability to form biofilm on biotic as well as abiotic surfaces [2–6]. Due to the common use of medical devices in modern medicine, Candida infections are often associated with biofilm formation. Candida spp. are ranked as the fourth leading cause of vascular catheter-related infections and the third leading cause of urinary catheter-related infections [7, 8]. Fungal cells existing in biofilm structure differ from their free-living counterparts. As far as clinical aspects are concerned, an important feature of fungal biofilm is its dramatically decreased susceptibility to antifungal drugs [9–18]. This may lead to chronic, refractory infections, with severe, even systemic course. In the device-related infections, device removal is often necessary for a cure. However, device removal can be difficult or impossible to perform or biofilm can be formed on the surface of a patient’s tissue. This is the reason why researching on
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the susceptibility of Candida spp. biofilms remains an actual issue. The resistance of Candida spp. to antifungals is puzzling, since free-living cells of the same strains can be susceptible to a range of antifungal drugs. The mechanism of Candida spp. biofilm resistance is still unclear. Based on existing literature, Candida spp. biofilm is consistently thought to be highly resistant to azoles [9–12, 14–18], and susceptible to echinocandins [13, 15, 18, 19]. In available literature, there are discrepancies in the results of the effects of amphotericin B on Candida spp. biofilms [12–18]. The resistant to amphotericin B Candida spp. biofilms [12, 14, 15, 17] as well as susceptible ones [13, 15, 16, 18] was reported. Furthermore, the majority of these reports describe the susceptibility of C. albicans biofilms only. There is relatively little about other Candida species. Amphotericin B is a polyene antifungal agent. It binds to ergosterol in fungal protoplasmatic membrane, which leads to the formation of transmembraneal pores and the leakage of cellular components. Unfortunately, its affinity to sterols is not selective to ergosterol and the drug also binds to cholesterol in mammalian cells. Therefore, amphotericin B is nephrotoxic and causes a wide range of adverse effects. Due to the introduction of newer, less toxic antifungals, amphotericin B is reserved for treatment of severe, life-threatening, invasive fungal infections. Its advantages comprise a wide spectrum of antifungal activity, including yeasts and molds, and fungicidal effects on susceptible fungi at clinically relevant concentrations [20]. Here, we report on the effect of amphotericin B on Candida spp. biofilms at different stages of maturation. Although C. albicans is still the predominant etiologic agent of candidiasis, other Candida species have emerged as significant pathogens [6, 14]. For the study, we employed C. albicans, C. parapsilosis, and C. glabrata, which are three the most commonly isolated fungal pathogens in our institution—Dr. Antoni Jurasz University Hospital No. 1 in Bydgoszcz, Poland.
Materials and Methods Strains A total of 78 Candida spp. strains were studied: 30 of C. albicans, 28 of C. parapsilosis, and 20 of C.
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glabrata. All the strains were isolated in The Department of Microbiology of Dr. A. Jurasz University Hospital No. 1 in Bydgoszcz, Nicolaus Copernicus University in Torun, Ludwik Rydygier Collegium Medicum in Bydgoszcz, in 2007–2010. One strain per patient was studied. Isolates were isolated from urine (n = 25), blood cultures (n = 10), bronchoalveolar lavage fluid (n = 7), middle ear discharge (n = 8), pharyngeal swab (n = 7), wound swab (n = 4), tip of vascular catheter (n = 3), peritoneal fluid (n = 5), gastrostomy swab (n = 2), and one strain per piece of drain, piece of surgical mesh, respiratory tract swab, insertion-site skin swab, peritoneal cavity swab, tracheotomy swab, and groin swab. The detailed data on the origin of the examined strains are given in Online Resource 1. C. albicans GDH 2346 served as a positive control. Strains were stored at -70 °C. Before conducting researches, the species identification was confirmed by means of germ tube tests [21] and ID 32 C tests carried out according to the manufacturer’s protocol (bioMe´rieux) [22]. Antifungal Susceptibility Testing Amphotericin B concentrations ranging from 0.03 to 128 lg/mL were tested. Planktonic minimal inhibitory concentrations (MICs) were determined by CLSI broth microdilution method [23, 24]. Sessile MICs (SMICs) were determined using a modification of methods described by Ramage and Lo´pez-Ribot [25] and Krom et al. [26]. Briefly, the strains were recovered from frozen storage. A loopful of the stock culture was streaked on Sabouraud dextrose agar (SDA, Becton–Dickinson) and incubated at 37 °C for 48–72 h. A single colony from the obtained culture was inoculated on SDA and incubated at 37 °C for 24 h. A single colony from a 24-h culture was inoculated in a yeast nitrogen base medium (YNB, Becton–Dickinson), supplemented with 0.25 % glucose (BTL) and incubated at 37 °C for 24 h on a rotary shaker (120 rpm). These conditions promote growing in the budding yeast phase. The resultant cultures were harvested (3,0009g, 5 min) and washed three times with a sterile phosphate-buffered saline (PBS, pH 7.0 ± 0.1) (BTL) and standardized to 0.5 McFarland in YNB with a densitometer. Biofilms formed in 96-well, flat-bottomed, sterile, polystyrene plates. Each well was filled with 100 lL
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of yeast cell suspensions or sterile YNB as negative controls, each in three repetitions, and the plates were incubated at 37 °C for 2, 6, or 24 h, in order to obtain biofilms at different stages of maturation. After incubation, suspensions were discarded and wells were rinsed gently and thoroughly in sterile PBS three times. Biofilm-coated wells were filled with 100 lL serial dilutions of amphotericin B in RPMI 1640 medium. Untreated biofilm wells and biofilm-free wells were also included to serve as positive and negative controls, respectively. These wells were filled with RPMI 1640 medium without antifungal drug. Microtiter plates were incubated for an additional 24 h at 35 °C. After 24 h, the medium was discarded and wells were rinsed gently and thoroughly in sterile PBS three times.
21 Table 1 Distribution of amphotericin B MICs for Candida spp. isolates (n = 78) Species
No. of isolates with indicated AMB MIC (lg/mL) B0.03
C. albicans (n = 30) C. parapsilosis (n = 28) C. glabrata (n = 20)
%A550untreated ¼
1
MIC50
MIC90
0.25
0.5
1
2
16
12
0.5
1
8
8
11
0.5
1
1
5
14
1
1
A550amphotericinB A550negativecontrol A550untreated 100 %:
MTT Reduction Assay
Statistics
The colorimetric assay, based on an MTT [3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] reduction, was employed to determine cell viability and thereby drug susceptibility of Candida cells in biofilms [26]. Briefly, the MTT (AlfaAesar) was prepared at 0.5 g/L in sterile, prewarmed to 37 °C PBS, containing 1 % glucose and 10 lM menadione (Sigma) dissolved in acetone. The solution was filter sterilized through a 0.22-mm pore-size filter. The MTT of 100 lL was added to each well. The plates were then incubated in the dark at 37 °C for 30 min to 2 h, depending on the examined strains. The product of the MTT reduction and the insoluble purple formazan crystals were solubilized in 100 lL acid isopropanol (5 % 1 M HCl in isopropanol). Efficient solubilization was achieved by vigorously shaking the plates for 15 min. The metabolic activity of Candida sessile cells was assessed quantitatively by measuring the absorbance in a microtiter plate reader at 550 nm, after transferring the solution to new 96-well plates. SMICs were determined at C50 % inhibition comparing to untreated biofilms.
Statistical analysis was performed by two-tailed Student’s t test and differences with P values of \0.05 were considered statistically significant.
Metabolic Activity The metabolic activities of Candida spp. biofilms affected by amphotericin B at 1 lg/mL were estimated as a percentage of the metabolic activities of untreated biofilms of particular Candida spp. strains, according to the following formula:
Results Planktonic MICs for all examined Candida spp. isolates were B1 lg/mL (Table 1). The distribution of amphotericin B SMICs for examined isolates is presented in Table 2. The amphotericin B SMIC50 and SMIC90 for C. albicans isolates were both 1 lg/mL for 2- and 6-h-old biofilms, 1 and 2 lg/mL, respectively, for 24-h-old biofilms. The amphotericin B SMIC50 and SMIC90 for C. parapsilosis isolates were 0.25 and 0.5 lg/mL, respectively, for 2-h-old biofilms, 0.5 and 1 lg/mL, respectively, for 6-h-old biofilms, 8 and 32 lg/mL, respectively, for mature biofilms. The amphotericin B SMIC50 and SMIC90 for C. glabrata were 0.5 and 1 lg/mL, respectively, for 2-h-old biofilms, and both 1 lg/mL for 6- and 24-h-old biofilms. Amphotericin B MIC for C. albicans GDH 2346 was 0.25 lg/mL, whereas SMICs for 2-, 6-, and 24-hold biofilms were 0.5, 0.5, and 1.0, respectively. All C. parapsilosis and C. glabrata and 96.7 % C. albicans examined strains displayed amphotericin B SMICs B1 lg/mL at the early stages of biofilm maturation; that is 2- and 6-h-old biofilms, whereas mature biofilms kept amphotericin B SMICs B1 lg/
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Table 2 Distribution of amphotericin B SMICs for Candida spp. isolates (n = 78) Species and age of biofilm
No. of isolates with indicated AMB SMIC (lg/mL) B0.03
0.06
0.125
0.25
0.5
1a
2
4
8
16
32
6
3
C. albicans (n = 30) 2 h old
4
22
1
6 h old
1
2
4
25
1
24 h old
4
19
6
1
2 2
2
6
1
1
C. parapsilosis (n = 28) 2 h old
7
9
9
6 h old
2
1 1
6
14
5
24 h old
2
1
2
2
2
1
5
7
7
3 3
5 5
12 10
C. glabrata (n = 20) 2 h old 6 h old 24 h old a
Amphotericin B concentration achieved in human body [22]
Table 3 The metabolic activity of Candida spp. (n = 78) biofilms affected by amphotericin B at 1 lg/mL Species
The metabolic activity of Candida spp. biofilm (%A550-untreated): 2-h old Range
C. albicans (n = 30)
6-h old Mean ± SD
Range
24-h old Mean ± SD
Range
Mean ± SD
13.0–79.5
30.7 ± 17.9
17.0–66.4
39.4 ± 11.9
27.1–81.3
49.4 ± 14.0
C. parapsilosis (n = 28)
3.5–47.8
15.8 ± 12.3
13.4–53.6
27.7 ± 12.8
28.0–112.4
71.7 ± 24.7
C. glabrata (n = 20)
5.8–54.4
21.8 ± 10.6
22.1–56.4
39.4 ± 10.9
16.8–89.2
42.7 ± 18.1
mL for 90.0 % of C. glabrata, 76.9 % of C. albicans and 32.1 % of C. parapsilosis examined strains. The statistically significant differences were observed between mature biofilms of C. albicans and C. parapsilosis (P = 0.0004), along with C. glabrata and C. parapsilosis (P \ 0.0001). The more mature the biofilm, the weaker effect of amphotericin B on metabolic status of Candida spp. sessile cells was observed. The highest metabolic activities of biofilms following amphotericin B treatment at 1 lg/mL were noticed for mature C. parapsilosis biofilms, ranging 28.0–112.4 % of the metabolic activities of untreated biofilms. The mean values of the rest of the metabolic activities were lower than 50.0 % (15.8–49.4 %), which indicates the average biofilm reductions about more than a half (50.6–84.2 %) due to amphotericin B at 1 lg/mL. Detailed data on the metabolic activities of biofilms treated with amphotericin B at 1 lg/mL are presented in Table 3.
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Statistically significant differences in the mean metabolic activities of Candida spp. biofilms were observed between 2-h-old biofilms of C. albicans and C. parapsilosis (P = 0.0006), 6-h-old biofilms of C. parapsilosis and C. albicans (P = 0.001), C. parapsilosis and C. glabrata (P = 0.004), 24-h-old biofilms of C. parapsilosis and C. albicans (P = 0.0002), C. parapsilosis and C. glabrata (P = 0.0001). These investigations indicate that amphotericin B changes the metabolic status of Candida spp. biofilms and this effect depends on species, strains, and stage of biofilm maturation.
Discussion Our investigations confirmed lower susceptibility to amphotericin B of all Candida examined isolates when grown as biofilms, compared with planktonic
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forms. From 32.1 to 90.0 % of tested isolates, depending on species of examined strains, displayed SMICs B1 lg/mL for mature biofilms. The concentration of amphotericin B obtained in the human body is established at 1–2 lg/mL [27]. The highest number of isolates with amphotericin B SMIC[1 lg/mL was observed among C. parapsilosis strains (18 strains; 67.9 %), whereas the lowest number of that was noticed among C. glabrata (10.0 %). Among C. albicans strains, seven out of 30 strains (23.3 %) presented amphotericin B SMIC[1 lg/mL for mature biofilm. The differences in the biofilm architecture of C. albicans, C. parapsilosis, and C. glabrata are documented in the existing literature [6, 11, 28]. The architecture of C. glabrata biofilm, which is thin and irregular, rather compact, composed of small population of blastospores embedded within an extracellular matrix might be partially responsible for its general susceptibility to amphotericin B. C. albicans strains develop in unique biphasic biofilm, different from that of other species. This is thick (450–550 lm) and complex, spatially organized structure comprising a basal blastospore layer with a dense overlaying mass of abundant pseudohyphae and hyphae partially embedded in an extracellular matrix comprised of cell wall-like compounds. In contrast, C. parapsilosis biofilms are less thick (75–125 lm) than these of C. albicans and are comprised exclusively of clumped blastospores with minimal, if any, extracellular matrix. This architecture does not explain the highest number of C. parapsilosis strains with SMIC [1 lg/ mL among three tested Candida species, or this correlation is not clear. Against C. parapsilosis strains, there were both very high and very low anti-biofilm activity of amphotericin B observed. Silva et al. [28] reported that biofilm formation by C. parapsilosis is strongly strain-depending. The selective predilection of C. parapsilosis strains for plastic biomaterials is of particular interest, since this enhances the ability of C. parapsilosis to colonize intravascular central lines and catheters. Resistance of Candida spp. biofilm to antifungals seems to be multifactorial phenomenon [9, 10, 12, 14, 29, 30]. This could be in part due to: (1) usually high density of fungal cells in biofilm, (2) protection from the immune system by exopolymer matrix, (3) slow penetration of an antifungal agent into biofilm, (4) surface-induced upregulation of genes encoding multidrug resistance (MDR) transporters, MDR1, CDR1,
23
CDR2, (5) downregulation of ergosterol biosynthetic gene and decreased ergosterol content, (6) slow growth of sessile cells, (7) presence of a small population of extremely resistant persister cells, and (8) drug–matrix interactions. Pfaller et al. [31] reported that C. parapsilosis strains derived from the blood samples produced biofilms significantly more often than these isolated from other specimens, 83 versus 53 %, respectively. Ru˚zicka et al. [17] also informed that C. parapsilosis strains derived from the blood samples developed biofilms significantly more often than these isolated from the skin, 59 versus 39 %, respectively. In our study, seven out of nine (77.8 %) C. parapsilosis strains isolated from blood demonstrated high amphotericin B SMICs—4–16 lg/mL for mature biofilm (Table 4). All C. parapsilosis strains isolated from peritoneal fluids, gastrostomy swabs, tracheotomy swabs, urine, and tips of vascular catheters displayed high amphotericin B SMICs—they are from 8 to 32 lg/mL for mature biofilm. In contrast, C. albicans strain derived from tip of vascular catheter expressed amphotericin B SMIC equals 1 lg/mL. C. parapsilosis strains derived from middle ear discharge and wound swabs presented low as well as high amphotericin B SMICs. In the available body of literature, there are discrepancies in the reported effects of amphotericin B on Candida spp. biofilms [12–18]. Ramage et al. [16] reported amphotericin B SMIC values for 24-hold biofilms of seven C. albicans strains ranging 0.25–0.5 lg/mL. Chandra et al. [12] described recalcitrant to amphotericin B C. albicans GDH 2346 biofilm, characterized by amphotericin B SMIC with a value of 8 lg/mL. Hawser et al. [14] also noticed low activity of amphotericin B against C. albicans GDH 2346, when amphotericin B SMIC values ranging 48.5–57.5 lg/mL were observed— depending on the employed method. However, these investigators treated biofilm with amphotericin B for 5 h only. Therefore, it is unreliable to compare these results with others. Interesting results were published by Kuhn et al. [15], in which SMIC values for two C. albicans strains and for two C. parapsilosis strains were 4 lg/mL and 8 lg/mL, respectively, when reference amphotericin B powder suitable for laboratory use was applied. Amphotericin B SMIC values ranged 0.25–1 lg/mL, when lipid formulations of amphotericin B, liposomal
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Peritoneal cavity swab
Peritoneal fluid
Abdominal cavity fluid
1
1
1
Urine
Pharyngeal swab
1
BAL
Groin swab C. glabrata (n = 20)
1
2
2
1
1
1
1
1
1
1
Tracheotomy swab
4
1 1
1
16
1
2
1
1
4
8
Tip of vascular catheter
1
2
1
4
1
1
1
1
1
1
2
2
2
Gastrostomy swab
Peritoneal fluid
Pharyngeal swab
Urine
Wound swab
Middle ear discharge
Blood
C. parapsilosis (n = 28)
Respiratory tract swab
1
1
Abdominal cavity fluid
Piece of surgical mesh
1
Tip of vascular catheter
1
12
1a
1
1
3
0.5
Pharyngeal swab
1
0.25
Middle ear discharge
1
0.125
2
1
B0.03
No. of isolates with indicated AMB SMIC (lg/mL)
BALb
Urine
C. albicans (n = 30)
Species and isolation site
Table 4 The distribution of amphotericin B SMICs for 24-h Candida spp. biofilms (n = 78) with respect to the origin of the strains
1
1
1
32
24 Mycopathologia (2014) 177:19–27
32
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1 1
1
0.5
1a
2
4
8
16
amphotericin B, and amphotericin B lipid complex were employed. Ru˚zicka et al. [17] described resistant to amphotericin B C. parapsilosis biofilms of 19 strains, with amphotericin B SMIC values ranging 2–16 lg/mL. The SMIC50 and SMIC90 were 4 and 8 lg/mL, respectively. However, the method used for biofilm susceptibility testing seems to be controversial. The cell viability indicator—resazurin—was added to biofilms simultaneously with antifungals. Therefore, resazurin could have been reduced before the antifungals affected usually high inoculum in biofilm. By contrast, Choi et al. [13] revealed relatively high activity of amphotericin B against mature Candida spp. biofilm. The SMIC50 and SMIC90 were 0.5 and 1 lg/mL, respectively, for C. albicans and C. glabrata, and 1 and 2 lg/mL, respectively, for C. parapsilosis. The lowest activity of amphotericin B was observed against C. parapsilosis biofilms—41.7 % of strains displayed SMIC values of 2 lg/mL. These results are consistent with ours. Similar results to ours where published by Chandra et al. [11]—2-, 4- and 8-h biofilms of C. albicans GDH 2346 displayed amphotericin B SMIC 0.5 lg/mL, but amphotericin B SMIC for 16-h biofilm reached 4 lg/ mL. Shuford et al. [19] revealed in vivo efficacy of amphotericin B in C. albicans biofilm elimination, applying experimental an intravascular catheter infection animal model. Their results also indicate the important effect of an individual factor in C. albicans biofilm eradication. In three out of 16 cases, amphotericin B treatment was not successful, although catheters were infected by the same strain and the same treatment was applied.
Bronchoalveolar lavage fluid
Piece of drain
Conclusions
b
a
Insertion-site skin swab
Blood
Amphotericin B concentration achieved in human body [22]
0.25 0.125 B0.03
No. of isolates with indicated AMB SMIC (lg/mL)
Species and isolation site
Table 4 continued
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Amphotericin B administration is limited due to the toxicity of its conventional formula and high prices of its less toxic lipid formulas. However, due to significant problems in Candida spp. biofilm eradication, the revealed activity of amphotericin B deserves additional researches, especially these in vivo, on animal models. Including different Candida species into researches on animal models, also these with high amphotericin B SMICs in vitro, could contribute to the
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current view on Candida spp. biofilm susceptibility to antifungals. Our investigations revealed that Candida spp. biofilm susceptibility to amphotericin B is species and strain depending. Therefore, making extrapolation about resistance of any particular Candida biofilm from other Candida strains resistance can be misleading. Our studies strongly indicate that amphotericin B should be considered as potential antibiofilm antifungal for conventional treatment and catheter lock solutions therapy. Acknowledgments This work was supported by the Nicolaus Copernicus University in Torun´, Ludwik Rydygier Collegium Medicum in Bydgoszcz (Grant Number 06/CM). Conflict of interest
None to declare.
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In Vitro Effect of Amphotericin B on Candida albicans, Candida glabrata and Candida parapsilosis Biofilm Formation Małgorzata Pra_zyn´ska • Eugenia Gospodarek
Received: 2 August 2013 / Accepted: 6 January 2014 / Published online: 17 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Candida spp. biofilm is considered highly resistant to conventional antifungals. The aim of this study was to investigate the in vitro effect of amphotericin B on Candida spp. biofilms at different stages of maturation. We investigated the activity of amphotericin B against 78 clinical isolates of Candida spp., representing three species, growing as planktonic and sessile cells, by a widely accepted broth microdilution method. The in vitro effect on sessile cell viability was evaluated by MTT reduction assay. All examined strains were susceptible to amphotericin B when grown as free-living cells. At the early stages of biofilm maturation 96.7–100.0 % strains, depending on species, displayed amphotericin B sessile minimal inhibitory concentration (SMIC) B1 lg/mL. Mature Candida spp. biofilm of 32.1–90.0 % strains displayed amphotericin B SMIC B1 lg/mL. Based on these results, amphotericin B displays species- and straindepending activity against Candida spp. biofilms.
Electronic supplementary material The online version of this article (doi:10.1007/s11046-014-9727-7) contains supplementary material, which is available to authorized users. M. Pra_zyn´ska (&) E. Gospodarek Department of Microbiology, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun´, 9 Maria Skłodowska-Curie Street, 85-094 Bydgoszcz, Poland e-mail: [email protected]
Keywords Antifungals Candidosis Polyenes Sessile cells
Introduction A biofilm is defined as a spatially organized hydrated community of microorganisms surrounded by a selfproduced extracellular polymeric matrix and adherent to a biotic or abiotic surface. This is considered the most prevalent growth form of microorganisms [1]. Fungi Candida spp., especially C. albicans, C. parapsilosis, C. glabrata, and C. tropicalis, possess a well-documented ability to form biofilm on biotic as well as abiotic surfaces [2–6]. Due to the common use of medical devices in modern medicine, Candida infections are often associated with biofilm formation. Candida spp. are ranked as the fourth leading cause of vascular catheter-related infections and the third leading cause of urinary catheter-related infections [7, 8]. Fungal cells existing in biofilm structure differ from their free-living counterparts. As far as clinical aspects are concerned, an important feature of fungal biofilm is its dramatically decreased susceptibility to antifungal drugs [9–18]. This may lead to chronic, refractory infections, with severe, even systemic course. In the device-related infections, device removal is often necessary for a cure. However, device removal can be difficult or impossible to perform or biofilm can be formed on the surface of a patient’s tissue. This is the reason why researching on
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the susceptibility of Candida spp. biofilms remains an actual issue. The resistance of Candida spp. to antifungals is puzzling, since free-living cells of the same strains can be susceptible to a range of antifungal drugs. The mechanism of Candida spp. biofilm resistance is still unclear. Based on existing literature, Candida spp. biofilm is consistently thought to be highly resistant to azoles [9–12, 14–18], and susceptible to echinocandins [13, 15, 18, 19]. In available literature, there are discrepancies in the results of the effects of amphotericin B on Candida spp. biofilms [12–18]. The resistant to amphotericin B Candida spp. biofilms [12, 14, 15, 17] as well as susceptible ones [13, 15, 16, 18] was reported. Furthermore, the majority of these reports describe the susceptibility of C. albicans biofilms only. There is relatively little about other Candida species. Amphotericin B is a polyene antifungal agent. It binds to ergosterol in fungal protoplasmatic membrane, which leads to the formation of transmembraneal pores and the leakage of cellular components. Unfortunately, its affinity to sterols is not selective to ergosterol and the drug also binds to cholesterol in mammalian cells. Therefore, amphotericin B is nephrotoxic and causes a wide range of adverse effects. Due to the introduction of newer, less toxic antifungals, amphotericin B is reserved for treatment of severe, life-threatening, invasive fungal infections. Its advantages comprise a wide spectrum of antifungal activity, including yeasts and molds, and fungicidal effects on susceptible fungi at clinically relevant concentrations [20]. Here, we report on the effect of amphotericin B on Candida spp. biofilms at different stages of maturation. Although C. albicans is still the predominant etiologic agent of candidiasis, other Candida species have emerged as significant pathogens [6, 14]. For the study, we employed C. albicans, C. parapsilosis, and C. glabrata, which are three the most commonly isolated fungal pathogens in our institution—Dr. Antoni Jurasz University Hospital No. 1 in Bydgoszcz, Poland.
Materials and Methods Strains A total of 78 Candida spp. strains were studied: 30 of C. albicans, 28 of C. parapsilosis, and 20 of C.
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glabrata. All the strains were isolated in The Department of Microbiology of Dr. A. Jurasz University Hospital No. 1 in Bydgoszcz, Nicolaus Copernicus University in Torun, Ludwik Rydygier Collegium Medicum in Bydgoszcz, in 2007–2010. One strain per patient was studied. Isolates were isolated from urine (n = 25), blood cultures (n = 10), bronchoalveolar lavage fluid (n = 7), middle ear discharge (n = 8), pharyngeal swab (n = 7), wound swab (n = 4), tip of vascular catheter (n = 3), peritoneal fluid (n = 5), gastrostomy swab (n = 2), and one strain per piece of drain, piece of surgical mesh, respiratory tract swab, insertion-site skin swab, peritoneal cavity swab, tracheotomy swab, and groin swab. The detailed data on the origin of the examined strains are given in Online Resource 1. C. albicans GDH 2346 served as a positive control. Strains were stored at -70 °C. Before conducting researches, the species identification was confirmed by means of germ tube tests [21] and ID 32 C tests carried out according to the manufacturer’s protocol (bioMe´rieux) [22]. Antifungal Susceptibility Testing Amphotericin B concentrations ranging from 0.03 to 128 lg/mL were tested. Planktonic minimal inhibitory concentrations (MICs) were determined by CLSI broth microdilution method [23, 24]. Sessile MICs (SMICs) were determined using a modification of methods described by Ramage and Lo´pez-Ribot [25] and Krom et al. [26]. Briefly, the strains were recovered from frozen storage. A loopful of the stock culture was streaked on Sabouraud dextrose agar (SDA, Becton–Dickinson) and incubated at 37 °C for 48–72 h. A single colony from the obtained culture was inoculated on SDA and incubated at 37 °C for 24 h. A single colony from a 24-h culture was inoculated in a yeast nitrogen base medium (YNB, Becton–Dickinson), supplemented with 0.25 % glucose (BTL) and incubated at 37 °C for 24 h on a rotary shaker (120 rpm). These conditions promote growing in the budding yeast phase. The resultant cultures were harvested (3,0009g, 5 min) and washed three times with a sterile phosphate-buffered saline (PBS, pH 7.0 ± 0.1) (BTL) and standardized to 0.5 McFarland in YNB with a densitometer. Biofilms formed in 96-well, flat-bottomed, sterile, polystyrene plates. Each well was filled with 100 lL
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of yeast cell suspensions or sterile YNB as negative controls, each in three repetitions, and the plates were incubated at 37 °C for 2, 6, or 24 h, in order to obtain biofilms at different stages of maturation. After incubation, suspensions were discarded and wells were rinsed gently and thoroughly in sterile PBS three times. Biofilm-coated wells were filled with 100 lL serial dilutions of amphotericin B in RPMI 1640 medium. Untreated biofilm wells and biofilm-free wells were also included to serve as positive and negative controls, respectively. These wells were filled with RPMI 1640 medium without antifungal drug. Microtiter plates were incubated for an additional 24 h at 35 °C. After 24 h, the medium was discarded and wells were rinsed gently and thoroughly in sterile PBS three times.
21 Table 1 Distribution of amphotericin B MICs for Candida spp. isolates (n = 78) Species
No. of isolates with indicated AMB MIC (lg/mL) B0.03
C. albicans (n = 30) C. parapsilosis (n = 28) C. glabrata (n = 20)
%A550untreated ¼
1
MIC50
MIC90
0.25
0.5
1
2
16
12
0.5
1
8
8
11
0.5
1
1
5
14
1
1
A550amphotericinB A550negativecontrol A550untreated 100 %:
MTT Reduction Assay
Statistics
The colorimetric assay, based on an MTT [3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] reduction, was employed to determine cell viability and thereby drug susceptibility of Candida cells in biofilms [26]. Briefly, the MTT (AlfaAesar) was prepared at 0.5 g/L in sterile, prewarmed to 37 °C PBS, containing 1 % glucose and 10 lM menadione (Sigma) dissolved in acetone. The solution was filter sterilized through a 0.22-mm pore-size filter. The MTT of 100 lL was added to each well. The plates were then incubated in the dark at 37 °C for 30 min to 2 h, depending on the examined strains. The product of the MTT reduction and the insoluble purple formazan crystals were solubilized in 100 lL acid isopropanol (5 % 1 M HCl in isopropanol). Efficient solubilization was achieved by vigorously shaking the plates for 15 min. The metabolic activity of Candida sessile cells was assessed quantitatively by measuring the absorbance in a microtiter plate reader at 550 nm, after transferring the solution to new 96-well plates. SMICs were determined at C50 % inhibition comparing to untreated biofilms.
Statistical analysis was performed by two-tailed Student’s t test and differences with P values of \0.05 were considered statistically significant.
Metabolic Activity The metabolic activities of Candida spp. biofilms affected by amphotericin B at 1 lg/mL were estimated as a percentage of the metabolic activities of untreated biofilms of particular Candida spp. strains, according to the following formula:
Results Planktonic MICs for all examined Candida spp. isolates were B1 lg/mL (Table 1). The distribution of amphotericin B SMICs for examined isolates is presented in Table 2. The amphotericin B SMIC50 and SMIC90 for C. albicans isolates were both 1 lg/mL for 2- and 6-h-old biofilms, 1 and 2 lg/mL, respectively, for 24-h-old biofilms. The amphotericin B SMIC50 and SMIC90 for C. parapsilosis isolates were 0.25 and 0.5 lg/mL, respectively, for 2-h-old biofilms, 0.5 and 1 lg/mL, respectively, for 6-h-old biofilms, 8 and 32 lg/mL, respectively, for mature biofilms. The amphotericin B SMIC50 and SMIC90 for C. glabrata were 0.5 and 1 lg/mL, respectively, for 2-h-old biofilms, and both 1 lg/mL for 6- and 24-h-old biofilms. Amphotericin B MIC for C. albicans GDH 2346 was 0.25 lg/mL, whereas SMICs for 2-, 6-, and 24-hold biofilms were 0.5, 0.5, and 1.0, respectively. All C. parapsilosis and C. glabrata and 96.7 % C. albicans examined strains displayed amphotericin B SMICs B1 lg/mL at the early stages of biofilm maturation; that is 2- and 6-h-old biofilms, whereas mature biofilms kept amphotericin B SMICs B1 lg/
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Table 2 Distribution of amphotericin B SMICs for Candida spp. isolates (n = 78) Species and age of biofilm
No. of isolates with indicated AMB SMIC (lg/mL) B0.03
0.06
0.125
0.25
0.5
1a
2
4
8
16
32
6
3
C. albicans (n = 30) 2 h old
4
22
1
6 h old
1
2
4
25
1
24 h old
4
19
6
1
2 2
2
6
1
1
C. parapsilosis (n = 28) 2 h old
7
9
9
6 h old
2
1 1
6
14
5
24 h old
2
1
2
2
2
1
5
7
7
3 3
5 5
12 10
C. glabrata (n = 20) 2 h old 6 h old 24 h old a
Amphotericin B concentration achieved in human body [22]
Table 3 The metabolic activity of Candida spp. (n = 78) biofilms affected by amphotericin B at 1 lg/mL Species
The metabolic activity of Candida spp. biofilm (%A550-untreated): 2-h old Range
C. albicans (n = 30)
6-h old Mean ± SD
Range
24-h old Mean ± SD
Range
Mean ± SD
13.0–79.5
30.7 ± 17.9
17.0–66.4
39.4 ± 11.9
27.1–81.3
49.4 ± 14.0
C. parapsilosis (n = 28)
3.5–47.8
15.8 ± 12.3
13.4–53.6
27.7 ± 12.8
28.0–112.4
71.7 ± 24.7
C. glabrata (n = 20)
5.8–54.4
21.8 ± 10.6
22.1–56.4
39.4 ± 10.9
16.8–89.2
42.7 ± 18.1
mL for 90.0 % of C. glabrata, 76.9 % of C. albicans and 32.1 % of C. parapsilosis examined strains. The statistically significant differences were observed between mature biofilms of C. albicans and C. parapsilosis (P = 0.0004), along with C. glabrata and C. parapsilosis (P \ 0.0001). The more mature the biofilm, the weaker effect of amphotericin B on metabolic status of Candida spp. sessile cells was observed. The highest metabolic activities of biofilms following amphotericin B treatment at 1 lg/mL were noticed for mature C. parapsilosis biofilms, ranging 28.0–112.4 % of the metabolic activities of untreated biofilms. The mean values of the rest of the metabolic activities were lower than 50.0 % (15.8–49.4 %), which indicates the average biofilm reductions about more than a half (50.6–84.2 %) due to amphotericin B at 1 lg/mL. Detailed data on the metabolic activities of biofilms treated with amphotericin B at 1 lg/mL are presented in Table 3.
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Statistically significant differences in the mean metabolic activities of Candida spp. biofilms were observed between 2-h-old biofilms of C. albicans and C. parapsilosis (P = 0.0006), 6-h-old biofilms of C. parapsilosis and C. albicans (P = 0.001), C. parapsilosis and C. glabrata (P = 0.004), 24-h-old biofilms of C. parapsilosis and C. albicans (P = 0.0002), C. parapsilosis and C. glabrata (P = 0.0001). These investigations indicate that amphotericin B changes the metabolic status of Candida spp. biofilms and this effect depends on species, strains, and stage of biofilm maturation.
Discussion Our investigations confirmed lower susceptibility to amphotericin B of all Candida examined isolates when grown as biofilms, compared with planktonic
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forms. From 32.1 to 90.0 % of tested isolates, depending on species of examined strains, displayed SMICs B1 lg/mL for mature biofilms. The concentration of amphotericin B obtained in the human body is established at 1–2 lg/mL [27]. The highest number of isolates with amphotericin B SMIC[1 lg/mL was observed among C. parapsilosis strains (18 strains; 67.9 %), whereas the lowest number of that was noticed among C. glabrata (10.0 %). Among C. albicans strains, seven out of 30 strains (23.3 %) presented amphotericin B SMIC[1 lg/mL for mature biofilm. The differences in the biofilm architecture of C. albicans, C. parapsilosis, and C. glabrata are documented in the existing literature [6, 11, 28]. The architecture of C. glabrata biofilm, which is thin and irregular, rather compact, composed of small population of blastospores embedded within an extracellular matrix might be partially responsible for its general susceptibility to amphotericin B. C. albicans strains develop in unique biphasic biofilm, different from that of other species. This is thick (450–550 lm) and complex, spatially organized structure comprising a basal blastospore layer with a dense overlaying mass of abundant pseudohyphae and hyphae partially embedded in an extracellular matrix comprised of cell wall-like compounds. In contrast, C. parapsilosis biofilms are less thick (75–125 lm) than these of C. albicans and are comprised exclusively of clumped blastospores with minimal, if any, extracellular matrix. This architecture does not explain the highest number of C. parapsilosis strains with SMIC [1 lg/ mL among three tested Candida species, or this correlation is not clear. Against C. parapsilosis strains, there were both very high and very low anti-biofilm activity of amphotericin B observed. Silva et al. [28] reported that biofilm formation by C. parapsilosis is strongly strain-depending. The selective predilection of C. parapsilosis strains for plastic biomaterials is of particular interest, since this enhances the ability of C. parapsilosis to colonize intravascular central lines and catheters. Resistance of Candida spp. biofilm to antifungals seems to be multifactorial phenomenon [9, 10, 12, 14, 29, 30]. This could be in part due to: (1) usually high density of fungal cells in biofilm, (2) protection from the immune system by exopolymer matrix, (3) slow penetration of an antifungal agent into biofilm, (4) surface-induced upregulation of genes encoding multidrug resistance (MDR) transporters, MDR1, CDR1,
23
CDR2, (5) downregulation of ergosterol biosynthetic gene and decreased ergosterol content, (6) slow growth of sessile cells, (7) presence of a small population of extremely resistant persister cells, and (8) drug–matrix interactions. Pfaller et al. [31] reported that C. parapsilosis strains derived from the blood samples produced biofilms significantly more often than these isolated from other specimens, 83 versus 53 %, respectively. Ru˚zicka et al. [17] also informed that C. parapsilosis strains derived from the blood samples developed biofilms significantly more often than these isolated from the skin, 59 versus 39 %, respectively. In our study, seven out of nine (77.8 %) C. parapsilosis strains isolated from blood demonstrated high amphotericin B SMICs—4–16 lg/mL for mature biofilm (Table 4). All C. parapsilosis strains isolated from peritoneal fluids, gastrostomy swabs, tracheotomy swabs, urine, and tips of vascular catheters displayed high amphotericin B SMICs—they are from 8 to 32 lg/mL for mature biofilm. In contrast, C. albicans strain derived from tip of vascular catheter expressed amphotericin B SMIC equals 1 lg/mL. C. parapsilosis strains derived from middle ear discharge and wound swabs presented low as well as high amphotericin B SMICs. In the available body of literature, there are discrepancies in the reported effects of amphotericin B on Candida spp. biofilms [12–18]. Ramage et al. [16] reported amphotericin B SMIC values for 24-hold biofilms of seven C. albicans strains ranging 0.25–0.5 lg/mL. Chandra et al. [12] described recalcitrant to amphotericin B C. albicans GDH 2346 biofilm, characterized by amphotericin B SMIC with a value of 8 lg/mL. Hawser et al. [14] also noticed low activity of amphotericin B against C. albicans GDH 2346, when amphotericin B SMIC values ranging 48.5–57.5 lg/mL were observed— depending on the employed method. However, these investigators treated biofilm with amphotericin B for 5 h only. Therefore, it is unreliable to compare these results with others. Interesting results were published by Kuhn et al. [15], in which SMIC values for two C. albicans strains and for two C. parapsilosis strains were 4 lg/mL and 8 lg/mL, respectively, when reference amphotericin B powder suitable for laboratory use was applied. Amphotericin B SMIC values ranged 0.25–1 lg/mL, when lipid formulations of amphotericin B, liposomal
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Peritoneal cavity swab
Peritoneal fluid
Abdominal cavity fluid
1
1
1
Urine
Pharyngeal swab
1
BAL
Groin swab C. glabrata (n = 20)
1
2
2
1
1
1
1
1
1
1
Tracheotomy swab
4
1 1
1
16
1
2
1
1
4
8
Tip of vascular catheter
1
2
1
4
1
1
1
1
1
1
2
2
2
Gastrostomy swab
Peritoneal fluid
Pharyngeal swab
Urine
Wound swab
Middle ear discharge
Blood
C. parapsilosis (n = 28)
Respiratory tract swab
1
1
Abdominal cavity fluid
Piece of surgical mesh
1
Tip of vascular catheter
1
12
1a
1
1
3
0.5
Pharyngeal swab
1
0.25
Middle ear discharge
1
0.125
2
1
B0.03
No. of isolates with indicated AMB SMIC (lg/mL)
BALb
Urine
C. albicans (n = 30)
Species and isolation site
Table 4 The distribution of amphotericin B SMICs for 24-h Candida spp. biofilms (n = 78) with respect to the origin of the strains
1
1
1
32
24 Mycopathologia (2014) 177:19–27
32
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1 1
1
0.5
1a
2
4
8
16
amphotericin B, and amphotericin B lipid complex were employed. Ru˚zicka et al. [17] described resistant to amphotericin B C. parapsilosis biofilms of 19 strains, with amphotericin B SMIC values ranging 2–16 lg/mL. The SMIC50 and SMIC90 were 4 and 8 lg/mL, respectively. However, the method used for biofilm susceptibility testing seems to be controversial. The cell viability indicator—resazurin—was added to biofilms simultaneously with antifungals. Therefore, resazurin could have been reduced before the antifungals affected usually high inoculum in biofilm. By contrast, Choi et al. [13] revealed relatively high activity of amphotericin B against mature Candida spp. biofilm. The SMIC50 and SMIC90 were 0.5 and 1 lg/mL, respectively, for C. albicans and C. glabrata, and 1 and 2 lg/mL, respectively, for C. parapsilosis. The lowest activity of amphotericin B was observed against C. parapsilosis biofilms—41.7 % of strains displayed SMIC values of 2 lg/mL. These results are consistent with ours. Similar results to ours where published by Chandra et al. [11]—2-, 4- and 8-h biofilms of C. albicans GDH 2346 displayed amphotericin B SMIC 0.5 lg/mL, but amphotericin B SMIC for 16-h biofilm reached 4 lg/ mL. Shuford et al. [19] revealed in vivo efficacy of amphotericin B in C. albicans biofilm elimination, applying experimental an intravascular catheter infection animal model. Their results also indicate the important effect of an individual factor in C. albicans biofilm eradication. In three out of 16 cases, amphotericin B treatment was not successful, although catheters were infected by the same strain and the same treatment was applied.
Bronchoalveolar lavage fluid
Piece of drain
Conclusions
b
a
Insertion-site skin swab
Blood
Amphotericin B concentration achieved in human body [22]
0.25 0.125 B0.03
No. of isolates with indicated AMB SMIC (lg/mL)
Species and isolation site
Table 4 continued
25
Amphotericin B administration is limited due to the toxicity of its conventional formula and high prices of its less toxic lipid formulas. However, due to significant problems in Candida spp. biofilm eradication, the revealed activity of amphotericin B deserves additional researches, especially these in vivo, on animal models. Including different Candida species into researches on animal models, also these with high amphotericin B SMICs in vitro, could contribute to the
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current view on Candida spp. biofilm susceptibility to antifungals. Our investigations revealed that Candida spp. biofilm susceptibility to amphotericin B is species and strain depending. Therefore, making extrapolation about resistance of any particular Candida biofilm from other Candida strains resistance can be misleading. Our studies strongly indicate that amphotericin B should be considered as potential antibiofilm antifungal for conventional treatment and catheter lock solutions therapy. Acknowledgments This work was supported by the Nicolaus Copernicus University in Torun´, Ludwik Rydygier Collegium Medicum in Bydgoszcz (Grant Number 06/CM). Conflict of interest
None to declare.
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