AAC Accepts, published online ahead of print on 23 June 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.02738-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Physiological Serum Concentrations of Micafungin Show Antifungal Activity Against Candida
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albicans and Candida parapsilosis Biofilms
3
Authors
4
Guembe M., PharmD, PhD1, 2 #
5
Guinea J., PharmD, PhD1, 2, 3,4 #
6
Marcos-Zambrano L.J., BSc1, 2
7
Fernández-Cruz A., MD, PhD1,2
8
Peláez T., PharmD, PhD1, 2, 3, 4
9
Muñoz P., MD, PhD1, 2, 3, 4
10
Bouza E., MD, PhD1, 2, 3, 4
11
1
12
Marañón, Madrid, Spain
13
2
Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
14
3
CIBER Enfermedades Respiratorias-CIBERES (CB06/06/0058), Madrid, Spain
15
4
Medicine Department, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain
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Running title: “Micafungin and Candida biofilm”
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Abstract word count: 76
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Text word count: 974
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Key words for indexing: Catheter-Related Candidemia. Antifungal lock therapy. Micafungin.
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Biofilm.
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#Corresponding authors:
22
María Guembe Ramírez, PharmD, PhD
23
Jesús Guinea Ortega, PharmD, PhD
24
Servicio de Microbiología Clínica y Enfermedades Infecciosas
25
Hospital General Universitario “Gregorio Marañón”
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C/. Dr. Esquerdo, 46
27
28007 Madrid, Spain
Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio
1
28
Phone: +34 91 586 84 53
29
Fax: +34 91 504 49 06
30
E-mails: [email protected], [email protected]
2
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ABSTRACT
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We assessed the in vitro activity of micafungin against preformed Candida biofilms by
33
measuring the concentration of drug causing the most fungal damage and inhibition of
34
regrowth. We studied 37 biofilm-producing Candida spp. strains from blood cultures.
35
We showed that micafungin was active against planktonic and sessile forms of C.
36
albicans strains and moderately active against C. parapsilosis sessile cells.
37
Concentrations of micafungin above 2 µg/mL were sufficiently high to inactivate
38
regrowth of Candida sessile cells.
3
39
Candida spp. cause approximately 8-15% of bloodstream infections and are the
40
third cause of these infections after coagulase-negative staphylococci and
41
Staphylococcus aureus [1-8].
42
A high number of episodes of candidemia originate in central venous catheters
43
[9, 10], leading to high rates of morbidity and mortality [11]. The ability of some
44
Candida strains to produce biofilms, especially C. albicans and C. parapsilosis, may
45
explain the high frequency of catheter-related candidemia (CRC) [12-22].
46
Recent in vitro and in vivo models designed to assess the antifungal susceptibility
47
of Candida biofilms [19, 21, 23-29] showed that the echinocandins, particularly
48
micafungin, had the highest antifungal activity [30-34]. However, the studies are
49
limited by the inclusion of a low number of clinical isolates; furthermore, the
50
concentration of antifungal necessary to inactivate sessile cells is unknown. Hence, it
51
seems reasonable to determine the micafungin concentration able to inactivate the
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Candida sessile cells.
53
Our objective was to assess the in vitro concentration of micafungin causing the
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most fungal damage against mature biofilms involving Candida strains isolated from
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candidemic patients.
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We collected 37 biofilm-producing Candida strains (C. albicans, 29; C.
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parapsilosis, 8) from 37 patients with candidemia admitted to Hospital Gregorio
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Marañón, a large tertiary hospital in Madrid, Spain. We studied the activity of
59
micafungin against planktonic and sessile forms of the isolates.
60 61
The crystal violet binding assay was used to assess biofilm production. A cut-off of OD ≥0.5 was used to define biofilm-forming isolates [35].
4
62
We defined catheter-related candidemia as an episode in which the same
63
Candida species was isolated both in peripheral blood and in a catheter segment
64
sample [36].
65
The MICs of planktonic isolates were determined using the EUCAST broth
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microdilution procedure [37]. Isolates were grown on Sabouraud dextrose agar plates
67
for 24 hours at 37°C. Serial two-fold micafungin dilutions ranging from 16 to 0.015
68
µg/mL were studied. The lowest concentration producing a ≥50% reduction in turbidity
69
compared with that of the antifungal-free control well was reported as the MIC. Sessile
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MICs (SMICs) were studied on biofilms formed in 96-well, flat-bottomed microtiter
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plates as described by other authors [38, 39]. The SMIC was assessed by measuring the
72
absorbance of each well at 492 nm with XTT to determine the lowest concentration
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associated with a 50% reduction in absorbance compared with the level for the control
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well (percent fungal damage = [1-X/C] x 100, where X is the absorbance of
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experimental wells and C is the absorbance of control wells).
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Candida biofilms were regrown using the same methodology as that used for the
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study of the SMICs, including 24-hour incubation (37°C) of the trays filled with 100 µL
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of YPD per well. We measured well absorbance at 492 nm to calculate the percentage
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of growth inhibition ([1-X/C] x 100, where X is the absorbance of the experimental
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wells and C is the absorbance of the control wells) to assess the inhibition of sessile cell
81
regrowth. We considered Candida biofilm regrowth as any absorbance compared to
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the negative control well. A calibration curve was constructed to study the number of
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cells that regrew in each well after exposure to micafungin.
84 85
Each experiment was tested in triplicate, and the average value was used for the analysis.
5
86
Of the 37 Candida strains, 19 (51.4%) were from patients with CRC. The
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micafungin MICs for planktonic cells of C. albicans were always ≤0.015 µg/mL whereas
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the MICs for C. parapsilosis ranged from ≤0.015 to 2 µg/mL. In contrast, the micafungin
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MICs for sessile cells of both C. albicans and C. parapsilosis ranged between ≤0.015
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and >16 (table 1).
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Fungal damage worsened with increasing micafungin concentrations (figure 1a).
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Both C. albicans and C. parapsilosis biofilms showed a reduction in metabolic activity
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≥50% at micafungin concentrations ≥2 µg/mL.
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Inhibition of regrowth for all Candida strains was > 80% after 24 hours of
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incubation with micafungin at concentrations ≥2 µg/mL. Inhibition of regrowth was
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>50% for C. albicans and C. parapsilosis at micafungin concentrations of 0.25 µg/mL
97
and 1 µg/mL, respectively (figure 1b, figures 2a and 2b). The number of cells that
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regrew after exposure to micafungin was studied (figures 2a and 2b). Both species
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showed a significant decrease in the number of cells/mL after 24 hours’ incubation
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with micafungin at concentrations ≥2 µg/mL. The number of cells/mL increased slightly
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when C. albicans and C. parapsilosis were incubated with micafungin at 16 µg/mL.
102
Our study showed that micafungin was active against both planktonic and sessile
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strains of C. albicans and moderately active against sessile strains of C. parapsilosis.
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Regrowth of strains from both species decreased decreased by >80% when they were
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exposed to concentrations of micafungin up to 2 µg/mL.
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As most candidemia episodes are catheter-related [9, 10], our results could be
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applied in patients with CRC. Guidelines recommend removing the catheter in patients
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with suspicion of CRC, although this issue remains controversial. Nucci et al.
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demonstrated that non-neutropenic patients treated with echinocandins or liposomal
6
110
amphotericin B did not benefit from early removal of the catheter [40]. Therefore, not
111
removing the catheter in patients with CRC may be an alternative approach
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(accompanied by antifungal lock therapy in combination with systemic antifungals)
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when the risk of replacing the catheter is greater than the benefit [41]. Our data show
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that physiological concentrations of micafungin of 4 µg/mL [42, 43] are active against
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biofilm, since metabolic activity decreased and fungal regrowth was inhibited in most
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of the preformed biofilms. Our in vitro observations also support the previous clinical
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observations reported by Nucci et al. [40].
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In summary, we provide new data on the in vitro antibiofilm activity of
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micafungin against Candida strains from patients with candidemia. Although
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micafungin MICs were higher for sessile cells than for planktonic cells, regrowth of the
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cells forming the biofilm was reduced more than 80% at physiological serum
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concentrations of 4 µg/mL. Our in vitro study supports that micafungin concentrations
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≥2 µg/mL may be sufficiently high to eradicate Candida biofilms.
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125 126 127 128
ACKNOWLEDGEMENTS
We thank Thomas O’Boyle for his help in the preparation of the manuscript.
129 130
Financial support. This work was supported by grants from Astellas Pharma, by grants
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from the Fondo de Investigación Sanitaria (PI11/00167), and by Fundación MUTUA
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Madrileña de Madrid. M. Guembe (CP13/00268), J. Guinea (MS09/00055), and L.J.
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Marcos-Zambrano (FI12/00265) are supported by the Fondo de Investigación Sanitaria.
134 135
Potential conflicts of interest. The authors declare no conflicts of interest.
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FIGURE LEGENDS
278
Figure 1. Mean percentage of the reduction in metabolic activity and regrowth of
279
Candida biofilms after exposure to micafungin
280
Figure 1a. Mean percentage reduction in metabolic activity (fungal damage) of
281
Candida biofilms after exposure to micafungin
282
Figure 1b. Mean percentage inhibition of regrowth of Candida biofilms after exposure
283
to different concentrations of micafungin
284
Figure 2. Correlation between absorbance of Candida sessile cells and number of
285
cells/mL after exposure to micafungin
286
Figure 2a. Correlation between absorbance of C. albicans sessile cells and number of
287
cells/mL after exposure to micafungin
288
Figure 2b. Correlation between absorbance of C. parapsilosis sessile cells and number
289
of cells/mL after exposure to micafungin
290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310
15
Figure 1a 1a. Mean percentage reduction in metabolic activity (fungal damage) of Candida biofilms after exposure to micafungin 80 70
% of fungal dam mage
60 50 C. albicans
40
C. parapsilosis 30 20 10 0 0.015
0.03
0.06
0.12
0.25
0.5
1
2
Micafungin concentrations (µg/mL)
4
8
16
Figure 1b 1b. Mean percentage inhibition of regrowth of Candida biofilms after exposure to different concentrations of micafungin
100 90 80
60 50
C. albicans
40
C. parapsilosis
30 20 10
Micafungin concentrations (µg/mL)
16
8
4
2
1
0. 5
0. 25
0. 12
0. 06
0. 03
0
0. 01 5
%o of re-growth inhibittion
70
Figure 2a. Correlation between absorbance of C. albicans sessile cells and number of cells/mL after exposure to micafungin
Absorbance
No. cells/mL /
0,8
30000000
0,7
25000000
0,6 20000000
0,5 0,4
15000000
Absorbance Cells/mL
03 0,3
10000000
0,2 5000000
0,1
16
4
8
1
2
0
0. 01 5 0. 03 0. 06 0. 12 0. 25 0. 50
0 Micafungin concentration (µg/mL)
Figure 2b. Correlation between absorbance of C. parapsilosis sessile cells and number of cells/mL after exposure to micafungin
Absorbance
No. cells/mL
1,2
45000000 40000000
1
35000000 30000000
0,8
25000000
0,6
20000000
04 0,4
15000000 10000000
0,2
5000000 16
4
8
1
2
0
0. 01 5 0. 03 0. 06 0. 12 0. 25 0. 50
0 Micafungin concentration (µg/mL)
Absorbance Cells/mL
Table 1. Distribution of micafungin MICs for the 37 planktonic and sessile Candida isolates. Species (No.)
No. of isolates in each MIC (µg/mL) 0.015
0.031 0.062 0.125 0.25
0.5
1
2
4
8
16
>16
Planktonic
29
-
-
-
-
-
-
-
-
-
-
-
Sessile
14
-
-
1
4
-
-
3
1
1
3
2
Planktonic
1
-
-
-
-
-
6
1
-
-
-
-
Sessile
-
-
-
-
-
-
2
1
3
-
-
2
C. albicans (29)
C. parapsilosis (8)
1
Physiological Serum Concentrations of Micafungin Show Antifungal Activity Against Candida
2
albicans and Candida parapsilosis Biofilms
3
Authors
4
Guembe M., PharmD, PhD1, 2 #
5
Guinea J., PharmD, PhD1, 2, 3,4 #
6
Marcos-Zambrano L.J., BSc1, 2
7
Fernández-Cruz A., MD, PhD1,2
8
Peláez T., PharmD, PhD1, 2, 3, 4
9
Muñoz P., MD, PhD1, 2, 3, 4
10
Bouza E., MD, PhD1, 2, 3, 4
11
1
12
Marañón, Madrid, Spain
13
2
Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
14
3
CIBER Enfermedades Respiratorias-CIBERES (CB06/06/0058), Madrid, Spain
15
4
Medicine Department, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain
16
Running title: “Micafungin and Candida biofilm”
17
Abstract word count: 76
18
Text word count: 974
19
Key words for indexing: Catheter-Related Candidemia. Antifungal lock therapy. Micafungin.
20
Biofilm.
21
#Corresponding authors:
22
María Guembe Ramírez, PharmD, PhD
23
Jesús Guinea Ortega, PharmD, PhD
24
Servicio de Microbiología Clínica y Enfermedades Infecciosas
25
Hospital General Universitario “Gregorio Marañón”
26
C/. Dr. Esquerdo, 46
27
28007 Madrid, Spain
Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio
1
28
Phone: +34 91 586 84 53
29
Fax: +34 91 504 49 06
30
E-mails: [email protected], [email protected]
2
31
ABSTRACT
32
We assessed the in vitro activity of micafungin against preformed Candida biofilms by
33
measuring the concentration of drug causing the most fungal damage and inhibition of
34
regrowth. We studied 37 biofilm-producing Candida spp. strains from blood cultures.
35
We showed that micafungin was active against planktonic and sessile forms of C.
36
albicans strains and moderately active against C. parapsilosis sessile cells.
37
Concentrations of micafungin above 2 µg/mL were sufficiently high to inactivate
38
regrowth of Candida sessile cells.
3
39
Candida spp. cause approximately 8-15% of bloodstream infections and are the
40
third cause of these infections after coagulase-negative staphylococci and
41
Staphylococcus aureus [1-8].
42
A high number of episodes of candidemia originate in central venous catheters
43
[9, 10], leading to high rates of morbidity and mortality [11]. The ability of some
44
Candida strains to produce biofilms, especially C. albicans and C. parapsilosis, may
45
explain the high frequency of catheter-related candidemia (CRC) [12-22].
46
Recent in vitro and in vivo models designed to assess the antifungal susceptibility
47
of Candida biofilms [19, 21, 23-29] showed that the echinocandins, particularly
48
micafungin, had the highest antifungal activity [30-34]. However, the studies are
49
limited by the inclusion of a low number of clinical isolates; furthermore, the
50
concentration of antifungal necessary to inactivate sessile cells is unknown. Hence, it
51
seems reasonable to determine the micafungin concentration able to inactivate the
52
Candida sessile cells.
53
Our objective was to assess the in vitro concentration of micafungin causing the
54
most fungal damage against mature biofilms involving Candida strains isolated from
55
candidemic patients.
56
We collected 37 biofilm-producing Candida strains (C. albicans, 29; C.
57
parapsilosis, 8) from 37 patients with candidemia admitted to Hospital Gregorio
58
Marañón, a large tertiary hospital in Madrid, Spain. We studied the activity of
59
micafungin against planktonic and sessile forms of the isolates.
60 61
The crystal violet binding assay was used to assess biofilm production. A cut-off of OD ≥0.5 was used to define biofilm-forming isolates [35].
4
62
We defined catheter-related candidemia as an episode in which the same
63
Candida species was isolated both in peripheral blood and in a catheter segment
64
sample [36].
65
The MICs of planktonic isolates were determined using the EUCAST broth
66
microdilution procedure [37]. Isolates were grown on Sabouraud dextrose agar plates
67
for 24 hours at 37°C. Serial two-fold micafungin dilutions ranging from 16 to 0.015
68
µg/mL were studied. The lowest concentration producing a ≥50% reduction in turbidity
69
compared with that of the antifungal-free control well was reported as the MIC. Sessile
70
MICs (SMICs) were studied on biofilms formed in 96-well, flat-bottomed microtiter
71
plates as described by other authors [38, 39]. The SMIC was assessed by measuring the
72
absorbance of each well at 492 nm with XTT to determine the lowest concentration
73
associated with a 50% reduction in absorbance compared with the level for the control
74
well (percent fungal damage = [1-X/C] x 100, where X is the absorbance of
75
experimental wells and C is the absorbance of control wells).
76
Candida biofilms were regrown using the same methodology as that used for the
77
study of the SMICs, including 24-hour incubation (37°C) of the trays filled with 100 µL
78
of YPD per well. We measured well absorbance at 492 nm to calculate the percentage
79
of growth inhibition ([1-X/C] x 100, where X is the absorbance of the experimental
80
wells and C is the absorbance of the control wells) to assess the inhibition of sessile cell
81
regrowth. We considered Candida biofilm regrowth as any absorbance compared to
82
the negative control well. A calibration curve was constructed to study the number of
83
cells that regrew in each well after exposure to micafungin.
84 85
Each experiment was tested in triplicate, and the average value was used for the analysis.
5
86
Of the 37 Candida strains, 19 (51.4%) were from patients with CRC. The
87
micafungin MICs for planktonic cells of C. albicans were always ≤0.015 µg/mL whereas
88
the MICs for C. parapsilosis ranged from ≤0.015 to 2 µg/mL. In contrast, the micafungin
89
MICs for sessile cells of both C. albicans and C. parapsilosis ranged between ≤0.015
90
and >16 (table 1).
91
Fungal damage worsened with increasing micafungin concentrations (figure 1a).
92
Both C. albicans and C. parapsilosis biofilms showed a reduction in metabolic activity
93
≥50% at micafungin concentrations ≥2 µg/mL.
94
Inhibition of regrowth for all Candida strains was > 80% after 24 hours of
95
incubation with micafungin at concentrations ≥2 µg/mL. Inhibition of regrowth was
96
>50% for C. albicans and C. parapsilosis at micafungin concentrations of 0.25 µg/mL
97
and 1 µg/mL, respectively (figure 1b, figures 2a and 2b). The number of cells that
98
regrew after exposure to micafungin was studied (figures 2a and 2b). Both species
99
showed a significant decrease in the number of cells/mL after 24 hours’ incubation
100
with micafungin at concentrations ≥2 µg/mL. The number of cells/mL increased slightly
101
when C. albicans and C. parapsilosis were incubated with micafungin at 16 µg/mL.
102
Our study showed that micafungin was active against both planktonic and sessile
103
strains of C. albicans and moderately active against sessile strains of C. parapsilosis.
104
Regrowth of strains from both species decreased decreased by >80% when they were
105
exposed to concentrations of micafungin up to 2 µg/mL.
106
As most candidemia episodes are catheter-related [9, 10], our results could be
107
applied in patients with CRC. Guidelines recommend removing the catheter in patients
108
with suspicion of CRC, although this issue remains controversial. Nucci et al.
109
demonstrated that non-neutropenic patients treated with echinocandins or liposomal
6
110
amphotericin B did not benefit from early removal of the catheter [40]. Therefore, not
111
removing the catheter in patients with CRC may be an alternative approach
112
(accompanied by antifungal lock therapy in combination with systemic antifungals)
113
when the risk of replacing the catheter is greater than the benefit [41]. Our data show
114
that physiological concentrations of micafungin of 4 µg/mL [42, 43] are active against
115
biofilm, since metabolic activity decreased and fungal regrowth was inhibited in most
116
of the preformed biofilms. Our in vitro observations also support the previous clinical
117
observations reported by Nucci et al. [40].
118
In summary, we provide new data on the in vitro antibiofilm activity of
119
micafungin against Candida strains from patients with candidemia. Although
120
micafungin MICs were higher for sessile cells than for planktonic cells, regrowth of the
121
cells forming the biofilm was reduced more than 80% at physiological serum
122
concentrations of 4 µg/mL. Our in vitro study supports that micafungin concentrations
123
≥2 µg/mL may be sufficiently high to eradicate Candida biofilms.
124
7
125 126 127 128
ACKNOWLEDGEMENTS
We thank Thomas O’Boyle for his help in the preparation of the manuscript.
129 130
Financial support. This work was supported by grants from Astellas Pharma, by grants
131
from the Fondo de Investigación Sanitaria (PI11/00167), and by Fundación MUTUA
132
Madrileña de Madrid. M. Guembe (CP13/00268), J. Guinea (MS09/00055), and L.J.
133
Marcos-Zambrano (FI12/00265) are supported by the Fondo de Investigación Sanitaria.
134 135
Potential conflicts of interest. The authors declare no conflicts of interest.
136
8
137
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277
FIGURE LEGENDS
278
Figure 1. Mean percentage of the reduction in metabolic activity and regrowth of
279
Candida biofilms after exposure to micafungin
280
Figure 1a. Mean percentage reduction in metabolic activity (fungal damage) of
281
Candida biofilms after exposure to micafungin
282
Figure 1b. Mean percentage inhibition of regrowth of Candida biofilms after exposure
283
to different concentrations of micafungin
284
Figure 2. Correlation between absorbance of Candida sessile cells and number of
285
cells/mL after exposure to micafungin
286
Figure 2a. Correlation between absorbance of C. albicans sessile cells and number of
287
cells/mL after exposure to micafungin
288
Figure 2b. Correlation between absorbance of C. parapsilosis sessile cells and number
289
of cells/mL after exposure to micafungin
290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310
15
Figure 1a 1a. Mean percentage reduction in metabolic activity (fungal damage) of Candida biofilms after exposure to micafungin 80 70
% of fungal dam mage
60 50 C. albicans
40
C. parapsilosis 30 20 10 0 0.015
0.03
0.06
0.12
0.25
0.5
1
2
Micafungin concentrations (µg/mL)
4
8
16
Figure 1b 1b. Mean percentage inhibition of regrowth of Candida biofilms after exposure to different concentrations of micafungin
100 90 80
60 50
C. albicans
40
C. parapsilosis
30 20 10
Micafungin concentrations (µg/mL)
16
8
4
2
1
0. 5
0. 25
0. 12
0. 06
0. 03
0
0. 01 5
%o of re-growth inhibittion
70
Figure 2a. Correlation between absorbance of C. albicans sessile cells and number of cells/mL after exposure to micafungin
Absorbance
No. cells/mL /
0,8
30000000
0,7
25000000
0,6 20000000
0,5 0,4
15000000
Absorbance Cells/mL
03 0,3
10000000
0,2 5000000
0,1
16
4
8
1
2
0
0. 01 5 0. 03 0. 06 0. 12 0. 25 0. 50
0 Micafungin concentration (µg/mL)
Figure 2b. Correlation between absorbance of C. parapsilosis sessile cells and number of cells/mL after exposure to micafungin
Absorbance
No. cells/mL
1,2
45000000 40000000
1
35000000 30000000
0,8
25000000
0,6
20000000
04 0,4
15000000 10000000
0,2
5000000 16
4
8
1
2
0
0. 01 5 0. 03 0. 06 0. 12 0. 25 0. 50
0 Micafungin concentration (µg/mL)
Absorbance Cells/mL
Table 1. Distribution of micafungin MICs for the 37 planktonic and sessile Candida isolates. Species (No.)
No. of isolates in each MIC (µg/mL) 0.015
0.031 0.062 0.125 0.25
0.5
1
2
4
8
16
>16
Planktonic
29
-
-
-
-
-
-
-
-
-
-
-
Sessile
14
-
-
1
4
-
-
3
1
1
3
2
Planktonic
1
-
-
-
-
-
6
1
-
-
-
-
Sessile
-
-
-
-
-
-
2
1
3
-
-
2
C. albicans (29)
C. parapsilosis (8)
