Journal of Medical Microbiology Papers in Press. Published February 5, 2015 as doi:10.1099/jmm.0.000030
Journal of Medical Microbiology Histoplasma capsulatum in planktonic and biofilm forms: in vitro susceptibility to amphotericin B, itraconazole and farnesol --Manuscript Draft-Manuscript Number:
JMM-D-14-00303R1
Full Title:
Histoplasma capsulatum in planktonic and biofilm forms: in vitro susceptibility to amphotericin B, itraconazole and farnesol
Short Title:
Antifungal susceptibility of H. capsulatum biofilm
Article Type:
Standard
Section/Category:
Antimicrobial agents and chemotherapy
Corresponding Author:
Raimunda Sâmia Nogueira Brilhante Federal University of Ceará Fortaleza, BRAZIL
First Author:
Raimunda Brilhante
Order of Authors:
Raimunda Brilhante Rita Amanda Chaves de Lima Francisca Jakelyne de Farias Marques Natalya Fechine Silva Erica Pacheco Caetano Débora de Souza Collares Maia Castelo-Branco Tereza de Jesus Pinheiro Gomes Bandeira José Luciano Bezerra Moreira Rossana de Aguiar Cordeiro André Jalles Monteiro Zoilo Pires de Camargo José Júlio Costa Sidrim Marcos Fábio Gadelha Rocha
Abstract:
It is believed that most microbial infections are caused by pathogens organized in biofilms. Recently, it was shown that the dimorphic fungus Histoplasma capsulatum, estimated to be the most common cause of fungal respiratory diseases, is also able to form biofilm. Although the antifungal therapy commonly used is effective, refractory cases and recurrences have been reported. In the search for new compounds with antimicrobial activity, the sesquiterpene farnesol has gained prominence for its antifungal action. This study aimed to evaluate the in vitro susceptibility of H. capsulatum var. capsulatum to the antifungal agents itraconazole and amphotericin B and farnesol, alone and combined, as well as to determine the in vitro antifungal activity of these compounds against biofilms of this pathogen. The results show that farnesol has antifungal activity against H. capsulatum in the yeast and filamentous phases, with MIC values ranging from 0.0078 to 0.00312 µM. A synergistic effect (FICI ≤ 0.5) between itraconazole and farnesol was found against 100% and 83.3% of the isolates in yeast and mycelial forms, respectively, while synergism between amphotericin B and farnesol was only observed against 37.5% and 44.4% of the isolates in yeast and filamentous forms, respectively. Afterwards, the antifungal drugs and farnesol alone and the combination of itraconazole and farnesol were tested against mature biofilms of H. capsulatum, through XTT metabolic assay, and the isolated antifungal drugs showed lower antibiofilm activity, when compared to farnesol alone and farnesol combined with itraconazole.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Brilhante et al., 2014 - JMM - Histoplasma Biofilm Final wi
1
1
Journal of Medical Microbiology.
2
Title: Histoplasma capsulatum in planktonic and biofilm forms: in vitro susceptibility
3
to amphotericin B, itraconazole and farnesol
4
Authors and affiliations: Raimunda Sâmia Nogueira Brilhante1,2*, Rita Amanda
5
Chaves de Lima1, Francisca Jakelyne de Farias Marques1, Natalya Fechine Silva2, Érica
6
Pacheco Caetano1, Débora de Souza Collares Maia Castelo-Branco1, Tereza de Jesus
7
Pinheiro Gomes Bandeira1,4, José Luciano Bezerra Moreira1, Rossana de Aguiar
8
Cordeiro1, André Jalles Monteiro5, Zoilo Pires de Camargo6, José Júlio Costa Sidrim1,2,
9
Marcos Fábio Gadelha Rocha1,3.
10
1
11
Microbiology, Federal University of Ceará, Fortaleza, Ceará, Brazil
12
2
13
Ceará, Brazil.
14
3
15
Ceará, Brazil.
16
4
17
5
18
Fortaleza, Ceará, Brazil.
19
6
20
São Paulo, São Paulo, Brazil
21
Running Title: Antifungal susceptibility of H. capsulatum biofilm
22
*Corresponding Author: R.S.N. Brilhante. Rua Barão de Canindé, 210; Montese.
23
CEP: 60.425-540. Fortaleza, CE, Brazil. Fax: 55 (85) 3295-1736 E mail:
24
[email protected]
25
Specialized Medical Mycology Center, Postgraduate Program in Medical
Postgraduate Program in Medical Sciences, Federal University of Ceará, Fortaleza,
Postgraduate Program in Veterinary Sciences, State University of Ceará, Fortaleza,
School of Medicine, Christus College, Fortaleza, Ceará, Brazil. Department of Statistics and Applied Mathematics, Federal University of Ceará,
Department of Microbiology, Immunology and Parasitology, Federal University of
2
26
Abstract
27
It is believed that most microbial infections are caused by pathogens organized in
28
biofilms. Recently, it was shown that the dimorphic fungus Histoplasma capsulatum,
29
estimated to be the most common cause of fungal respiratory diseases, is also able to
30
form biofilm. Although the antifungal therapy commonly used is effective, refractory
31
cases and recurrences have been reported. In the search for new compounds with
32
antimicrobial activity, the sesquiterpene farnesol has gained prominence for its
33
antifungal action. This study aimed to evaluate the in vitro susceptibility of H.
34
capsulatum var. capsulatum to the antifungal agents itraconazole and amphotericin B
35
and farnesol, alone and combined, as well as to determine the in vitro antifungal activity
36
of these compounds against biofilms of this pathogen. The results show that farnesol
37
has antifungal activity against H. capsulatum in the yeast and filamentous phases, with
38
MIC values ranging from 0.0078 to 0.00312 µM. A synergistic effect (FICI ≤ 0.5)
39
between itraconazole and farnesol was found against 100% and 83.3% of the isolates in
40
yeast and mycelial forms, respectively, while synergism between amphotericin B and
41
farnesol was only observed against 37.5% and 44.4% of the isolates in yeast and
42
filamentous forms, respectively. Afterwards, the antifungal drugs and farnesol alone and
43
the combination of itraconazole and farnesol were tested against mature biofilms of H.
44
capsulatum, through XTT metabolic assay, and the isolated antifungal drugs showed
45
lower antibiofilm activity, when compared to farnesol alone and farnesol combined with
46
itraconazole. In conclusion, farnesol showed promising results as an antifungal agent
47
against H. capsulatum and also showed adjuvant action, especially when combined with
48
itraconazole, increasing the fungal susceptibility to this drug.
49
Keywords: farnesol, H. capsulatum, biofilm, susceptibility test
50
3
51
1. Introduction
52
Microorganisms in biofilms grow in multicellular communities and produce an
53
extracellular matrix that in nature provides a protective niche against aggressive
54
environmental factors such as changes in pH, osmolarity and dehydration, as well as
55
exposure to predators, chemical agents, among others (Mittelman, 1998). In the host,
56
biofilms have been shown to play critical role in the development of infection, being
57
considered an important virulence factor (Phillips & Schultz, 2012). The association in
58
biofilms has a great medical importance as it confers greater resistance to specific
59
mediators of the immune response and increased resistance to antimicrobial agents. This
60
is the main reason why biofilm-associated infections are often refractory to
61
conventional antibiotic therapy (Martinez & Fries, 2010).
62
Currently, it is believed that most microbial infections in humans are caused by
63
pathogens organized in biofilms. Therefore, research in mycology has increasingly
64
focused on biofilm phenotyping (Martinez & Fries, 2010). A wide variety of clinically
65
important fungi have demonstrated the ability to colonize surfaces and form biofilms,
66
including the genera Candida (Ganguly & Mitchell, 2011), Aspergillus (Müller et al.,
67
2011), Coccidioides (Davis et al., 2002), Cryptococcus (Martinez et al., 2008),
68
Pneumocystis (Cushion et al., 2009) and Trichosporon (Di Bonaventura et al., 2006).
69
Recently, it was shown that the dimorphic fungi Histoplasma capsulatum, estimated to
70
be the most common cause of fungal respiratory diseases, is also able to form biofilm in
71
vitro(Pitangui et al., 2012).
72
In addition, a possible association between the ability of H. capsulatum to form
73
biofilms and the pattern of yeast adhesion to epithelial cells was also described.
74
Verstrepen and Klis (2006) emphasized that the ability of biofilm formation is
4
75
associated with the adhesion property of some fungi. The pattern of infection of H.
76
capsulatum in epithelial cells was characterized as a compact mass of yeast cells, which
77
possibly leads to the formation of a complex three-dimensional architecture of biofilms
78
and promotes the internalization of yeast into host cells. It is believed that this finding
79
could be related to the ability of the H. capsulatum yeasts to avoid the immune system
80
cells (Pitangui et al., 2012). It is noteworthy that no information on the antifungal
81
susceptibility of H. capsulatum biofilms has been reported.
82
Histoplasmosis treatment depends on the severity of the disease and risk factors
83
inherent to the patient. In general, the therapy indicated for mild to moderate cases is to
84
prescribe azoles such as itraconazole, while the use of amphotericin B is indicated in
85
serious cases. Although the antifungal therapy commonly used is usually effective,
86
refractory cases and relapses have been described (Wheat et al., 2009). Many studies
87
have sought new compounds with antimicrobial activity. In this perspective, the
88
sesquiterpene farnesol has gained prominence due to its high antimicrobial activity
89
against bacteria and fungi (Inoue et al., 2004; Jabra-Rizk et al., 2006; Brilhante et al.,
90
2012; Brilhante et al., 2013). Its potential antifungal activity has already been
91
demonstrated against different species of Candida (Cordeiro et al., 2013) and
92
Cryptococcus (Cordeiro et al., 2012), as well as against Coccidioides posadasii
93
(Brilhante et al., 2013) and Paracoccidioides brasiliensis (Derengowski et al., 2009).
94
Given the above, the present study aimed to evaluate the in vitro susceptibility of
95
Histoplasma capsulatum var. capsulatum to the antifungals itraconazole and
96
amphotericin B and the compound farnesol, alone and combined, as well as to
97
determine the in vitro antifungal activity of these compounds against H. capsulatum var.
98
capsulatum biofilms.
5
99
100
2. Material and methods 2.1 Fungal culture
101
A total of 18 isolates of H. capsulatum var. capsulatum (18 in filamentous phase
102
and 8 in yeast phase) were included in this study, all belonging to the Fungal Culture
103
Collection of the Specialized Medical Mycology Center (CEMM, Federal University of
104
Ceará, Brazil). The procedures were performed in a class II biological safety cabinet in
105
a biosafety level 3 laboratory.
106
2.2 Antimicrobial agents
107
The stock solutions of the tested antimicrobial agents, itraconazole, amphotericin
108
B and farnesol (Sigma-Aldrich Co, USA), were prepared with DMSO (Sigma-Aldrich
109
Co, USA), as solvent, according to the method M27-A3, preconized by CLSI.
110
Subsequently, the solutions were prepared in RPMI 1640 medium with L-glutamine
111
buffered to pH 7.0 with 0.165M MOPS. The concentration range tested for these drugs
112
alone was from 0.0004 to 0.5 µg/mL for itraconazole, from 0.0039 to 2 µg/mL for
113
amphotericin B and from 0.0009 to 0.5 µM for farnesol. MIC values obtained with
114
individual drugs were used to determine the highest concentration of these drugs tested
115
in combination. Thus, in combination the concentration ranges varied from FICI
139
Journal of Medical Microbiology Histoplasma capsulatum in planktonic and biofilm forms: in vitro susceptibility to amphotericin B, itraconazole and farnesol --Manuscript Draft-Manuscript Number:
JMM-D-14-00303R1
Full Title:
Histoplasma capsulatum in planktonic and biofilm forms: in vitro susceptibility to amphotericin B, itraconazole and farnesol
Short Title:
Antifungal susceptibility of H. capsulatum biofilm
Article Type:
Standard
Section/Category:
Antimicrobial agents and chemotherapy
Corresponding Author:
Raimunda Sâmia Nogueira Brilhante Federal University of Ceará Fortaleza, BRAZIL
First Author:
Raimunda Brilhante
Order of Authors:
Raimunda Brilhante Rita Amanda Chaves de Lima Francisca Jakelyne de Farias Marques Natalya Fechine Silva Erica Pacheco Caetano Débora de Souza Collares Maia Castelo-Branco Tereza de Jesus Pinheiro Gomes Bandeira José Luciano Bezerra Moreira Rossana de Aguiar Cordeiro André Jalles Monteiro Zoilo Pires de Camargo José Júlio Costa Sidrim Marcos Fábio Gadelha Rocha
Abstract:
It is believed that most microbial infections are caused by pathogens organized in biofilms. Recently, it was shown that the dimorphic fungus Histoplasma capsulatum, estimated to be the most common cause of fungal respiratory diseases, is also able to form biofilm. Although the antifungal therapy commonly used is effective, refractory cases and recurrences have been reported. In the search for new compounds with antimicrobial activity, the sesquiterpene farnesol has gained prominence for its antifungal action. This study aimed to evaluate the in vitro susceptibility of H. capsulatum var. capsulatum to the antifungal agents itraconazole and amphotericin B and farnesol, alone and combined, as well as to determine the in vitro antifungal activity of these compounds against biofilms of this pathogen. The results show that farnesol has antifungal activity against H. capsulatum in the yeast and filamentous phases, with MIC values ranging from 0.0078 to 0.00312 µM. A synergistic effect (FICI ≤ 0.5) between itraconazole and farnesol was found against 100% and 83.3% of the isolates in yeast and mycelial forms, respectively, while synergism between amphotericin B and farnesol was only observed against 37.5% and 44.4% of the isolates in yeast and filamentous forms, respectively. Afterwards, the antifungal drugs and farnesol alone and the combination of itraconazole and farnesol were tested against mature biofilms of H. capsulatum, through XTT metabolic assay, and the isolated antifungal drugs showed lower antibiofilm activity, when compared to farnesol alone and farnesol combined with itraconazole.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Brilhante et al., 2014 - JMM - Histoplasma Biofilm Final wi
1
1
Journal of Medical Microbiology.
2
Title: Histoplasma capsulatum in planktonic and biofilm forms: in vitro susceptibility
3
to amphotericin B, itraconazole and farnesol
4
Authors and affiliations: Raimunda Sâmia Nogueira Brilhante1,2*, Rita Amanda
5
Chaves de Lima1, Francisca Jakelyne de Farias Marques1, Natalya Fechine Silva2, Érica
6
Pacheco Caetano1, Débora de Souza Collares Maia Castelo-Branco1, Tereza de Jesus
7
Pinheiro Gomes Bandeira1,4, José Luciano Bezerra Moreira1, Rossana de Aguiar
8
Cordeiro1, André Jalles Monteiro5, Zoilo Pires de Camargo6, José Júlio Costa Sidrim1,2,
9
Marcos Fábio Gadelha Rocha1,3.
10
1
11
Microbiology, Federal University of Ceará, Fortaleza, Ceará, Brazil
12
2
13
Ceará, Brazil.
14
3
15
Ceará, Brazil.
16
4
17
5
18
Fortaleza, Ceará, Brazil.
19
6
20
São Paulo, São Paulo, Brazil
21
Running Title: Antifungal susceptibility of H. capsulatum biofilm
22
*Corresponding Author: R.S.N. Brilhante. Rua Barão de Canindé, 210; Montese.
23
CEP: 60.425-540. Fortaleza, CE, Brazil. Fax: 55 (85) 3295-1736 E mail:
24
[email protected]
25
Specialized Medical Mycology Center, Postgraduate Program in Medical
Postgraduate Program in Medical Sciences, Federal University of Ceará, Fortaleza,
Postgraduate Program in Veterinary Sciences, State University of Ceará, Fortaleza,
School of Medicine, Christus College, Fortaleza, Ceará, Brazil. Department of Statistics and Applied Mathematics, Federal University of Ceará,
Department of Microbiology, Immunology and Parasitology, Federal University of
2
26
Abstract
27
It is believed that most microbial infections are caused by pathogens organized in
28
biofilms. Recently, it was shown that the dimorphic fungus Histoplasma capsulatum,
29
estimated to be the most common cause of fungal respiratory diseases, is also able to
30
form biofilm. Although the antifungal therapy commonly used is effective, refractory
31
cases and recurrences have been reported. In the search for new compounds with
32
antimicrobial activity, the sesquiterpene farnesol has gained prominence for its
33
antifungal action. This study aimed to evaluate the in vitro susceptibility of H.
34
capsulatum var. capsulatum to the antifungal agents itraconazole and amphotericin B
35
and farnesol, alone and combined, as well as to determine the in vitro antifungal activity
36
of these compounds against biofilms of this pathogen. The results show that farnesol
37
has antifungal activity against H. capsulatum in the yeast and filamentous phases, with
38
MIC values ranging from 0.0078 to 0.00312 µM. A synergistic effect (FICI ≤ 0.5)
39
between itraconazole and farnesol was found against 100% and 83.3% of the isolates in
40
yeast and mycelial forms, respectively, while synergism between amphotericin B and
41
farnesol was only observed against 37.5% and 44.4% of the isolates in yeast and
42
filamentous forms, respectively. Afterwards, the antifungal drugs and farnesol alone and
43
the combination of itraconazole and farnesol were tested against mature biofilms of H.
44
capsulatum, through XTT metabolic assay, and the isolated antifungal drugs showed
45
lower antibiofilm activity, when compared to farnesol alone and farnesol combined with
46
itraconazole. In conclusion, farnesol showed promising results as an antifungal agent
47
against H. capsulatum and also showed adjuvant action, especially when combined with
48
itraconazole, increasing the fungal susceptibility to this drug.
49
Keywords: farnesol, H. capsulatum, biofilm, susceptibility test
50
3
51
1. Introduction
52
Microorganisms in biofilms grow in multicellular communities and produce an
53
extracellular matrix that in nature provides a protective niche against aggressive
54
environmental factors such as changes in pH, osmolarity and dehydration, as well as
55
exposure to predators, chemical agents, among others (Mittelman, 1998). In the host,
56
biofilms have been shown to play critical role in the development of infection, being
57
considered an important virulence factor (Phillips & Schultz, 2012). The association in
58
biofilms has a great medical importance as it confers greater resistance to specific
59
mediators of the immune response and increased resistance to antimicrobial agents. This
60
is the main reason why biofilm-associated infections are often refractory to
61
conventional antibiotic therapy (Martinez & Fries, 2010).
62
Currently, it is believed that most microbial infections in humans are caused by
63
pathogens organized in biofilms. Therefore, research in mycology has increasingly
64
focused on biofilm phenotyping (Martinez & Fries, 2010). A wide variety of clinically
65
important fungi have demonstrated the ability to colonize surfaces and form biofilms,
66
including the genera Candida (Ganguly & Mitchell, 2011), Aspergillus (Müller et al.,
67
2011), Coccidioides (Davis et al., 2002), Cryptococcus (Martinez et al., 2008),
68
Pneumocystis (Cushion et al., 2009) and Trichosporon (Di Bonaventura et al., 2006).
69
Recently, it was shown that the dimorphic fungi Histoplasma capsulatum, estimated to
70
be the most common cause of fungal respiratory diseases, is also able to form biofilm in
71
vitro(Pitangui et al., 2012).
72
In addition, a possible association between the ability of H. capsulatum to form
73
biofilms and the pattern of yeast adhesion to epithelial cells was also described.
74
Verstrepen and Klis (2006) emphasized that the ability of biofilm formation is
4
75
associated with the adhesion property of some fungi. The pattern of infection of H.
76
capsulatum in epithelial cells was characterized as a compact mass of yeast cells, which
77
possibly leads to the formation of a complex three-dimensional architecture of biofilms
78
and promotes the internalization of yeast into host cells. It is believed that this finding
79
could be related to the ability of the H. capsulatum yeasts to avoid the immune system
80
cells (Pitangui et al., 2012). It is noteworthy that no information on the antifungal
81
susceptibility of H. capsulatum biofilms has been reported.
82
Histoplasmosis treatment depends on the severity of the disease and risk factors
83
inherent to the patient. In general, the therapy indicated for mild to moderate cases is to
84
prescribe azoles such as itraconazole, while the use of amphotericin B is indicated in
85
serious cases. Although the antifungal therapy commonly used is usually effective,
86
refractory cases and relapses have been described (Wheat et al., 2009). Many studies
87
have sought new compounds with antimicrobial activity. In this perspective, the
88
sesquiterpene farnesol has gained prominence due to its high antimicrobial activity
89
against bacteria and fungi (Inoue et al., 2004; Jabra-Rizk et al., 2006; Brilhante et al.,
90
2012; Brilhante et al., 2013). Its potential antifungal activity has already been
91
demonstrated against different species of Candida (Cordeiro et al., 2013) and
92
Cryptococcus (Cordeiro et al., 2012), as well as against Coccidioides posadasii
93
(Brilhante et al., 2013) and Paracoccidioides brasiliensis (Derengowski et al., 2009).
94
Given the above, the present study aimed to evaluate the in vitro susceptibility of
95
Histoplasma capsulatum var. capsulatum to the antifungals itraconazole and
96
amphotericin B and the compound farnesol, alone and combined, as well as to
97
determine the in vitro antifungal activity of these compounds against H. capsulatum var.
98
capsulatum biofilms.
5
99
100
2. Material and methods 2.1 Fungal culture
101
A total of 18 isolates of H. capsulatum var. capsulatum (18 in filamentous phase
102
and 8 in yeast phase) were included in this study, all belonging to the Fungal Culture
103
Collection of the Specialized Medical Mycology Center (CEMM, Federal University of
104
Ceará, Brazil). The procedures were performed in a class II biological safety cabinet in
105
a biosafety level 3 laboratory.
106
2.2 Antimicrobial agents
107
The stock solutions of the tested antimicrobial agents, itraconazole, amphotericin
108
B and farnesol (Sigma-Aldrich Co, USA), were prepared with DMSO (Sigma-Aldrich
109
Co, USA), as solvent, according to the method M27-A3, preconized by CLSI.
110
Subsequently, the solutions were prepared in RPMI 1640 medium with L-glutamine
111
buffered to pH 7.0 with 0.165M MOPS. The concentration range tested for these drugs
112
alone was from 0.0004 to 0.5 µg/mL for itraconazole, from 0.0039 to 2 µg/mL for
113
amphotericin B and from 0.0009 to 0.5 µM for farnesol. MIC values obtained with
114
individual drugs were used to determine the highest concentration of these drugs tested
115
in combination. Thus, in combination the concentration ranges varied from FICI
139
