Fungal Biology 122 (2018) 563e569

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Fungal Biology
journal homepage: www.elsevier.com/locate/funbio

Species of the Metarhizium anisopliae complex with diverse ecological

niches display different susceptibilities to antifungal agents
Guilherme T.P. Brancini a, Ludmilla Tonani a, Drauzio E.N. Rangel b, Donald W. Roberts c,
Gilberto U.L. Braga a, *
a
Departamento de Ana
lises Clínicas, Toxicolo

gicas e Bromatolo

gicas, Faculdade de Ci^
encias Farmac^ ~o Preto, Universidade de Sa
euticas de Ribeira ~o Paulo,
Ribeira~o Preto, SP 14040-903, Brazil
b
Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goia
s, Goia
^nia, GO 74605-050, Brazil
c
Department of Biology, Utah State University, Logan, UT 84322-5305, USA

a r t i c l e i n f o a b s t r a c t

Article history: Species of the Metarhizium anisopliae complex are globally ubiquitous soil-inhabiting and predominantly
Received 19 October 2017 insect-pathogenic fungi. The Metarhizium genus contains species ranging from specialists, such as
Received in revised form Metarhizium acridum that only infects acridids, to generalists, such as M. anisopliae, Metarhizium brun-
4 December 2017
neum, and Metarhizium robertsii that infect a broad range of insects and can also colonize plant roots.
Accepted 6 December 2017
There is little information available about the susceptibility of Metarhizium species to clinical and non-
Available online 14 December 2017
clinical antifungal agents. We determined the susceptibility of 16 isolates comprising four Metarhizium
Corresponding Editor: Simon V. Avery species with different ecological niches to seven clinical (amphotericin B, ciclopirox olamine, fluconazole,
griseofulvin, itraconazole, tebinafine, and voriconazole) and one non-clinical (benomyl) antifungal
Keywords: agents. All isolates of the specialist M. acridum were clearly more susceptible to most antifungals than the
Antifungal susceptibility isolates of the generalists M. anisopliae sensu lato, M. brunneum, and M. robertsii. All isolates of
Insect-pathogenic fungi M. anisopliae, M. brunneum, and M. robertsii were resistant to fluconazole and some were also resistant to
Metarhizium acridum amphotericin B. The marked differences in susceptibility between the specialist M. acridum and the
Metarhizium brunneum
generalist Metarhizium species suggest that this characteristic is associated with their different ecological
Metarhizium robertsii
niches, and may assist in devising rational antifungal treatments for the rare cases of mycoses caused by
Metarhizium species.
© 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

1. Introduction Barelli et al., 2016). A phylogenomic analysis revealed that Meta-

rhizium is related to the mutualistic plant endophyte fungus Epi-
The taxonomy of the genus Metarhizium has been continuously chloe festucae [divergence time 88e114 million years (MY)] and to
revised through multilocus phylogenetic analyses, which allowed the wheat head blight fungus Fusarium graminearum (divergence
the description of novel species and definition of novel species time 144e187 MY) suggesting a possible origin of plant association
limits (Bishoff et al., 2009; Kepler et al., 2014; Chen et al., 2017; in this genus (Spatafora et al., 2007; Gao et al., 2011). Phylogenetic
Rehner and Kepler, 2017). Species of the Metarhizium anisopliae- studies are helping to elucidate the ecology and life histories of
group have cosmopolitan distribution and exhibit great metabolic Metarhizium-group species as entomopathogens and/or endo-
and ecologic versatility (Roberts and St. Leger, 2004; St. Leger, 2008; phytes soil-adapted fungi (Gao et al., 2011; Hu et al., 2014; Rehner
Behie et al., 2012; Hu et al., 2014; Lacey et al., 2015; Rehner and and Kepler, 2017). The genus Metarhizium contains species ranging
Kepler, 2017). The majority of Metarhizium species are entomo- from specialists, such as Metarhizium acridum and Metarhizium
pathogens that can also colonize plant roots (Hu and St. Leger, album, with very narrow host ranges, to generalists, such as
2002; St. Leger, 2008; Wyrebek et al., 2011; Liao et al., 2014; M. anisopliae, Metarhizium brunneum, and Metarhizium robertsii,
that parasite a broad range of insects representing more than seven
orders (Hu et al., 2014). Generalist species such as M. robertsii,
M. anisopliae, and M. brunneum can also colonize the roots of plants
* Corresponding author. Fax: þ55 16 3315 4723. and are common components of the rhizosphere (St. Leger, 2008;
E-mail address: gbraga@fcfrp.usp.br (G.U.L. Braga).

https://doi.org/10.1016/j.funbio.2017.12.004
1878-6146/© 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
564 G.T.P. Brancini et al. / Fungal Biology 122 (2018) 563e569

Vega et al., 2009; Behie et al., 2012; Liao et al., 2014). Hu et al. (2014) The aim of this study was to determine the susceptibilities of 16
used genomic analyses of seven Metarhizium species to demon- isolates spanning four species of Metarhizium (M. acridum,
strate that generalists evolved from specialists via transitional M. anisopliae, M. brunneum, and M. robertsii) to eight antifungal
species with intermediate host ranges. Besides the insect speciali- agents with different mechanisms of action. Two model ascomy-
zation, a plant-rhizosphere-specific association was also described cetes, Aspergillus nidulans and Aspergillus flavus, were also included
in Metarhizium species (Wyrebek et al., 2011) and plant relation- in the susceptibility tests. Differences in the susceptibility profiles
ships, rather than insect host, have been suggested as major driving of the Metarhizium species were discussed in the light of their
factor in the divergence of the genus Metarhizium (Wyrebek and diverse ecological niches and evolutionary stories.
Bidochka, 2013; Barelli et al., 2016).
Metarhizium spp. have been used almost worldwide to control 2. Materials and methods
agricultural insect pests and disease vectors (Lacey et al., 2015).
Licensed commercial products containing conidia and/or mycelia 2.1. Fungal species and isolates
of the fungus are available for application in the field for control
of insect and tick pests, and in-house use against insects such as Metarhizium isolates were obtained from the United States
cockroaches, flies, fleas, and termites (Roberts and St. Leger, Department of Agriculture/Agricultural Research Service (USDA/
2004; Samish et al., 2004; Faria and Wraight, 2007; Lacey et al., ARS) Collection of Entomopathogenic Fungal Cultures (ARSEF),
2015). Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA
Although Metarhizium species were previously not considered and from the Department of Entomology Collection of Entomopa-
pathogenic for humans or domestic animals, starting in the late thogenic Fungal Cultures, Universidade de S~ ao Paulo, Piracicaba,
1990's rare cases of mycosis caused by these fungi in both S~
ao Paulo, Brazil. Three fungal isolates were obtained from the
immunocompromised and immunocompetent individuals have American Type Culture Collection (ATCC), Manassas, VA. Candida
been reported. An extensive literature review showed that only 19 krusei ATCC 6258 was used as a quality-control strain; and A. flavus
human and one cat occurrences of Metarhizium spp. infections ATCC 204304 and A. nidulans ATCC 10074 were used as reference
have been reported between 1997 and 2015 (Nourrisson et al., strains. The geographic origins of the Metarhizium isolates and the
2017). For example, a case of invasive and disseminated mycosis substrates/insects from which they were isolated are listed in
caused by Metarhizium guizhouense was described in an immu- Table 1.
nocompromised child in Australia (Burgner et al., 1998); two cases
of sinusitis caused by M. anisopliae sensu lato were reported in 2.2. Antifungal agents
immunocompetent individuals (Revankar et al., 1999) and a case of
invasive rhinitis caused by M. anisopliae sensu lato was reported in Standard powders of amphotericin B, benomyl [methyl 1-
a cat (Muir et al., 1998). Skin- and eye-related infections include: a (butylcarbamoyl)-2-benzimidazolecarbamate], ciclopirox olamine,
case of keratitis caused by M. anisopliae sensu lato in an immu- fluconazole, griseofulvin, itraconazole, and terbinafine were ob-
nocompetent female patient (Jani et al., 2001); a case of keratitis tained from Sigma (Sigma Chemical Co., St. Louis, MO) and vor-
caused by M. anisopliae in an immunocompetent young man in iconazole from Pfizer (Pfizer Pharmaceutical, New York, NY).
Colombia (Cepero de Garcia et al., 1997); a case of a disseminated
skin infection in an immunocompromised adult patient (Osorio 2.3. Conidia production
et al., 2007); a cutaneous infection with M. anisopliae in a patient
with hypohidrotic ectodermal dysplasia and immunodeficiency Fungi were grown on 23 mL of PDAY [potato dextrose agar
(Marsh et al., 2008); and a case of sclerokeratitis caused by medium (PDA) (Acumedia, Lansing, MI) supplemented with 1 g L1
M. anisopliae in a 76-year-old patient (Eguchi et al., 2015). Recently, yeast extract (Difco Laboratories, Detroit, MI)], pH 5.5, in 100-mm
Nourrisson et al. (2017) reported eight new cases of Metarhizium Petri dishes in the dark, at 28  C for 5 d (A. nidulans and A. flavus)
infection in humans (three cases from France and five from or 12 d (Metarhizium). For inoculum preparation, conidia were
Australia). The strains isolated from these eight cases and three harvested, suspended in 0.01 % (v/v) Tween 80® (Sigma) solution
others from cases that had already been published and reported as and the concentration of the suspension was adjusted to
M. anisopliae were molecularly identified as M. robertsii (six), 106 conidia mL1 (based on hemocytometer counts).
M. guizhouense (three), M. brunneum (one) and M. pingshaense
(one). In none of these cases mycosis was associated with the use 2.4. Antifungal susceptibility testing
of the fungi as a bioinsecticide.
The increased exposure of humans and animals to the fungus 2.4.1. Minimal inhibitory concentration (MIC) determination
due to its commercial utilization and the growing number of Antifungal susceptibility testing of C. krusei was performed by
immunocompromised individuals have created a scenario that may the reference method for broth dilution antifungal susceptibility
favor an increase in the occurrence of mycosis caused by Meta- testing of yeasts described in the Clinical and Laboratory Standards
rhizium spp. (Roberts and St. Leger, 2004; Nourrisson et al., 2017). Institute (CLSI) document M27-A3 (CLSI, 2008b) and susceptibility
Unfortunately, there is currently little information available about testing of Metarhizium spp., A. flavus, and A. nidulans was performed
the susceptibility of Metarhizium species and its isolates, including based on the CLSI M38-A2 protocol (CLSI, 2008a). Antifungal
commercial strains, to antifungals and no antifungal recommen- powders were diluted at concentrations 100 fold higher than the
dations have been proposed to these fungi (Nourrisson et al., 2017). final concentrations in 100 % dimethyl sulfoxide (Sigma), followed
Determining the antifungal susceptibility profiles of the isolates of by further dilution (1:50) in the CLSI standard RPMI 1640 medium
Metarhizium species is not only important to guide the therapy in without bicarbonate (GIBCO, Grand Island, NY) buffered to pH 7.0
the rare cases of mycoses caused by these fungi, but also to design with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma). The
better selective media and to choose appropriate selection markers concentrations of all antifungal drugs ranged from 0.01 to
in transformation experiments with Metarhizium spp., since most 100 mg mL1. Conidial suspensions were diluted 1:50 in RPMI to a
markers used are genes that confer resistance to antifungal agents final inoculum concentration of 2  104 conidia mL1. Wells of 96-
(Goettel et al., 1990; Valadares-Inglis and Inglis, 1997; Cao et al., well microdilution plates were loaded with 100 mL of 2  drug
2007; Duarte et al., 2007). preparation, to which 100 mL of the conidial inoculum suspensions
 G.T.P. Brancini et al. / Fungal Biology 122 (2018) 563e569 565

Table 1 3. Results
Fungal species and isolates used in this study.

Specie/isolate Host/substrate Geographic origin The MIC values for the quality control strain C. krusei ATCC 6258
Aspergillus flavus
and for the reference strain A. flavus ATCC 204304 were within the
ATCC 204304 Human sputum USA expected values (Barry et al., 2000; CLSI, 2008b; Espinel-Ingroff
Aspergillus nidulans et al., 2011). The MIC of itraconazole and amphotericin B for the
ATCC 10074 Unknown Belgium A. nidulans ATCC 10074 were within the ranges reported for clinical
Metarhizium robertsii
and non-clinical isolates of A. nidulans (Table 2) (Espinel-Ingroff,
ARSEF 23 Conoderus sp. USA
[Coleoptera: Elateridae] 2001a,b; Araujo et al., 2007; Espinel-Ingroff, 2008; Espinel-Ingroff
ARSEF 2575 Curculio caryae USA et al., 2010; Espinel-Ingroff et al., 2011).
[Coleoptera: Curculionidae] All the isolates of M. anisopliae, M. anisopliae sensu lato,
Metarhizium brunneum
M. brunneum, and M. robertsii were more resistant to ciclopirox
ARSEF 1095 Carpocapsa pomonella Austria
[Lepidoptera: Olethreutidae]
olamine and to the triazoles fluconazole and itraconazole than the
Metarhizium anisopliae sensu lato isolates of M. acridum (Table 2). Most of the isolates of M. acridum
ARSEF 5749 Schistocerca piceifrons Mexico were also less resistant to amphotericin B and benomyl than the
[Orthoptera: Acrididae] isolates of the other Metarhizium spp. All the M. brunneum (MIC-
0 ¼ 30 mg mL1), M. robertsii (MIC-0 ¼ 67 mg mL1), and
Metarhizium anisopliae
ESALQ 9 Mahanarva posticata Brazil
[Homoptera: Cercopidae] M. anisopliae sensu lato ARSEF 5749 (MIC-0 ¼ 57 mg mL1) isolates
ESALQ 865 [Homoptera] Brazil were much more resistant to amphotericin B than the M. anisopliae
ESALQ 866 Atta sp. Brazil isolates (MIC-0  2 mg mL1). Griseofulvin did not completely
[Hymenoptera: Formicidae] inhibit the growth of any isolate of Metarhizium spp. (MIC-
ESALQ 1037 Solenopsis invicta Brazil
[Hymenoptera: Formicidae]
0 > 100 mg mL1 for all isolates). However, based on MIC-50 for
ESALQ 1104 Soil Brazil griseofulvin, all the Metarhizium spp. isolates (MIC-
ESALQ PL43 Mahanarva posticata Brazil 50 ¼ 4e6 mg mL1) were much less resistant than A. nidulans and
[Homoptera: Cercopidae] A. flavus isolates (MIC-50 > 100 mg mL1). Isolates of all species
were susceptible to terbinafine (MIC-0 ¼ 0.1e0.5 mg mL1).
Metarhizium acridum
ARSEF 324 Austracris guttulosa Australia
[Orthoptera: Acrididae] Upon germination analysis, amphotericin B, griseofulvin, and
ARSEF 3391 Zonocerus elegans Tanzania the triazoles fluconazole, itraconazole, and voriconazole did not
[Orthoptera: Pyrgomorphidae] inhibit the germination of any Metarhizium isolate. Ciclopirox ola-
ARSEF 3609 Patanga succincta Thailand mine was the only drug that inhibited the germination of all Met-
[Orthoptera: Acrididae]
ARSEF 5734 [Orthoptera: Acrididae] Madagascar
arhizium isolates tested and of Aspergillus. Griseofulvin did not
ARSEF 6421 Kraussaria angulifera Senegal inhibit the germination of any of the Metarhizium or A. nidulans
[Orthoptera: Acrididae] isolates (data not shown). Benomyl had a more pronounced effect
ARSEF 7486 Ornithacris cavroisi Niger on the germination of M. acridum than on the other Metarhizium
[Orthoptera: Acrididae]
species. At a concentration of 1 mg mL1 benomyl, conidia of
M. acridum presented germ tubes of increased thickness when
compared to those on media without benomyl (Fig S1). This is
probably a consequence of the antifungal actin-binding mechanism
(2  104 conidia mL1) was added. Growth and sterility controls of action.
were included for each strain tested. Microdilution plates were Based on MFC analysis, itraconazole killed all the M. acridum
incubated at 28  C, in the dark, and visually examined after 72 h of (MFC ¼ 0.5 mg mL1 for all isolates), A. nidulans and A. flavus
incubation. The Minimal Inhibitory Concentration (MIC)-0 was (MFC ¼ 0.5 mg mL1 for both) isolates, but did not kill the isolates of
defined as the lowest concentration that prevented any discernible other Metarhizium species (MFC > 100 mg mL1 for most of the
growth, or at least 90 % reduction in the growth relative to growth isolates). Amphotericin B killed all the isolates of M. acridum and
of the untreated control. The wells in which mycelial growth was M. anisopliae but not M. anisopliae sensu lato isolate ARSEF 5749,
fully inhibited were observed with an inverted microscope M. brunneum, and M. robertsii isolates (Table 2). Griseofunvin had
(400  magnification) to confirm that conidia had not germinated. no fungicidal activity against either Metarhizium or Aspergillus
When total growth inhibition was not observed, an MIC, designated species. Ciclopirox olamine was fungicidal to all species tested.
MIC-50, was defined as the lowest concentration that caused
approximately 50 % or more reduction in growth relative to the 4. Discussion
control. The mean MIC of three independent experiments was used
to compare the susceptibilities of the various isolates. Interpreta- We tested the susceptibilities of 16 non-clinical wild-type iso-
tive MIC breakpoints for Metarhizium species have not been defined lates of entomopathogenic fungi comprising four Metarhizium
by CLSI. Fungi were classified according to obtained MIC values species with different ecological niches to eight antifungal agents
as follows: susceptible when MIC 1 mg mL1, intermediate when with different mechanisms of action. In terms of antifungal class,
MIC ¼ 2 mg mL1, and resistant when MIC 2 mg mL1 (CLSI, 2008a). our analysis comprised polyenes (amphotericin B), hydroxypyr-
idones (ciclopirox olamine), benzofurans (griseofulvin), allylamines
2.4.2. Minimal fungicidal concentration (MFC) determination (terbinafine), benzimidazoles (benomyl), and triazoles (flucona-
The in vitro fungicidal activity of each agent was determined by zole, itraconazole, and voriconazole). Seven of the antifungals have
the Minimal fungicidal concentration (MFC) method as described been used for therapeutic purposes and one, benomyl, to control
by Espinel-Ingroff (1998). Briefly, 10 ml from each well that showed phytopathogenic fungi and as a research tool in fungal genetics and
complete inhibition were spread on PDAY plates. Then, plates were cell biology (Odds et al., 2003; Hsueh et al., 2005; Rathinasamy and
incubated at 28  C and examined after 3 d for fungal growth. The Panda, 2006). Isolates of the insect generalists and rhizosphere
MFC was the lowest concentration that showed fewer than three competent M. anisopliae, M. anisopliae sensu lato, M. brunneum, and
colonies. M. robertsii exhibited susceptibility profiles that clearly differed
566 G.T.P. Brancini et al. / Fungal Biology 122 (2018) 563e569

Table 2
Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC, between parentheses) for Metarhizium spp., Aspergillus flavus and Aspergillus nidulans
isolates. MIC-0 is the lowest concentration inhibiting growth by at least 90 %. MIC-50 is the lowest concentration inhibiting growth by 50 %. Each value is the mean of three
independent experiments.

Species/strains Polyene Hydroxypyridone Triazole Benzofuran Allylamine Benzimidazole

Amphotericin B Ciclopirox Fluconazole Itraconazole Voriconazole Griseofulvin Terbinafine Benomyl

olamine

Aspergillus flavus
ATCC 204304 MIC-0 ¼ 0.5 (0.5) 5 (10) >100 (>100) 0.5 (0.5) 1 (4) >100 (>100) 0.5 (0.7) 1 (1)
MIC-50 ¼ 0.1 2 >100 0.1 0.5 >100 0.1 >0.5
Aspergillus nidulans
ATCC 10074 MIC-0 ¼ 2 (2) 0.5 (1) 100 (>100) 0.5 (0.5) 0.5 (0.5) >100 (>100) 0.5 (0.5) 1 (1)
MIC-50 ¼ 1 0.1 50 0.1 0.1 >100 0.1 >0.5
Metarhizium acridum
ARSEF 324 MIC-0 ¼ 1.3 (5) 0.8 (50) 50 (>100) 0.5 (0.5) 0.5 (3.5) >100 (>100) 0.5 (>100) 1 (1)
MIC-50 ¼ 0.5 0.5 9.2 0.1 0.1 5 0.1 >0.5
ARSEF 3391 MIC-0 ¼ 0.5 (1) 0.5 (2) 50 (>100) 0.5 (0.5) 0.1 (1.5) >100 (>100) 0.8 (2) 2 (>100)
MIC-50 ¼ 0.1 >0.1 13.3 0.1 <0.1 4 0.5 1
ARSEF 3609 MIC-0 ¼ 0.5 (1) 0.5 (0.5) 50 (>100) 0.5 (0.5) 0.4 (3.5) >100 (>100) 0.1 (>100) 1 (1)
MIC-50 ¼ 0.1 >0.1 9.2 0.1 0.1 4 <0.1 >0.5
ARSEF 5734 MIC-0 ¼ 0.5 (2) 0.7 (1) 7.5 (>100) 0.5 (0.5) 0.2 (1) >100 (>100) 0.5 (>100) 1.3 (2)
MIC-50 ¼ 0.1 >0.1 2 0.1 <0.1 5 0.1 >0.5
ARSEF 6421 MIC-0 ¼ 1.3 (5) 0.7 (2) 5 (>100) 0.5 (0.5) 0.1 (1) >100 (>100) 0.1 (>100) 2 (>7.5)
MIC-50 ¼ 0.5 >0.1 1 0.1 <0.1 5 <0.1 1
ARSEF 7486 MIC-0 ¼ 1.3 (5) 0.5 (2) 20 (>100) 0.5 (0.5) 0.1 (0.5) >100 (>100) 0.5 (>100) 1.7 (5)
MIC-50 ¼ 0.5 >0.1 7.5 0.1 <0.1 5 0.1 1
Metarhizium anisopliae sensu lato
ARSEF 5749 MIC-0 ¼ 56.7 (>100) 5 (5) >100 (>100) >100 (>100) 4 (>100) >100 (>100) 0.5 (0.5) 2 (2)
MIC-50 ¼ 6.7 >2 >100 0.5 1.7 5 0.1 >1
Metarhizium anisopliae
ESALQ 9 MIC-0 ¼ 2 (7.5) 10 (50) >100 (>100) >100 (>100) 1 (1) >100 (>100) 0.5 (1.2) 5 (5)
MIC-50 ¼ 0.7 5 >100 0.5 0.5 5.8 0.1 2
ESALQ 865 MIC-0 ¼ 2 (7.5) 10 (50) >100 (>100) >100 (>100) 1 (1.5) >100 (>100) 0.5 (1.3) 5 (5)
MIC-50 ¼ 0.7 5 >100 0.5 0.5 5.8 0.1 2
ESALQ 866 MIC-0 ¼ 1.3 (7.5) 7.5 (20) >100 (>100) >100 (>100) 2 (7.5) >100 (>100) 0.5 (1) 2 (2)
MIC-50 ¼ 0.5 5 >100 0.5 1 5 0.1 >1
ESALQ 1037 MIC-0 ¼ 2 (7.5) 10 (50) >100 (>100) >100 (>100) 1.3 (2) >100 (>100) 0.5 (1.7) 5 (5)
MIC-50 ¼ 0.8 5 >100 0.5 0.5 5.8 0.1 2
ESALQ 1104 MIC-0 ¼ 1 (5) 5 (10) >100 (>100) >100 (>100) 1.3 (>100) >100 (>100) 0.5 (1.7) 2 (2)
MIC-50 ¼ 0.5 >2 >100 0.5 0.5 5 0.1 >1
ESALQ PL43 MIC-0 ¼ 2 (7.5) 9.2 (20) >100 (>100) >100 (>100) 1 (1) >100 (>100) 0.5 (2) 5 (5)
MIC-50 ¼ 0.7 5 >100 0.5 0.5 5.8 0.1 2
Metarhizium brunneum
ARSEF 1095 MIC-0 ¼ 30 (50) 5 (7.5) >100 (>100) 100 (100) 0.8 (7.5) >100 (>100) 0.5 (>100) 2 (5)
MIC-50 ¼ 5 2 40 0.5 0.5 5 0.1 >1
Metarhizium robertsii
ARSEF 23 MIC-0 ¼ 66.6 (100) 5 (10) >100 (>100) >100 (>100) 2 (>100) >100 (>100) 0.2 (>100) 2 (5)
MIC-50 ¼ 7.5 2 8.7 0.5 1 5 <0.1 >1
ARSEF 2575 MIC-0 ¼ 66.7 (100) 6.7 (10) >100 (>100) >100 (>100) 1.3 (5) >100 (>100) 0.4 (0.7) 2 (5)
MIC-50 ¼ 7.5 5 >100 0.5 >0.5 5 <0.1 >1

from those of the acridid specialist M. acridum (Table 2). Resistance MIC for amphotericin B was >16 mg mL1 for each of the five iso-
of M. acridum isolates to most of the antifungal agents tested was lates. In the present study, we determined that all the M. anisopliae
lower than that of the other Metarhizium species. To our knowl- isolates are intermediately susceptible to amphothericin B (MIC-
edge, there are no published reports on the susceptibility of 0 ¼ 1e2 mg mL1) while all the isolates of M. brunneum and
M. acridum isolates to any therapeutic antifungal drug. M. robertsii are resistant (MIC-0 ¼ 30e67 mg mL1). Burgner et al.
Breakpoints have not been established for Metarhizium or other (1998), using a tablet diffusion method, observed that a clinical
moulds (CLSI, 2008a). However, as MIC below 1 mg mL1 are usually isolate of Metarhizium [at that time identified as M. anisopliae var.
reported for most Aspergillus spp. with amphotericin B, itracona- anisopliae and recently identified as M. guizhouense by Nourrisson
zole, and voriconazole (as is the case of the present study), fungal et al. (2017)] that had caused disseminated invasive mycosis in a
isolates were usually grouped as susceptible (MIC  1 mg mL1), severely immunocompromised child was resistant to itraconazole,
intermediate (MIC ¼ 2 mg mL1), and resistant (MIC  2 mg mL1) fluconazole, ketoconazole, and 5-fluorocytosine, but was suscepti-
(CLSI, 2008a). The results of the susceptibility tests for the envi- ble to amphotericin B. Marsh et al. (2008) reported that a clinical
ronmental isolates of Metarhizium spp. evaluated in the present isolate of Metarhizium (at that time identified as M. anisopliae var.
study agree with those previously reported for clinical isolates anisopliae) that had caused a cutaneous infection in a severely
of Metarhizium spp. Revankar et al. (1999), using the modified immunocompromised child was resistant to amphotericin B
National Committee for Clinical Laboratory Standards (NCCLS, (MIC > 16 mg mL1), susceptible to caspofungin
currectly CLSI) macrobroth method, tested the sensitivity of five (MIC ¼ 0.25 mg mL1) and intermediately susceptible to vor-
human isolates of Metarhizium (at that time identified as iconazole and posaconazole (MICs ¼ 2 mg mL1). Recently,
M. anisopliae var. anisopliae). Results suggested that the fungi may Nourrisson et al. (2017) evaluated the susceptibility of five clinical
be resistant to amphotericin B, 5-flucytosine, and fluconazole. The isolates of M. robertsii, three of M. guizhouense, one of M. brunneum,
 G.T.P. Brancini et al. / Fungal Biology 122 (2018) 563e569 567

and one of M. pingshaense to amphotericin B, voriconazole, and species causing infection, use of terbinafine or other allylamine
itraconazole using a broth microdilution technique according to antifungals appears to be a safer approach than opting for triazoles
guidelines of the Antifungal Susceptibility Testing Subcommittee of or polyenes.
the European Committee on Antibiotic Susceptibility Testing According to Hu et al. (2014) the higher detoxification capacity
(EUCAST) for filamentous fungi. All isolates had high MIC values of the generalists contributes to their broad host range. We hy-
for itraconazole (MIC > 8 mg mL1) and for amphotericin B pothesize that the increased resistance to xenobiotics also con-
(MIC > 16 mg mL1). tributes to rhizosphere competence of the generalist species by
In addition to being less resistant to most of the antifungals allowing them to better inactivate toxins produced by host plants
tested in the present study, previous results have shown that and by other soil and rhizosphere-inhabiting microorganisms. For
M. acridum is also less resistant to other xenobiotics when compared example, we have found that the generalist and rhizosphere
to other Metarhizium species. For example, M. acridum was found to competent M. anisopliae, M. brunneum, and M. robertsii are much
be less resistant to menadione than M. anisopliae, M. brunneum, and more resistant than M. acridum to amphotericin B which is pro-
M. robertsii (Azevedo et al., 2014), less resistant to N-dodecylgua- duced by the soil inhabiting bacteria Streptomyces nodosus (Caffrey
nidine monoacetate (dodine) than M. anisopliae and M. robertsii et al., 2001).
(Rangel et al., 2010a), less resistant to thiabendazole than M. robertsii Unlike with the antifungal-chemical challenges reported here
and M. brunneum, and less resistant to 4-nitroquinoline-1-oxide (4- and elsewhere, M. acridum is much more tolerant to physical
NQO) than other Metarhizium species (data not published). stressors than other Metarhizium species. We and others reported
A recent genomic study with seven Metarhizium species showed an increased tolerance of M. acridum isolates to ultraviolet-A, ul-
that the evolutionary trajectory of Metarhizium spp. was from traviolet-B, and solar radiation, and also to elevated temperatures
specialists via intermediate host range to generalist and the esti- when compared to other Metarhizium species (Braga et al., 2001a,b;
mated divergence time between M. acridum and the other species is Rangel et al., 2005; Fernandes et al., 2010; Rangel et al., 2010b;
48 million years (Hu et al., 2014). Analysis of protein families Keyser et al., 2014). Most likely, the higher tolerance of
showed protein-family expansion associated with Metarhizium M. acridum to solar radiation and elevated temperatures results
speciation (Hu et al., 2014; Pattemore et al., 2014). A marked from its coevolution with the acridid host. Acridids spend most of
expansion in families of genes that contributed to detoxification, their life cycle on the aerial parts of plants and exhibit behaviors
such as dehydrogenases and cytochrome P450 enzymes, and that expose their pathogens and parasites to higher temperature
transporters was observed in the generalist species relative to the and solar irradiance (Elliot et al., 2002; Ouedraogo et al., 2004). It is
specialist M. acridum. For example, M. acridum has only 37 cyto- remarkable that, due to their diverse evolutionary histories and
chrome P450 genes while 61, 63, 67, and 63 genes are present in the ecological niches, the species of the generalists and rhizosphere
genomes of M. anisopliae, M. brunneum, M. guizhouense, and competent entomopathogens have diverged from the acridid
M. robertsii, respectively (Hu et al., 2014). We hypothesize that the specialist M. acridum to tolerate chemical and physicals stressors.
reduced number of genes involved in detoxification and transport Only 19 cases of human mycoses due to Metarhizium spp. have
of xenobiotics is one of the factors responsible for the increased been reported between 1997 and 2015 (Nourrisson et al., 2017). In
susceptibility to antifungals and other xenobiotics observed in the cases in which the species were determined based on DNA
M. acridum. sequencing, the causal agents were M. brunneum, M. guizhouense,
In this regard, we observed an interesting pattern when sus- M. pingshaense, and M. robertsii; which are the species we found to
ceptibilities to triazoles (fluconazole, itraconazole, and vor- be the more resistant to most of the clinical antifungals tested. In
iconazole) and to allylamines (terbinafine) were compared for general, isolates of Metarhizium spp. grow poorly at temperatures
M. acridum and other Metarhizium isolates. Both of these antifungal above 35  C (Rangel et al., 2010b; Keyser et al., 2014). The sub-
classes act by inhibiting ergosterol biosynthesis, but triazoles optimally high body temperature of most mammals is probably
inhibit lanosterol 14 a-demethylase while allylamines inhibit involved in constraining the number of clinical cases of Meta-
squalene epoxidase. Isolates of M. acridum were more susceptible to rhizium infections (Nourrisson et al., 2017). However, it is intriguing
triazole antifungals when compared to other Metarhizium isolates, that none of the human mycoses reported to date was caused by
but the same was not observed for the allylamine terbinafine, for M. acridum which is the most thermotolerant species in the genus.
which similar susceptibilities were found (Table 2). One possible We are tempted to hypothesize that one of the reasons for this
explanation for this result is how resistance to these antifungal difference in the ability to cause human infection is related to the
classes is achieved in fungi. On the one hand, resistance mecha- presence (or absence thereafter) of destruxin gene clusters. Gene
nisms for triazoles are multiple and include modifications on the clusters for the synthesis of the cyclic depsipeptide destruxin are
target enzyme, overexpression of target enzyme gene, and over- absent from the genomes of the specialist M. acridum, but are found
expression of genes coding for efflux pumps (Franz et al., 1998; on generalists M. anisopliae, M. brunneum, M. guizhouense,
Robbins et al., 2017). On the other hand, the major resistance M. pingshaense, and M. robertsii (Wang et al., 2012; Hu et al., 2014).
mechanism to allylamines seems to be single point mutations on Ficheux et al. (2013) have reported that the cyclic depsipeptides
the squalene epoxidase gene (Leber et al., 2003). Recently, Yamada beauvericin and enniatin b, produced by Fusarium species, nega-
et al. (2017) analyzed 2056 Trichophyton clinical isolates and found tively interact with the human immune system, mostly interfering
decreased susceptibility to terbinafine in 17. All isolates displaying with monocyte differentiation into macrophages and also hinder-
resistance harbored either one of four different single point mu- ing dendritic cells maturation process. The authors conclude that,
tations on the squalene epoxidase gene. Also, introducing these in the event of infection, these depsipeptides could cause a
point mutations into squalene epoxidase genes on terbinafine- decrease in immune response (Ficheux et al., 2013). In a different
sensitive dermatophytes conferred terbinafine resistance (Yamada study, Wu et al. (2013) reported that beauvericin suppresses human
et al., 2017). Therefore, it is reasonable to suppose that, during T-cell differentiation and induces T-cell apoptotic response (Wu
Metarhizium evolutionary process, genome modifications could et al., 2013). Therefore, it is reasonable to suppose that destruxins
have led to one or more of the multiple resistance mechanisms to produced by nonspecialized Metarhizium species could exert
triazoles while leaving the squalene epoxidase gene intact, thus inhibitory effects on human immunity in the same way they do
making terbinafine susceptibility equal across the genus. In an with insect immunity (Huxham et al., 1989). We take the oppor-
event where it is not possible to determine the precise Metarhizium tunity to stress the need that future studies investigate the effects
568 G.T.P. Brancini et al. / Fungal Biology 122 (2018) 563e569

of destruxins on the human immune response as this information is Chen, W.H., Han, Y.F., Liang, J.D., Liang, Z.Q., Jin, D.C., 2017. Metarhizium den-
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essential in understanding the few yet relevant cases of human
Mycosphere 8, 31e37.
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of Filamentous Fungi. Approved Standard, CLSI document M38-A2, second ed.
Clinical and Laboratory Standards Institute, Wayne.
5. Conclusion CLSI, 2008b. Reference Method for Broth Dilution Antifungal Susceptibility Testing
of Yeasts. Approved Standard, CLSI document M27-A3, third ed. Clinical and
Laboratory Standards Institute, Wayne.
We have determined that species of the acridid specialist
Duarte, R.T.D., Staats, C.C., Fungaro, M.H.P., Schrank, A., Vainsten, M.H., Furlaneto-
M. acridum have different susceptibility profiles and are less resis- Maia, L., Nakamura, C.V., de Souza, W., Furlaneto, M.C., 2007. Development of a
tant to several antifungal agents than those of the generalist and simple and rapid Agrobacterium tumefaciens-mediated transformation system
for the entomopathogenic fungus Metarhizium anisopliae var. acridum. Lett.
rhizosphere competent M. anisopliae, M. brunneum, and M. robertsii.
Appl. Microbiol. 44, 248e254.
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best antifungals to treat the rare cases of human mycosis caused by fungal sclerokeratitis caused by Metarhizium anisopliae: a case report and
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Espinel-Ingroff, A., 1998. Comparison of in vitro activities of the new triazole
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Espinel-Ingroff, A., 2001a. Germinated and nongerminated conidial suspensions for
This work was supported by grants from The State of Sa ~o Paulo testing of susceptibilities of Aspergillus spp. to amphotericin B, itraconazole,
Research Foundation (FAPESP) to G.U.L.B (2016/11386-5), and to posaconazole, ravuconazole, and voriconazole. Antimicrob. Agents Chemother.
D.E.N.R (2010/06374-1) and from The Brazilian National Council for 45, 605e607.
Espinel-Ingroff, A., 2001b. Comparison of the E-test with the NCCLS M-38-P method
Scientific and Technological Development (CNPq) to G.U.L.B for antifungal susceptibility testing of common and emerging pathogenic fila-
(308505/2015-8) and to D.E.N.R. (478899/2010-6 and PQ1D mentous fungi. J. Clin. Microbiol. 39, 1360e1367.
308436/2014-8). This work was also facilitated by grants in support Espinel-Ingroff, A., Cuenca-Estrella, M., Fothergill, A., Fuller, J., Ghannoum, M.,
Johnson, E., Pelaez, T., Pfaller, M.A., Turnidge, J., 2011. Wild-type MIC distribu-
of the International Symposium on Fungal Stress (ISFUS) 2017 tions and epidemiological cutoffs values for amphotericin B and Aspergillus spp.
meeting from the Coordenaça ~o de Aperfeiçoamento de Pessoal de for the CLSI broth microdilution method (M38-A2 Document). Antimicrob.
Nível Superior (CAPES) (PAEP 88887.126652/2017-00) and by a Agents Chemother. 55, 5150e5154.
Espinel-Ingroff, A., Diekema, D.J., Fothergill, A., Johnson, E., Pelaez, T., Pfaller, M.A.,
grant from the Fundaç~
ao de Amparo  a Pesquisa do Estado de Goia
s
Rinaldi, M.G., Canton, E., Turnidge, J., 2010. Wild-type MIC distributions and
(201710267000110). epidemiological cutoff values for the triazoles and six Aspergillus spp. for the
CLSI broth microdilution method (M38-A2 Document). J. Clin. Microbiol. 48,
3251e3257.
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https://doi.org/10.1016/j.funbio.2017.12.004. Faria, M.R., Wraight, S.P., 2007. Mycoinsecticides and mycoacaricides: a compre-
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