Hindawi Publishing Corporation

BioMed Research International

Volume 2013, Article ID 835081, 8 pages
http://dx.doi.org/10.1155/2013/835081

Research Article
Anti-Candida Properties of Urauchimycins from Actinobacteria
Associated with Trachymyrmex Ants

Thais D. Mendes,1,2 Warley S. Borges,3,4 Andre Rodrigues,1,5 Scott E. Solomon,6

Paulo C. Vieira,4 Marta C. T. Duarte,7 and Fernando C. Pagnocca1,5
1
Center for the Study of Social Insects, São Paulo State University (UNESP), 13506-900 Rio Claro, SP, Brazil
2
EMBRAPA Agroenergy, Parque Estação Biológica, 70770-901 Brası́lia, DF, Brazil
3
Chemistry Departament, Federal University of Espı́rito Santo (UFES), 29075-910 Vitória, ES, Brazil
4
Chemistry Department, Federal University of São Carlos (UFSCar), 18052-780 São Carlos, SP, Brazil
5
Department of Biochemistry and Microbiology, São Paulo State University (UNESP), 13506-900 Rio Claro, SP, Brazil
6
Department of Ecology and Evolutionary Biology, Rice University, Houston, TX, USA
7
Division of Microbiology, Center for Chemistry, Biology and Agriculture Research (CPQBA/UNICAMP),
13081-970 Paulı́nia, SP, Brazil

Correspondence should be addressed to Fernando C. Pagnocca; pagnocca@rc.unesp.br

Received 12 November 2012; Revised 29 January 2013; Accepted 2 February 2013

Academic Editor: Manish Bodas

Copyright © 2013 Thais D. Mendes et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

After decades of intensive searching for antimicrobial compounds derived from actinobacteria, the frequency of isolation of new
molecules has decreased. To cope with this concern, studies have focused on the exploitation of actinobacteria from unexplored
environments and actinobacteria symbionts of plants and animals. In this study, twenty-four actinobacteria strains isolated from
workers of Trachymyrmex ants were evaluated for antifungal activity towards a variety of Candida species. Results revealed that
seven strains inhibited the tested Candida species. Streptomyces sp. TD025 presented potent and broad spectrum of inhibition of
Candida and was selected for the isolation of bioactive molecules. From liquid shake culture of this bacterium, we isolated the
rare antimycin urauchimycins A and B. For the first time, these molecules were evaluated for antifungal activity against medically
important Candida species. Both antimycins showed antifungal activity, especially urauchimycin B. This compound inhibited the
growth of all Candida species tested, with minimum inhibitory concentration values equivalent to the antifungal nystatin. Our
results concur with the predictions that the attine ant-microbe symbiosis may be a source of bioactive metabolites for biotechnology
and medical applications.

1. Introduction Actinobacteria are widely known for their ability to pro-

duce bioactive secondary metabolites, especially compounds
The increased resistance of microorganisms to antibiotics is a with antimicrobial activity. These bacteria are responsible for
problem of public health [1]. The increasing number of fungal producing two-thirds of the commercially available antibi-
species that can infect humans, particularly immunocompro- otics [2, 3]. Most actinobacteria species explored commer-
mised individuals, further reinforces this concern. A limited cially were isolated from the soil. However, after decades of
number of antifungal agents are commercially available when bioprospecting actinobacteria from this environment, it is
compared to antibacterial drugs. This scenario motivates the becoming more difficult to obtain strains producing novel
search for new bioactive compounds in various biological bioactive metabolites [4]. Thus, many companies have turned
systems using several approaches, including metagenomics the search for microbial producers of novel antifungal com-
and microbial genome-mining. pounds to other environments such as hydrothermal vents,
2 BioMed Research International

marine environments, tropical rain forests, and microbial were carefully excavated in order to reach the first fungus
symbionts associated with plants and animals hosts [5, 6]. garden chamber. Fungus garden with the tending workers
For example, the occurrence of actinobacteria associated with and brood was sampled using an alcohol-flamed spoon and
marine sponges and the fact that such strains produce com- stored in sterile plastic containers. All containers were kept
pounds with antimicrobial activity confirms this potential [7– in a cooler during transport to the laboratory where they
9]. In addition, endophytic actinobacteria are also explored were maintained at 25∘ C.
for their capacity to produce antimicrobial compounds [10– From each colony, we randomly selected four workers
12]. for actinobacteria isolation. Then, the propleural plates
Several studies have focused on the association between were scraped with a sterile needle under a low power
actinobacteria and insects from an ecological perspective stereomicroscope. All ants used in the present study had a
[13–25]. On the other hand, few studies have focused on visible, whitish covering on the propleural plates. Scrapings
the multitude of chemical compounds that are involved were plated on SCN agar (in g ⋅ L−1 : 10.0 starch, 0.3 casein,
in such interactions [26]. The best studied example is the 2.0 KNO3 , 2.0 NaCl, 2.0 K2 HPO4 , 0.05 MgSO4 ⋅ 7 H2 O,
symbiosis between actinobacteria and fungus-growing ants 0.02 CaCO3 , 0.01 FeSO4 ⋅ 7H2 O and 18.0 agar supplemented
(Hymenoptera: Formicidae: tribe Attini). In this association, with 0.05 Nystatin) [36]. After scraping, the entire body
the actinobacteria are found on the ants’ integument and of all workers was inoculated on SCN agar. All plates
produce antimicrobial compounds that help the ants to were incubated at 25∘ C for 30 days. From each sampled
suppress the microfungus Escovopsis sp. [13, 14]. This fungus Trachymyrmex colony, one representative strain was
is considered a specialized parasite of the ant cultivar and selected from each morphotype obtained. The strains were
causes negative impacts to the ant colony [27]. subcultured in YMA (in g ⋅ L−1 : 3.0 yeast extract, 3.0 malt
Actinobacteria isolated from the integument of attine extract, 5.0 peptone, 10.0 glucose, 18.0 agar) and stored at
ants are generally classified in the genus Pseudonocardia and −80∘ C in 15% glycerol.
Streptomyces. Bioactive molecules have already been isolated We used a molecular approach to provide taxonomic
and characterized from actinobacteria isolated from several affiliation to actinobacteria strains. Genomic DNA was
attine genera [26]. Pseudonocardia isolated from Acromyrmex extracted following the method of Sampaio et al. [37]. We
octospinosus and Apterostigma dentigerum are known to pro- carried out 16S rDNA PCR with the universal primers
duce several compounds like (i) dentigerumycin, a complex 27F (5󸀠 -AGAGTTTGATCATGGCTCAG-3󸀠 ) and 1492R (5󸀠 -
compound active against Candida albicans and Escovopsis TACGGTTACCTTGTTACGACTT-3󸀠 ) [38]. Reactions were
[28]; (ii) a nystatin-like antifungal [29]; (iii) the novel quinone conducted in a final volume of 25 𝜇L and contained 1 𝜇L
pseudonocardones A–C active against the malaria causal of diluted DNA template (1 : 10), 2.0 𝜇L of each primer
agent Plasmodium berghei [30]; (iv) the already known antibi- (10 mM), 2.5 𝜇L of 10X buffer, 1.0 𝜇L of MgCl2 (50 mM),
otics 6-deoxy-8-O-methylrabelomycin and X-14 881, both 4.0 𝜇L of dNTPs (1.25 mM each), 0.2 𝜇L of Taq polymerase
active against Bacillus subtilis and P. berghei [30]. In addition (5 U/𝜇L), and 12.3 𝜇L of ultrapure water. Amplicons were
to Pseudonocardia, actinobacteria in the genus Streptomyces cleaned up with GFX PCR DNA and Gel Band Purifica-
are also found on the integument of Acromyrmex workers tion Kit (GE Healthcare). Forward and reverse sequences
and were shown to produce (i) candicidin, active against were generated using the same primers, along with an
Escovopsis sp. [29, 31–34], (ii) antimycins active against internal primer U519F (5󸀠 -CAGCMGCCGCGGTAATWC-
Escovopsis sp. [32–34], and (iii) actinomycin D, actinomycin 3󸀠 ). Sequences were generated using BigDye Terminator v.3.1
X2 and valinomycin that are active against B. subtilis [32]. Cycle Sequencing Kit (Life Technologies) in an ABI 3130
Poulsen [35] suggested that the attine ant-microbe asso- sequencer and manually edited in BioEdit v. 7.1.3 [39]. Contigs
ciation is little explored regarding the search for new were compared with those available in the databases NCBI-
antimicrobials. The author highlights the various symbiotic GenBank (http://blast.ncbi.nlm.nih.gov/) and Ribossomal
associations between attine ants and microorganisms as a Database Project (RDP, http://rdp.cme.msu.edu/). Sequences
promising source for drug discovery, especially those with generated in the present study were deposited in NCBI-
antimicrobial activity. Here, we explored the antimicrobial GenBank (accessions KC480554-KC480557).
potential of actinobacteria isolated from the integument of A phylogenetic analysis was carried out in order to deter-
Trachymyrmex fungus-growing ants and demonstrate the mine the taxonomic affiliation of strain TD025. Sequences
action against different medically important Candida species. of closest relatives were retrieved from the NCBI-GenBank
We also report two previously described urauchimycins from and the RDP Project and aligned in ClustalW. Phylogenetic
a Streptomyces strain and emphasize the newly discovered reconstruction was performed using the neighbor-joining
anti-Candida activity of these compounds. algorithm implemented in PAUP v. 4.0 [40]. Genetic dis-
tances were calculated using the Kimura 2-parameter model
of nucleotide substitution [41]. Robustness of the relation-
2. Material and Methods ships was estimated from 1000 bootstrap pseudoreplicates.

2.1. Actinobacteria Isolation and Identification. Twelve

Trachymyrmex colonies were collected in different Brazilian 2.2. Organic Extracts and Antifungal Assays. All actinobac-
biomes (see Table S1 in Supplementary Material available teria were grown in Erlenmeyer flasks (250 mL) containing
online at http://dx.doi.org/10.1155/2013/835081). Colonies 50 mL of modified MPE medium (in g ⋅ L−1 : 5.0 soy flour,
BioMed Research International 3

20.0 glucose, 5.0 of NaCl, 4.0 CaCO3 ) [42]. Each flask was compounds, they were evaluated for antimicrobial activity
inoculated with five mycelium disks (1 cm in diameter) cut following the method described previously. Besides the MIC
from a previously grown culture and then incubated at determination, we also evaluated the minimum fungicidal
28∘ C for two days on an orbital shaker at 150 rpm. From concentration (MFC). The MFC was determined by inoculat-
this culture, 10 mL was inoculated in two Erlenmeyer flasks ing Sabouraud dextrose medium with 10 𝜇L of the contents
(250 mL) containing 100 mL of the same medium and incu- of each of the wells where there was growth inhibition of
bated for five days on the same conditions. After incubation, yeast, the MFC was defined as the lowest concentration of the
the fermented broth was separated from the mycelium by substance capable of preventing the onset of colony forming
centrifugation and partitioned three times with ethyl acetate units.
(EtOAc). The organic solvent was evaporated under vacuum,
and the EtOAc extracts were diluted in RPMI-1640 culture
broth containing 10% DMSO and used in the antimicrobial 3. Results and Discussion
assays.
3.1. Actinobacteria Isolation and Identification. Several acti-
The antimicrobial activity of the extracts was evalu-
nobacteria colonies were observed after incubation of iso-
ated against the yeasts Candida albicans CBS 562, Candida
lation plates. We selected just one morphotype of each per
dubliniensis CBS 7987, Candida glabrata CBS 138, Candida
ant colony, rendering a total of 24 strains ouf of 12 Tra-
krusei CBS 573, Candida parapsilosis CBS 604, and Candida
chymyrmex spp. nests (Table S1). Four actinobacteria genera
tropicalis CBS 94. The minimum inhibitory concentration
were identified and Streptomyces was the most abundant
(MIC) was determined using the microdilution method
taxon (Table 1), corresponding to 66.67% of the strains. It
according to the M27-A2 standard of the Clinical and Lab-
is assumed that the main actinobacteria associated with the
oratory Standards Institute [43].
integument of attine workers is the genus Pseudonocardia
[14–16, 20]. However, several authors have demonstrated the
2.3. Isolation and Characterization of the Bioactive Com- isolation of actinobacteria other than Pseudonocardia on the
pounds of Strain TD025. The actinobacteria strain that exhib- integument of attine ants [17–19, 21, 22, 29, 31]. The prevalence
ited both a broad antifungal spectrum and lower MIC values of Streptomyces and absence of Pseudonocardia among our
was strain TD025. In order to identify the compounds isolates may be due to the culture medium used [44]. The
responsible for the observed results, the strain was cultured in SCN medium is suitable for the isolation of fast-growing
5 L of modified MPE medium and the extracts were obtained actinobacteria, but according to other authors [13–16], the use
as described. of a low-nutrient medium, such as chitin agar, may provide
The fermented broth (5 L) was separated from the cells by the recovery of Pseudonocardia strains.
centrifugation and portioned three times with ethyl acetate In eight out of 12 ant colonies, we obtained more than
(EtOAc). The organic solvent was evaporated under vacuum. one actinobacteria morphotype (Table S1). From colony
The crude extract, a dark green oil (1.40 g), was separated CTL080820-02, the two morphotypes isolated from two
by means of column chromatography using silica gel 60 different workers were identified as the same actinobacteria
eluted with n-hexane/EtOAc as the elution gradient, yielding species (Tables S1 and 1). On the other hand, different
8 fractions. All fractions were submitted to antimicrobial actinobacteria species were isolated in the seven remaining
assays against C. albicans following the procedure described colonies. We also observed the occurrence of different acti-
above. The most active fraction (67.5 mg) was subjected to nobacteria strains in a single worker (Tables S1 and 1). This
preparative TLC (thin layer chromatography) eluted with result demonstrates the diversity of actinobacteria present on
n-hexane/EtOAc (7 : 3) two times, yielding 3 subfractions. the integument of these ants (Table S1, nests SES080911-04
The subfractions obtained were submitted to antimicrobial and SES080924-01).
assays against C. albicans. The most active subfraction was The 16S rDNA sequence of strain TD025 showed 99%
submitted to semipreparative HPLC separations carried out similarity with sequences of several species of the genus
in a Shimadzu (LC-6AD apparatus, Japan) multisolvent Streptomyces deposited in the databases. For a better charac-
delivery system, Shimadzu SPD-M10Avp Photodiode Array terization, we performed a phylogenetic analysis (Figure 1).
Detector, and an Intel Celeron computer for analytical system The result suggests a differentiated phylogenetic position for
control, data collection and processing (software Class-VP). strain TD025 when compared with the remaining sequences.
The separation was carried out using VP 250/10 NUCLEOSIL This preliminary analysis allowed us to assign this strain
120-5 C18 column eluted with acetonitrile/water/acetic acid as belonging to the genus Streptomyces, with S. cirratus as
(50 : 50 : 0.01) at a flow rate of 3 mL ⋅ min−1 , yielding com- the closest relative strain (Figure 1). However, more refined
pounds 1 and 2. The isolated molecules were characterized by phylogenetic analyses, along with morphological and physi-
1
H and 13 C NMR spectroscopic experiments recorded on a ological studies, are necessary to ensure the identification of
BRUKER DRX-400 spectrometer with CDCl3 as solvent and TD025 to the species level.
TMS as internal standard.
3.2. Screening for Antifungal Activity. Our screening for
2.4. Minimum Inhibitory Concentration and Minimum Fungi- antifungal activity revealed that seven out of 24 extracts
cide Concentration of Bioactive Compounds. After isola- (29.16%) inhibited the growth of at least one Candida
tion and determination of the structure of the targeted species. C. albicans was the most sensitive yeast and was
4 BioMed Research International

Table 1: Actinobacteria identification according to 16S rDNA sequencing.

1
Isolate Id bp NCBI-GenBank closest relative Coverage % Accession #
TD016 1251 Nocardia neocaledoniensis DSM 44717 100 100 JF797311
TD017 1248 Nocardia neocaledoniensis DSM 44717 100 100 JF797311
TD018 1184 Streptomyces zaomyceticus NRLL B-2038 99 100 NR044144
TD019 1342 Streptomyces alivochromogenes NBRC 3404 99 100 AB184761
TD020 1167 Streptomyces sannanensis NBRC 14239 99 99 NR041160
TD021 1250 Streptomyces mauvecolor NBRC 13854 100 99 NR041154
TD022 1254 Streptomyces lydicus CGMCC 4.1412 100 100 JN566018
TD023 1246 Streptomyces sp. QZGY-A17 100 100 JQ812074
TD025 1333 Streptomyces sp. QLS92 100 99 JQ838121
Streptomyces cirratus 100 99 JQ222143
TD027 1342 Streptomyces alivochromogenes NBRC 3404 99 100 AB184761
TD028 1353 Actinoplanes ferrugineus 100 99 AB048221
TD030 1255 Streptomyces sp. CA13 100 99 AB622252
TD032 1263 Streptomyces chartreusis NBRC 12753 100 100 NR041216
TD033 1278 Streptomyces griseoplanus 99 100 HQ699516
TD034 1283 Amycolatopsis decaplanina DSM 44594 100 100 NR025562
TD035 1320 Streptomyces atriruber NRLL B-24676 100 99 FJ169330
TD045 1183 Amycolatopsis equina 100 99 HQ021204
TD047 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251
TD049 1260 Streptomyces luteogriseus NBRC 13402 100 99 NR041128
TD050 1261 Streptomyces rubiginosohelvolus NBRC 12912 100 100 NR041093
TD051 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251
TD053 1265 Streptomyces kunmingensis NBRC14463 99 98 AB184597
TD055 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251
TD058 1257 Streptomyces globisporus KCTC 9026 100 100 HQ995504
1
bp: base pair.

inhibited by seven extracts with MIC ranging between 10 Compounds 1 and 2 exhibited typical NMR data of
and 1000 𝜇g ⋅ mL−1 (Table 2). The yeasts C. glabrata and C. urauchimycins (Figures S1 and S2). Their NMR data are in
tropicalis were the most resistant strains, being inhibited by agreement with those previously reported by Imamura et al.
one and two actinobacteria extracts, respectively, with MIC [46]. Although these urauchimycins (1 and 2) have already
values of 1000 𝜇g ⋅ mL−1 (Table 2). been isolated, they have never been tested on various species
Except for strain TD034 identified as Amycolatopsis of Candida as carried out in the present study.
decaplanina (Table 1), the other actinobacteria exhibiting The 13 C NMR spectrum of 1 showed 22 carbon signals:
antimicrobial activity were identified as belonging to the four carbonyls (𝛿 179.0, 𝛿 170.6, 𝛿 169.8, and 𝛿 158.7), three
genus Streptomyces. This genus is recognized as the largest quaternary sp2 carbons (𝛿 150.6, 𝛿 127.4, and 𝛿 112.8), three
producer of antibiotics because from approximately 3,000 methine aromatic carbons (𝛿 124.8, 𝛿 120.6, and 𝛿 119.0), two
known antibiotics obtained from actinobacteria, the genus sp3 methylene group (𝛿 35.6 and 𝛿 30.5), three sp3 methine
Streptomyces contributes with 90% of this total [45]. groups (𝛿 54.0, 𝛿 50.1, and 𝛿 32.4), three oxymethinic groups
The extracts of Streptomyces sp. TD025 and Streptomyces (𝛿 77.1, 𝛿 76.3, and 𝛿 70.9), and four methyl groups (𝛿 18.4,
crystallinus TD027 showed activity against all yeast strains 𝛿 18.4, 𝛿 15.1, and 𝛿 11.4). Two carboxylic carbons 𝛿 170.6
except for C. glabrata. These extracts were effective against and 𝛿 173.9 showed correlations with different hydrogens of
C. albicans and C. krusei and showed low activity against the structure, showing a dilactone system of nine members,
C. tropicalis (Table 2). More interestingly, both strains were typical of the antimycin class.
isolated from the same colony but from independent workers The 1 H NMR spectrum showed a singlet at 𝛿 8.50,
(Table 1). Because lower MICs were obtained for the extract assigned to a hydrogen bounded to a carbonyl group and
of Streptomyces sp. TD025, this strain was selected to verify three aromatic hydrogens at 𝛿 8.55 (dd, J 8.1 and 1.2 Hz), 𝛿 7.24
the chemical compounds responsible for the antimicrobial (dd, J 8.1 and 1.2 Hz), and d 6.92 (t, J 8.1 Hz), suggesting a 1,2,3
activity. trisubstituted aromatic ring. The substance was identified as
urauchimycin A by comparison with the literature data [46].
3.3. Bioactive Compounds of Streptomyces sp. TD025. Chro- The 1 H and 13 C NMR spectram of 2 were very similar to
matographic procedures revealed that EtOAc extract from those observed for compound 1. Differences were observed in
TD025 contains two compounds (1 and 2, Figure 2). chemical shifts of the hydrogen of the methyl and methylene
BioMed Research International 5

TD025 (KC480562)
100
Streptomyces sp. (JQ838121)
100
73
Streptomyces cirratus (JQ422143)

65
Streptomyces sp. (EF093114)

74 Streptomyces spiroverticillatus (NR041214)

Streptomyces griseobrunneus (AB249912)

98

Streptomyces albolongus (NR041144)

100

Streptomyces flavogriseus (NR028988)

58

71 Streptomyces griseolus (NR041207)

Streptomyces cinereorectus (NR041173)

86
Streptomyces badius (NR043350)
58

68 Streptomyces rubinosohelvolus (NR041093)

Streptomyces globisporus (NR044145)

Streptomyces crystallinus (NR041177)

0.001

Figure 1: Phylogenetic relationships of strain TD025 (in bold) isolated from the integument of Trachymyrmex sp. The phylogeny was inferred
from 16S rDNA sequences retrieved from the NCBI-GenBank using the neighbor-joining algorithm and the Kimura 2-parameter model of
nucleotide substitution. Numbers in parentheses correspond to GenBank accessions. Numbers on branches indicate the bootstrap support
after 1,000 pseudoreplicates. The scale bar denotes the number of substitutions per site.

groups of the side chain. Based on published data [46] Urauchimycins A and B were previously isolated from
compound 2 was identified as Urauchimycin B, an isomer of Streptomyces sp. Ni-80 isolated from a marine sponge in
compound 1. Urauchicove, Irimore, Japan. These substances were the
Urauchimycins belong to the antimycin class, a group first antimycins having an odd number of carbons and a
of well-known antifungals. Antimycins act by inhibiting the branching side chain [46]. Imamura et al. [46] suggested that
electron flow in the mitochondrial respiratory chain [47]. such structures are the result of an evolution of actinobacteria
Antimycins have been previously identified in Streptomyces in the marine environment, which could have resulted in a
isolated from the integument of attine ants [32–34]. Schoe- change in their secondary metabolism.
nian and colleagues [32] detected the well-know antimycins In 2006, two new urauchimycins were described: urau-
A1–A4 in 50% of the actinobacteria identified as Streptomyces chimycin C, isolated from Streptomyces sp. B1751 from marine
isolated from workers of several Acromyrmex species. These sediment, and urauchimycin D, isolated from Streptomyces
data along with the rare antimycins identified in the present sp. AdM21 from soil [48]. In the study by Imamura and
study indicate that this chemical class is often produced coworkers [46], the urauchimycins A and B inhibited the
by Streptomyces associated with attine ants. Compounds morphological differentiation of C. albicans up to a con-
belonging to this class may have an important role in the centration of 10 𝜇g ⋅ mL−1 . Urauchimycins C and D showed
attine ant-microbe association. no inhibitory activity against C. albicans, Mucor miehei, and
Another antifungal compound widely distributed in bacteria [48].
Streptomyces associated with attine ants is candicidin The study of antimicrobial activity of urauchimycins A
[31–34], which was not detected in Streptomyces sp. TD025. It and B was restricted to C. albicans in the work by Imamura
is possible that candicidin was lost in one of the purification and colleagues [46]. The reisolation of these molecules in the
steps of the AcOEt extract or it is not produced by this strain. present study allowed a better evaluation of its spectrum of
6 BioMed Research International

Table 2: Minimum inhibitory concentrations (𝜇g⋅mL−1 ) of actinobacteria extracts towards different medically important Candida species.

C. albicans C. dubliniensis C. glabrata C. krusei C. parapsilosis C. tropicalis

Isolate ID
CBS 562 CBS 7987 CBS 138 CBS 573 CBS 604 CBS 94
TD016 ∗ ∗ ∗ ∗ ∗ ∗
TD017 ∗ ∗ ∗ ∗ ∗ ∗
TD018 ∗ ∗ ∗ ∗ ∗ ∗
TD019 60 900 ∗ 40 80 1000
TD020 ∗ ∗ ∗ ∗ ∗ ∗
TD021 ∗ ∗ ∗ ∗ ∗ ∗
TD022 800 ∗ 1000 ∗ ∗ ∗
TD023 ∗ ∗ ∗ ∗ ∗ ∗
TD025 40 700 ∗ 15 125 1000
TD027 60 1000 ∗ 15 200 ∗
TD028 ∗ ∗ ∗ ∗ ∗ ∗
TD030 ∗ ∗ ∗ ∗ ∗ ∗
TD032 1000 ∗ ∗ ∗ ∗
TD033 10 800 ∗ 125 200 ∗
TD034 500 ∗ ∗ ∗ ∗
TD035 ∗ ∗ ∗ ∗ ∗ ∗
TD045 ∗ ∗ ∗ ∗ ∗ ∗
TD047 ∗ ∗ ∗ ∗ ∗ ∗
TD049 ∗ ∗ ∗ ∗ ∗ ∗
TD050 ∗ ∗ ∗ ∗ ∗ ∗
TD051 ∗ ∗ ∗ ∗ ∗ ∗
TD053 ∗ ∗ ∗ ∗ ∗ ∗
TD055 ∗ ∗ ∗ ∗ ∗ ∗
TD058 ∗ ∗ ∗ ∗ ∗ ∗
∗
Minimum inhibitory concentration > 1000 𝜇g⋅mL−1 .

O O
O O
H
N H
N O N
H H N O
H O OH H
O OH
OH O
O OH O
O

1 2

Figure 2: Chemical structures of compounds isolated from Streptomyces sp. TD025. (1) urauchimycin A; (2) urauchimycin B.

activity. The urauchimycins from Streptomyces sp. TD025 pre- antifungal agents that complement or substitute for the scarce
sented MIC values equivalent to the reference antifungal nys- products available on the market, it is interesting and nec-
tatin for C. albicans and C. glabrata (Table 3). Urauchimycin essary to determine the toxicity presented by urauchimycin
B showed inhibitory activity against all Candida strains eval- B, to assess whether it can be used as an antifungal agent for
uated, showing MIC similar to those provided by nystatin. humans and animals. In addition, evaluation of the isolated
Urauchimycin B showed a broad spectrum of activity compound against Candida species resistant to commercially
against Candida spp. with MIC values equivalent to the available antifungal agents should be performed to confirm
antifungal nystatin, which indicates the potential for medical the potential of this relatively unexplored antifungal.
use. For many years, antimycins were used for the treatment Here we show that Trachymyrmex ants, one attine genus
of human infections, but due to its mechanism of action understudied with respect to its microbial symbionts, har-
and associated side effects, its use in human treatment was bor antimicrobial-producing actinobacteria. As observed by
discontinued [47]. However, with the pressing need for new other authors [28–33], the present study demonstrates that
BioMed Research International 7

Table 3: Minimum inhibitory concentration (MIC) and minimum thank two anonymous referees for helpful comments on this
fungicide concentrations (MFC) (𝜇g⋅mL−1 ) of Urauchimycins A and paper.
B obtained from Streptomyces sp. TD025 in comparison with the
antifungal Nystatin.
References
Urauchimycin A Urauchimycin B Nystatin
Candida species [1] J. Travis, “Reviving the antibiotic miracle?” Nature, vol. 264, no.
MIC MFC MIC MFC MIC MFC
5157, pp. 360–362, 1994.
C. albicans 1 ∗ 1 3 1 2
[2] S. Miyadoh, “Research on antibiotic screening in Japan over
C. dubliniensis 800 ∗ 2 3 1 2
the last decade: a producing microorganism approach,” Actino-
C. glabrata 2 15,6 2 2 1 1 mycetologica, vol. 7, no. 2, pp. 100–106, 1993.
C. krusei 15,6 15,6 2 3 2 3
[3] G. L. Challis and D. A. Hopwood, “Synergy and contingency
C. parapsilosis ∗ ∗ 2 2 1 2 as driving forces for the evolution of multiple secondary
C. tropicalis ∗ ∗ 2 2 4 4 metabolite production by Streptomyces species,” Proceedings of
∗
>1000 𝜇g⋅mL−1 . the National Academy of Sciences of the United States of America,
vol. 100, no. 24, pp. 14555–14561, 2003.
[4] M. Goodfellow and H. P. Fiedler, “A guide to successful bio-
actinobacteria of attine ants are able to produce antifungal prospecting: informed by actinobacterial systematics,” Antonie
compounds active against other fungal species and not only van Leeuwenhoek, vol. 98, no. 2, pp. 119–142, 2010.
against the specific fungal parasite Escovopsis. [5] A. C. W. Waugh and P. F. Long, “Prospects for generating new
Moreover, our study corroborates previous work [35] that antibiotics,” Science Progress, vol. 85, no. 1, pp. 73–88, 2002.
suggests the attine ant-microbe association is a promising [6] J. Clardy, M. A. Fischbach, and C. T. Walsh, “New antibiotics
source of microorganisms that produce active metabolites. from bacterial natural products,” Nature Biotechnology, vol. 24,
The few recent studies that focused on the chemical charac- no. 12, pp. 1541–1550, 2006.
terization of bioactive compounds produced by actinobacte- [7] T. K. Kim, A. K. Hewavitharana, P. N. Shaw, and J. A. Fuerst,
ria associated with attine ants support the potential isolation “Discovery of a new source of rifamycin antibiotics in marine
of novel molecules with biological activity [28–32]. Thus, sponge actinobacteria by phylogenetic prediction,” Applied and
an exploration program of isolation of bioactive molecules Environmental Microbiology, vol. 72, no. 3, pp. 2118–2125, 2006.
from actinobacteria from attine ants certainly will result [8] R. Gandhimathi, M. Arunkumar, J. Selvin et al., “Antimicrobial
in the discovery of novel compounds with activity against potential of sponge associated marine actinomycetes,” Journal
microorganisms that are potentially pathogenic to humans. de Mycologie Medicale, vol. 18, no. 1, pp. 16–22, 2008.
[9] E. J. Choi, H. C. Kwon, J. Ham, and H. O. Yang, “6-Hydrox-
ymethyl-1-phenazine-carboxamide and 1,6-phenazinedim-
4. Conclusion ethanol from a marine bacterium, Brevibacterium sp. KMD
003, associated with marine purple vase sponge,” Journal of
As suggested by Poulsen [35], we found that the integument
Antibiotics, vol. 62, no. 11, pp. 621–624, 2009.
of Trachymrymex ants is a potential source for the isolation
of actinobacteria that produce bioactive molecules. The isola- [10] B. Bieber, J. Nuske, M. Ritzau, and U. Grafe, “Alnumycin a
tion of Urauchimycins A and B enabled, for the first time, the new naphthoquinone antibiotic produced by an endophytic
Streptomyces sp.,” Journal of Antibiotics, vol. 51, no. 3, pp. 381–
evaluation of their activity against various Candida species.
382, 1998.
Urauchimycin B showed a broad spectrum of activity and
MIC values equivalent to the reference antifungal nystatin. [11] U. F. Castillo, G. A. Strobel, E. J. Ford et al., “Munumbicins,
wide-spectrum antibiotics produced by Streptomyces NRRL
Toxicity studies and in vivo activity should be carried out
30562, endophytic on Kennedia nigriscans,” Microbiology, vol.
in order to verify the potential use of this molecule in the
148, no. 9, pp. 2675–2685, 2002.
treatment of fungal infections.
[12] D. Ezra, U. F. Castillo, G. A. Strobel et al., “Coronamycins,
peptide antibiotics produced by a verticillate Streptomyces sp.
Acknowledgments (MSU-2110) endophytic on Monstera sp.,” Microbiology, vol. 150,
no. 4, pp. 785–793, 2004.
The authors thank FAPESP (Fundação de Amparo à Pesquisa [13] C. R. Currie, J. A. Scottt, R. C. Summerbell, and D. Malloch,
do Estado de São Paulo), CNPq/INCT (Conselho Nacional de “Fungus-growing ants use antibiotic-producing bacteria to
Desenvolvimento Cientı́fico e Tecnológico), and NSF IRFP control garden parasites,” Nature, vol. 398, no. 6729, pp. 701–704,
(United States National Science Foundation International 1999.
Research Fellowship Program no. 07012333) for financial [14] C. R. Currie, J. A. Scott, R. C. Summerbell, and D. Malloch,
support. The authors also thank CAPES (Coordenação de “Corrigendum: fungus-growing ants use antibiotic-producing
Aperfeiçoamento de Pessoal de Ensino Superior) for provid- bacteria to control garden parasites,” Nature, vol. 423, no. 6938,
ing a scholarship to the first author. This work was conducted p. 461, 2003.
under collecting permit number 14789-1 issued by the “Insti- [15] M. J. Cafaro, M. Poulsen, A. E. F. Little et al., “Specificity in
tuto Brasileiro do Meio Ambiente e dos Recursos Naturais the symbiotic association between fungus-growing ants and
Renováveis” (IBAMA) and the “Instituto Chico Mendes de protective Pseudonocardia bacteria,” Proceedings of the Royal
Conservação da Biodiversidade” (ICMBio). The authors also Society B, vol. 278, no. 1713, pp. 1814–1822, 2011.
8 BioMed Research International

[16] M. J. Cafaro and C. R. Currie, “Phylogenetic analysis of mutual- [32] I. I. Schoenian, M. M. Spiteller, M. J. Manoj et al., “Chemical
istic filamentous bacteria associated with fungus-growing ants,” basis of the synergism and antagonism in microbial communi-
Canadian Journal of Microbiology, vol. 51, no. 6, pp. 441–446, ties in the nests of leaf-cutting ants,” Proceedings of the National
2005. Academy of Sciences of the United States of America, vol. 108, no.
[17] C. Kost, T. Lakatos, I. Böttcher, W. R. Arendholz, M. Redenbach, 5, pp. 1955–1960, 2011.
and R. Wirth, “Non-specific association between filamentous [33] R. F. Seipke, J. Barke, C. Brearley et al., “A single Streptomyces
bacteria and fungus-growing ants,” Naturwissenschaften, vol. symbiont makes multiple antifungals to support the fungus
94, no. 10, pp. 821–828, 2007. farming ant Acromyrmex octospinosus,” PLoS ONE, vol. 6, no.
[18] U. G. Mueller, D. Dash, C. Rabeling, and A. Rodrigues, 8, Article ID e22028, 8 pages, 2011.
“Coevolution between attine ants and actinomycete bacteria: a [34] F. R. Seipke, S. Gruschow, R. J. Goss, and M. I. Hutchings, “Iso-
reevaluation,” Evolution, vol. 62, no. 11, pp. 2894–2912, 2008. lating antifungals from fungus-growing ant symbionts using a
genome-guided chemistry approach,” Methods in Enzymology,
[19] U. G. Mueller, “Symbiont recruitment versus ant-symbiont co-
vol. 517, pp. 47–70, 2012.
evolution in the attine ant-microbe symbiosis,” Current Opinion
in Microbiology, vol. 15, no. 3, pp. 269–277, 2012. [35] M. Poulsen, “Biomedical exploitation of the fungus-growing ant
symbiosis,” Drug News and Perspectives, vol. 23, no. 3, pp. 203–
[20] M. Poulsen, M. Cafaro, J. J. Boomsma, and C. R. Currie, 210, 2010.
“Specificity of the mutualistic association between actinomycete
bacteria and two sympatric species of Acromyrmex leaf-cutting [36] E. Küster and S. T. Williams, “Selection of media for isolation of
ants,” Molecular Ecology, vol. 14, no. 11, pp. 3597–3604, 2005. streptomycetes,” Nature, vol. 202, no. 4935, pp. 928–929, 1964.
[37] J. P. Sampaio, M. Gadanho, S. Santos et al., “Polyphasic
[21] T. D. Zucchi, A. S. Guidolin, and F. L. Cônsoli, “Isolation
taxonomy of the basidiomycetous yeast genus Rhodosporid-
and characterization of actinobacteria ectosymbionts from
ium: Rhodosporidium kratochvilovae and related anamorphic
Acromyrmex subterraneus brunneus (Hymenoptera, Formici-
species,” International Journal of Systematic and Evolutionary
dae),” Microbiological Research, vol. 166, no. 1, pp. 68–76, 2010.
Microbiology, vol. 51, no. 2, pp. 687–697, 2001.
[22] R. Sen, H. D. Ishak, D. Estrada, S. E. Dowd, E. Hong, and U. G. [38] D. J. Lane, “16S/23S RNAr sequencing,” in Nucleic Acid Tech-
Mueller, “Generalized antifungal activity and 454-screening of niques in Bacterial Systematic, E. Stackebrandt and M. Goodfel-
Pseudonocardia and Amycolatopsis bacteria in nests of fungus- low, Eds., pp. 115–175, John Willey, New York, NY, USA, 1991.
growing ants,” Proceedings of the National Academy of Sciences
[39] T. A. Hall, “BioEdit: a user-friendly biological sequence align-
of the United States of America, vol. 106, no. 42, pp. 17805–17810,
ment editor and analysis program for Windows 95/97/NT,”
2009.
Nucleic Acids Symposium Series, vol. 41, pp. 95–98, 1999.
[23] M. Kaltenpoth, W. Göttler, G. Herzner, and E. Strohm, “Sym-
[40] D. L. Swofford, PAUP∗ : Phylogenetic Analysis Using Parsimony
biotic bacteria protect wasp larvae from fungal infestation,”
(∗ : and other Methods), Version 4, Sinauer Associates, Sunder-
Current Biology, vol. 15, no. 5, pp. 475–479, 2005.
land, Mass, USA, 2002.
[24] M. Kaltenpoth, W. Goettler, C. Dale et al., “Candidatus [41] M. Kimura, “A simple method for estimating evolutionary rates
Streptomyces philanthi, an endosymbiotic streptomycete in the of base substitutions through comparative studies of nucleotide
antennae of Philanthus digger wasps,” International Journal of sequences,” Journal of Molecular Evolution, vol. 16, no. 2, pp. 111–
Systematic and Evolutionary Microbiology, vol. 56, no. 6, pp. 120, 1980.
1403–1411, 2006.
[42] S. M. Bomfim, Isolamento de metabólitos antifúngicos de Strep-
[25] J. J. Scott, D. C. Oh, M. C. Yuceer, K. D. Klepzig, J. Clardy, and tomyces sp., UFPEDA, 3347, endófito de Momordica charantia L.,
C. R. Currie, “Bacterial protection of beetle-fungus mutualism,” (Curcubitaceae) [M.S. thesis], Universidade Federal de Pernam-
Science, vol. 322, no. 5898, p. 63, 2008. buco, Recife, Brazil, 2008.
[26] R. V. vander Meer, “Ant interactions with soil organisms and [43] CLSI (Clinical and Laboratory Standards Institute), “Norma
associated semiochemicals,” Journal of Chemical Ecology, vol. Aprovada M27-A2,” Método de referência para testes de
38, no. 6, pp. 728–745, 2012. diluição em caldo para determinação da sensibilidade de
[27] C. R. Currie, “Prevalence and impact of a virulent parasite on a leveduras à terapia antifúngica, 2002.
tripartite mutualism,” Oecologia, vol. 128, no. 1, pp. 99–106, 2001. [44] C. N. Seong, J. H. Choi, and K. S. Baik, “An improved selective
[28] D. C. Oh, M. Poulsen, C. R. Currie et al., “Dentigerumycin: a isolation of rare Actinomycetes from forest soil,” Journal of
bacterial mediator of an ant-fungus symbiosis,” Nature Chemi- Microbiology, vol. 39, no. 1, pp. 17–23, 2001.
cal Biology, vol. 5, no. 6, pp. 391–393, 2009. [45] M. G. Watve, R. Tickoo, M. M. Jog, and B. D. Bhole, “How many
[29] J. Barke, R. F. Seipke, S. Grüschow et al., “A mixed community antibiotics are produced by the genus Streptomyces?” Archives of
of actinomycetes produce multiple antibiotics for the fungus Microbiology, vol. 176, no. 5, pp. 386–390, 2001.
farming ant Acromyrmex octospinosus,” BMC Biology, vol. 8, [46] N. Imamura, M. Nishijima, K. Adachi, and H. Sano, “Novel
article 109, 2010. antimycin antibiotics, urauchimycins A and B, produced by
marine actinomycete,” Journal of Antibiotics, vol. 46, no. 2, pp.
[30] G. Carr, E. R. Derbyshire, E. Caldera et al., “Antibiotic and
241–246, 1993.
antimalarial quinones from fungus-growing ant-associated
Pseudonocardia sp.,” Journal of Natural Products, vol. 75, no. 10, [47] C. J. Barrow, J. J. Oleynek, H. H. Sun et al., “Antimycins,
pp. 1806–1809, 2012. inhibitors of ATP-citrate lyase, from a Streptomyces sp.,” Journal
of Antibiotics, vol. 50, no. 9, pp. 729–733, 1997.
[31] S. Haeder, R. Wirth, H. Herz, and D. Spiteller, “Candicidin-
producing Streptomyces support leaf-cutting ants to protect [48] C. B. F. Yao, M. Schiebel, E. Helmke, H. Anke, and H. Laatsch,
their fungus garden against the pathogenic fungus Escovopsis,” “Prefluostatin and new urauchimycin derivatives produced by
Proceedings of the National Academy of Sciences of the United Streptomycete isolates,” Zeitschrift fur Naturforschung B, vol. 61,
States of America, vol. 106, no. 12, pp. 4742–4746, 2009. no. 3, pp. 320–325, 2006.