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
The increased resistance of microorganisms to antibiotics is a problem of public health [
Actinobacteria are widely known for their ability to produce bioactive secondary metabolites, especially compounds with antimicrobial activity. These bacteria are responsible for producing two-thirds of the commercially available antibiotics [
Several studies have focused on the association between actinobacteria and insects from an ecological perspective [
Actinobacteria isolated from the integument of attine ants are generally classified in the genus
Poulsen [
Twelve
From each colony, we randomly selected four workers for actinobacteria isolation. Then, the propleural plates were scraped with a sterile needle under a low power stereomicroscope. All ants used in the present study had a visible, whitish covering on the propleural plates. Scrapings were plated on SCN agar (in g · L−1: 10.0 starch, 0.3 casein, 2.0 KNO3, 2.0 NaCl, 2.0 K2HPO4, 0.05 MgSO4 · 7 H2O, 0.02 CaCO3, 0.01 FeSO4 · 7H2O and 18.0 agar supplemented with 0.05 Nystatin) [
We used a molecular approach to provide taxonomic affiliation to actinobacteria strains. Genomic DNA was extracted following the method of Sampaio et al. [
A phylogenetic analysis was carried out in order to determine the taxonomic affiliation of strain TD025. Sequences of closest relatives were retrieved from the NCBI-GenBank and the RDP Project and aligned in ClustalW. Phylogenetic reconstruction was performed using the neighbor-joining algorithm implemented in PAUP v. 4.0 [
All actinobacteria were grown in Erlenmeyer flasks (250 mL) containing 50 mL of modified MPE medium (in g · L−1: 5.0 soy flour, 20.0 glucose, 5.0 of NaCl, 4.0 CaCO3) [
The antimicrobial activity of the extracts was evaluated against the yeasts
The actinobacteria strain that exhibited both a broad antifungal spectrum and lower MIC values was strain TD025. In order to identify the compounds responsible for the observed results, the strain was cultured in 5 L of modified MPE medium and the extracts were obtained as described.
The fermented broth (5 L) was separated from the cells by centrifugation and portioned three times with ethyl acetate (EtOAc). The organic solvent was evaporated under vacuum. The crude extract, a dark green oil (1.40 g), was separated by means of column chromatography using silica gel 60 eluted with n-hexane/EtOAc as the elution gradient, yielding 8 fractions. All fractions were submitted to antimicrobial assays against
After isolation and determination of the structure of the targeted compounds, they were evaluated for antimicrobial activity following the method described previously. Besides the MIC determination, we also evaluated the minimum fungicidal concentration (MFC). The MFC was determined by inoculating Sabouraud dextrose medium with 10
Several actinobacteria colonies were observed after incubation of isolation plates. We selected just one morphotype of each per ant colony, rendering a total of 24 strains ouf of 12
Actinobacteria identification according to 16S rDNA sequencing.
Isolate Id | bp1 | NCBI-GenBank closest relative | Coverage | % | Accession # |
---|---|---|---|---|---|
TD016 | 1251 |
|
100 | 100 | JF797311 |
TD017 | 1248 |
|
100 | 100 | JF797311 |
TD018 | 1184 |
|
99 | 100 | NR044144 |
TD019 | 1342 |
|
99 | 100 | AB184761 |
TD020 | 1167 |
|
99 | 99 | NR041160 |
TD021 | 1250 |
|
100 | 99 | NR041154 |
TD022 | 1254 |
|
100 | 100 | JN566018 |
TD023 | 1246 |
|
100 | 100 | JQ812074 |
TD025 | 1333 |
|
100 | 99 | JQ838121 |
|
100 | 99 | JQ222143 | ||
TD027 | 1342 |
|
99 | 100 | AB184761 |
TD028 | 1353 |
|
100 | 99 | AB048221 |
TD030 | 1255 |
|
100 | 99 | AB622252 |
TD032 | 1263 |
|
100 | 100 | NR041216 |
TD033 | 1278 |
|
99 | 100 | HQ699516 |
TD034 | 1283 |
|
100 | 100 | NR025562 |
TD035 | 1320 |
|
100 | 99 | FJ169330 |
TD045 | 1183 |
|
100 | 99 | HQ021204 |
TD047 | 1173 |
|
100 | 100 | AB327251 |
TD049 | 1260 |
|
100 | 99 | NR041128 |
TD050 | 1261 |
|
100 | 100 | NR041093 |
TD051 | 1173 |
|
100 | 100 | AB327251 |
TD053 | 1265 |
|
99 | 98 | AB184597 |
TD055 | 1173 |
|
100 | 100 | AB327251 |
TD058 | 1257 |
|
100 | 100 | HQ995504 |
In eight out of 12 ant colonies, we obtained more than one actinobacteria morphotype (Table S1). From colony CTL080820-02, the two morphotypes isolated from two different workers were identified as the same actinobacteria species (Tables S1 and
The 16S rDNA sequence of strain TD025 showed 99% similarity with sequences of several species of the genus
Phylogenetic relationships of strain TD025 (in bold) isolated from the integument of
Our screening for antifungal activity revealed that seven out of 24 extracts (29.16%) inhibited the growth of at least one
Minimum inhibitory concentrations (
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 | * | * | * | * | * | * |
Except for strain TD034 identified as
The extracts of
Chromatographic procedures revealed that EtOAc extract from TD025 contains two compounds (
Chemical structures of compounds isolated from
The 13C NMR spectrum of
The 1H NMR spectrum showed a singlet at
The 1H and 13C NMR spectram of
Urauchimycins belong to the antimycin class, a group of well-known antifungals. Antimycins act by inhibiting the electron flow in the mitochondrial respiratory chain [
Another antifungal compound widely distributed in
Urauchimycins A and B were previously isolated from
In 2006, two new urauchimycins were described: urauchimycin C, isolated from
The study of antimicrobial activity of urauchimycins A and B was restricted to
Minimum inhibitory concentration (MIC) and minimum fungicide concentrations (MFC) (
Candida species | Urauchimycin A | Urauchimycin B | Nystatin | |||
---|---|---|---|---|---|---|
MIC | MFC | MIC | MFC | MIC | MFC | |
|
1 | * | 1 | 3 | 1 | 2 |
|
800 | * | 2 | 3 | 1 | 2 |
|
2 | 15,6 | 2 | 2 | 1 | 1 |
|
15,6 | 15,6 | 2 | 3 | 2 | 3 |
|
* | * | 2 | 2 | 1 | 2 |
|
* | * | 2 | 2 | 4 | 4 |
Urauchimycin B showed a broad spectrum of activity against
Here we show that
Moreover, our study corroborates previous work [
As suggested by Poulsen [
The authors thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), CNPq/INCT (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and NSF IRFP (United States National Science Foundation International Research Fellowship Program no. 07012333) for financial support. The authors also thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior) for providing a scholarship to the first author. This work was conducted under collecting permit number 14789-1 issued by the “Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis” (IBAMA) and the “Instituto Chico Mendes de Conservação da Biodiversidade” (ICMBio). The authors also thank two anonymous referees for helpful comments on this paper.