Antimicrobial and antibiofilm potential of Streptomyces sp. metabolites against Staphylococcus aureus

Potencial antimicrobiano e antibiofilme dos metabólitos de Streptomyces sp. contra Staphylococcus aureus

Abstract

This study evaluated the antimicrobial and antibiofilm activities of metabolites produced by a Streptomyces strain isolated from impacted soil against Staphylococcus aureus. The study employed the Streptomyces sp. SLA3 strain. Secondary metabolites were obtained through submerged fermentation followed by liquid-liquid extraction. Chemical characterization was performed using gas chromatography coupled with mass spectrometry (GC-MS). The pathogenic models included S. aureus ATCC 25923 and five clinical isolates. Antimicrobial activity was assessed by the agar diffusion method and determination of minimum inhibitory concentration (MIC). Antibiofilm activity was evaluated through inhibition of biofilm formation and eradication of preformed biofilms. Toxicity evaluation was conducted using Tenebrio molitor larvae and hemolytic activity assays. Chemical characterization of the extract revealed a predominance of 2,4-bis(1,1-dimethylethyl)phenol and long-chain saturated fatty acids (myristic, palmitic, and stearic acids). Inhibition halos reached approximately 35 mm, and MIC values ranged from 1,000 to 2,000 μg/mL. The S. aureus CIBC 9722 strain exhibited significant inhibition of biofilm formation (p < 0.05). For S. aureus ATCC 25923, S. aureus CIBC 9722, S. aureus CIBC 20, and S. aureus CIBC 14, all extract concentrations promoted a significant reduction in preformed biofilms (p < 0.05). In the toxicity assay using T. molitor larvae, groups treated with extract concentrations of 1,000-500 µg/mL exhibited a survival rate of 75%. The effective concentration required to induce 50% hemolysis (HC₅₀) was estimated at 329.9 µg/mL. These findings highlight the biotechnological potential of metabolites from Streptomyces sp.

Keywords:
actinobacteria; antimicrobial; bioactives; secondary metabolites

Resumo

Este estudo avaliou as atividades antimicrobiana e antibiofilme de metabólitos produzidos por uma cepa de Streptomyces isolada de solo impactado contra Staphylococcus aureus. Foi utilizada a cepa Streptomyces sp. SLA3. Os metabólitos secundários foram obtidos por fermentação submersa, seguida de extração líquido-líquido. A caracterização química foi realizada por cromatografia gasosa acoplada à espectrometria de massas (GC-MS). Os modelos patogênicos incluíram S. aureus ATCC 25923 e cinco isolados clínicos. A atividade antimicrobiana foi avaliada pelo método de difusão em ágar e pela determinação da concentração inibitória mínima (CIM). A atividade antibiofilme foi analisada por meio da inibição da formação de biofilmes e da erradicação de biofilmes pré-formados. A toxicidade foi avaliada utilizando larvas de Tenebrio molitor e ensaios de atividade hemolítica. A caracterização química do extrato revelou predominância de 2,4-bis(1,1-dimetiletil)fenol e ácidos graxos saturados de cadeia longa (mirístico, palmítico e esteárico). Os halos de inibição atingiram aproximadamente 35 mm, e os valores de CIM variaram de 1.000 a 2.000 μg/mL. A cepa S. aureus CIBC 9722 apresentou inibição significativa da formação de biofilme (p < 0,05). Para S. aureus ATCC 25923, S. aureus CIBC 9722, S. aureus CIBC 20 e S. aureus CIBC 14, todas as concentrações do extrato promoveram redução significativa de biofilmes pré-formados (p < 0,05). No ensaio de toxicidade com larvas de T. molitor, os grupos tratados com concentrações de 1.000-500 µg/mL apresentaram taxa de sobrevivência de 75%. A concentração eficaz para induzir 50% de hemólise (EC₅₀) foi estimada em 329,9 µg/mL. Esses resultados evidenciam o potencial biotecnológico dos metabólitos de Streptomyces sp.

Palavras-chave:
actinobactérias; antimicrobiano; bioativos; metabólitos secundários

1. Introduction

Actinobacteria are filamentous, spore-forming Gram-positive microorganisms with wide geographic distribution and the ability to inhabit a broad range of ecosystems, with soil as their primary habitat, where they play a key ecological role (Amaral et al., 2023). The genus Streptomyces stands out due to its remarkable secondary metabolism, which confers considerable biotechnological and antimicrobial potential (Tyc et al., 2017; Amorim et al., 2020; Rosa et al., 2023; Dornelas et al., 2023).

Among the microorganisms that cause infections, Staphylococcus aureus stands out as a Gram-positive cocci group. One of the main strategies employed by this pathogen is biofilm formation, aggregates of microbial cells embedded in a heterogeneous extracellular matrix with distinct physiological characteristics that hinder immune responses and increase resistance to a wide range of antimicrobials. Methicillin-resistant strains (S. aureus MRSA) are of particular concern due to their complex defense mechanisms, which confer resistance to multiple antibiotics (Satpathy et al., 2016; Rashid et al., 2023; Al-Fawares et al., 2024).

Although Streptomyces species are well known for producing antimicrobial compounds, they have also garnered increasing attention for their ability to synthesize substances with antibiofilm properties. This enhances their scientific and practical relevance, making them promising candidates for the control of resistant pathogens (Matsumoto and Takahashi, 2017; Quinn et al., 2020). This study investigated the antimicrobial and antibiofilm activity of metabolites produced by Streptomyces strains isolated from impacted soil, against Staphylococcus aureus.

2. Material and Methods

2.1. Streptomyces sp. and bacterial strains

All bacterial strains were obtained from the Laboratório de Eletroquímica e Biotecnologia da Universidade Ceuma. The Streptomyces strain SLA3, isolated from landfill-impacted soil in Apicum-Açu, Maranhão (Santos et al., 2024), was identified by morphological traits typical of the genus. The pathogenic strains included Staphylococcus aureus ATCC 25923 and clinical blood isolates (CIBC 9722, 4102, 20, 14, and 4).

2.2. Submerged fermentation and crude extract production

To obtain active metabolites from Streptomyces sp. SLA3, submerged fermentation was conducted in 250 mL Erlenmeyer flasks containing 50 mL of PD broth supplemented with 2% glucose and five inoculated PD agar blocks, incubated at 30 °C for 15 days under agitation (Amorim et al., 2020). Metabolites were extracted by liquid-liquid partitioning with ethyl acetate (1:1), following centrifugation (2,500 rpm, 10 min) and filtration (Trisuwan et al., 2008). The organic phase was recovered, solvent evaporated, and the extract resuspended in 1% DMSO to a final concentration of 10,000 μg/mL.

2.3. Gas chromatography coupled to mass spectrometry (GC-MS)

2.3.1. Headspace solid-phase microextraction (HS-SPME)

Based on sample chemistry, a 100 μm DVB/CAR/PDMS fiber (Supelco) was used. Fermentation occurred in 20 mL vials containing 500 μL of extract and 500 μL of 0.25% (v/v) saline to reduce surface tension and enhance headspace enrichment of volatiles, minimizing metabolite losses. Vials were sealed (PTFE cap, silicone septum), magnetically stirred in a water bath at 79 °C for 30 min, with 2 min equilibration, followed by headspace SPME according to Ramos et al. (2009). After extraction, the fiber was thermally desorbed in the GC injector for 4 min, as described by Ramos et al. (2020).

2.3.2. Chromatographic analysis by GC-MS and quantification of volatile compounds

The identification and quantification of volatile compounds from solid-state fermentations were performed by GC (GCMS-QP2010 Plus, Shimadzu) using a RESTEK RTX-5 column (30 m × 0.25 mm × 0.25 μm), with injector and FID at 250 °C in splitless mode. SPME headspace samples were injected under a temperature program of 100 °C (4 min), increased to 150 °C at 10 °C/min (4 min), to 200 °C at 20 °C/min (4 min), and to 230 °C at 10 °C/min (4 min). Nitrogen was used as carrier gas at 1.2 mL/min, with a total run time of 28 min.

2.4. Antimicrobial activity

2.4.1. Agar well diffusion assay

The agar well diffusion assay followed Bauer et al. (1966). Secondary metabolites from Streptomyces sp. SLA3 (1,000 µg/mL in 1% DMSO) were tested against 0.5 McFarland-standardized bacteria inoculated on Mueller-Hinton agar. Wells (5 mm) received 100 µL of the solutions and plates were incubated at 37 °C for 24 h in triplicate, using chloramphenicol (30 µg) as positive control. Antimicrobial activity was determined by inhibition zone diameters according to CLSI (2019).

2.4.2. Determination of minimum inhibitory concentration (MIC) by microdilution assay

Minimum Inhibitory Concentration (MIC) was determined by a 1:2 serial microdilution assay in 96-well plates (Zgoda and Porter, 2001) using Streptomyces sp. SLA3 metabolites (2,000 µg/mL) in MH broth. Inocula (0.5 McFarland; 10 µL) were added, with chloramphenicol (30 µg) as positive control, and plates were incubated at 37 °C for 24 h in triplicate. MIC was defined as the lowest concentration inhibiting visible growth and confirmed with 0.1% resazurin after a further 24 h, where a purple-to-pink change indicated growth.

2.5. Antibiofilm activity

2.5.1. Inhibition of biofilm formation on polystyrene plates (crystal violet assay)

The assay was conducted in 96-well microplates using bacterial suspensions (0.5 McFarland) in TSB with extract dilutions (MIC, 1/2 MIC, 1/4 MIC). After 24 h at 37 °C, wells were washed with PBS, fixed with methanol, stained with crystal violet, and the dye was solubilized with acetic acid. Absorbance was read at 570 nm, and biofilm production was classified by comparing isolate optical density (ODi) with the negative control (ODc) as non-, weak, moderate, or strong producer (Gomes et al., 2013).

2.5.2. Eradication of pre-formed biofilms on polystyrene plates (crystal violet assay)

This assay was performed in 96-well microtiter plates. Pathogenic suspensions (0.5 McFarland; OD 0.2 at 580 nm) were inoculated in TSB (180 μL) and incubated for 24 h at 37 °C. After washing, TSB and extract dilutions (2×MIC and MIC) were added and incubated for a further 24 h. Biofilms were then fixed with methanol, stained with crystal violet, solubilized with acetic acid, and the absorbance was read at 570 nm. Biofilm eradication was classified as previously described (Gomes et al., 2013).

2.6. Toxicity assessment

2.6.1. Toxicity in Tenebrio molitor larvae

Tenebrio molitor larvae (100-200 mg) were obtained commercially and distributed into three groups (n=10), each inoculated with 10 µL of Streptomyces sp. SLA3 metabolites at MIC, 1/2 MIC, and 1/4 MIC, following Amorim et al. (2020). PBS served as the negative control. Larvae were monitored for 5 days to assess survival and mortality based on responsiveness and melanization.

2.6.2. Hemolytic activity assay

An aliquot of 4 mL of commercially obtained sheep blood was washed with saline (0.9% NaCl) by centrifugation (3,000 rpm, 5 min), and the erythrocytes were resuspended in PBS to obtain a 1% (v/v) suspension. Equal volumes of this suspension and serial dilutions (1:2; 2,000-15.6 µg/mL) of secondary metabolite extracts from Streptomyces sp. SLA3 were incubated at 37 °C for 60 min under agitation, centrifuged, and the supernatant absorbance was read at 540 nm. Saline and distilled water were used as minimum and maximum hemolytic controls, and the mean hemolytic concentration (HC₅₀) was calculated by nonlinear regression (Yang et al., 2005).

2.7. Statistical analysis

Data were analyzed using GraphPad Prism 8. Differences between groups were assessed by ANOVA followed by Dunnett's multiple comparisons test; survival curves (T. molitor) by Kaplan-Meier with log-rank test; and HC₅₀ by nonlinear regression. Results were considered significant at p < 0.05.

3. Results

3.1. Gas chromatography coupled to mass spectrometry (GC-MS)ra

The analysis of the crude extract of Streptomyces sp. SLA3 by GC-MS enabled the elucidation of the metabolic profile of the isolated strain (Figure 1). The total ion chromatogram (TIC) revealed the presence of major constituents eluted between 17 and 25 minutes, characteristic of metabolites with medium to low polarity.

Figure 1
Chemical characterization by gas chromatography coupled to mass spectrometry (GC-MS) of the secondary metabolite extract of Streptomyces sp. SLA3.

Spectral evaluation indicated the predominance of the lipophilic phenolic compound 2,4-bis(1,1-dimethylethyl)phenol, corresponding to peak 5, eluted at a retention time (RT) of approximately 17.4 min, and representing the highest relative abundance in the sample. Additionally, a homologous series of long-chain saturated fatty acids was detected. Tetradecanoic acid (myristic acid) was identified in peak 7 (RT ~20.5 min), followed by n-hexadecanoic acid (palmitic acid) in peak 12 (RT ~22.7 min), and octadecanoic acid (stearic acid), corresponding to peak 15 (RT ~24.9 min). Compound identification was performed through comparison of the obtained fragmentation patterns with reference mass spectral libraries.

3.2. Antimicrobial activity

The agar diffusion method was employed as a preliminary test to evaluate the antibacterial activity of Streptomyces sp. SLA3, revealing visible inhibition zones against the S. aureus strains tested. The halos formed measured over 25 mm in diameter, indicating the susceptibility of the pathogens to SLA-derived metabolites. Furthermore, Streptomyces sp. SLA3 exhibited MIC values ranging from 1,000 µg/mL to 2,000 µg/mL against the tested strains. The inhibition zones and MIC values for each bacterial strain are presented in Table 1.

Table 1
Antimicrobial activity of the secondary metabolite extract from the Streptomyces sp. SLA3 isolate, obtained from the Apicum-Açu landfill soil, Maranhão, Brazil, against Staphylococcus aureus.

Microrganisms	Agar Diffusion (±SD)	Microdilution
S. aureus ATCC 25923	28 mm (± 3.60)	2,000 µg/ mL
S. aureus CIBC 14	33.6 mm (± 3.51)	1,000 µg/ mL
S. aureus CIBC 9722	26.6 mm (± 1.15)	1,500 µg/ mL
S. aureus CIBC 20	33.6 mm (± 0.57)	2,000 µg/ mL
S. aureus CIBC 4201	35 mm (± 1.00)	2,000 µg/ mL
S. aureus CIBC 4	33.6 mm (± 0.57)	2,000 µg/ mL

• SD: Standard Deviation; ATCC: American Type Culture Collection; CIBC: Clinical Isolate Blood Culture.

3.3. Antibiofilm activity

Regarding biofilm formation, all strains of S. aureus demonstrated the ability to produce biofilms on polystyrene surfaces, albeit with varying adherence intensities. This variation enabled the comparison of the reduction percentage (%) of biofilm formation or potential increase in the presence and absence of the secondary metabolite extract from Streptomyces sp. SLA3. Adherence was classified as strong for S. aureus CIBC 14 and as moderate for all other tested strains: S. aureus ATCC 25923, S. aureus CIBC 9722, S. aureus CIBC 20, S. aureus CIBC 4201, and S. aureus CIBC 4.

The activity of the extract against biofilm formation inhibition demonstrated variable effects among samples as well as across the tested concentrations, revealing distinct response profiles (Figure 2). The S. aureus CIBC 9722 strain exhibited inhibition of biofilm formation under the influence of the extract, with significance at subinhibitory concentrations (p < 0.05). An opposite pattern was observed for the isolates S. aureus ATCC 25923, S. aureus CIBC 20, S. aureus CIBC 14, S. aureus CIBC 4201, and S. aureus CIBC 4, which showed a significant increase in biofilm biomass compared to the PC at all concentrations (p < 0.05). Overall, the inhibition data indicate that the extract exhibits a complex extract-bacterium interaction, in which most strains responded with an increase in biofilm biomass.

Figure 2
Inhibition of Staphylococcus aureus biofilm formation with interference from secondary metabolites of Streptomyces sp. SLA3 isolated from landfill soil in Apicum-Açu, Maranhão, Brazil. ATCC: American Type Culture Collection; MIC: Minimum Inhibitory Concentration; CIBC: Clinical Isolate Blood Culture. PC: Positive Control. Statistically significant difference compared to the positive control (PC), where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (One-Way ANOVA / Dunnett's multiple comparisons test).

The ability of the extract to eradicate mature biofilms was also evaluated under the same experimental conditions. Overall, a more consistent profile was observed among the sample, with a significant reduction (p < 0.05) in biofilm biomass at the suprainhibitory concentration (Figure 3). For the sample S. aureus ATCC 25923, S. aureus CIBC 9722, S. aureus CIBC 20, and S. aureus CIBC 14, all extract concentrations promoted a significant reduction in preformed biofilm biomass, indicating strong antibiofilm activity against mature structures (p < 0.05). The sample S. aureus CIBC 4201 exhibited a non-significant reduction at the suprainhibitory concentration but showed a significant increase in biomass formation at the MIC concentration (p = 0.0274). For the sample S. aureus CIBC 4, there was a non-significant increase in mature biofilm production. The data demonstrate that the extract performs better in eradicating preformed biofilms than in inhibiting their initial formation, particularly for susceptible clinical strains.

Figure 3
Eradication of Staphylococcus aureus pre-formed biofilm with interference from secondary metabolites of Streptomyces sp. SLA3 isolated from landfill soil in Apicum-Açu, Maranhão, Brazil. ATCC: American Type Culture Collection; MIC: Minimum Inhibitory Concentration; CIBC: Clinical Isolate Blood Culture. PC: Positive Control. Statistically significant difference compared to the positive control (PC), where *p < 0.05, ***p < 0.001, and ****p < 0.0001 (One-Way ANOVA / Dunnett's multiple comparisons test).

3.4. Toxicity assessment

In the toxicity assay conducted with T. molitor larvae, the groups treated with extract concentrations of 1000 µg/mL and 500 µg/mL showed a survival rate of 75%, comparable to the control group, which exhibited an 80% survival rate. These results indicate that the extract does not present significant toxicity (p = 0.5351) at these concentrations (Figure 4). Conversely, the concentration of 2000 µg/mL resulted in a substantially lower survival rate of only 40%, suggesting a toxic effect at higher doses.

Figure 4
Comparison of the survival curve of Tenebrio molitor larvae in response to the secondary metabolite extract of Streptomyces sp. SLA3. Statistically significant difference when p < 0.05 (Log-Rank test (Mantel-Cox)).

In the hemolytic activity toxicity assessment, the extract exhibited low toxicity, with hemolytic variations ranging from 12.63% to 12.80% at the tested concentrations (62.5-2000 µg/mL). The effective concentration required to induce 50% hemolysis (HC₅₀) was estimated at 329.9 µg/mL.

4. Discussion

The increasing prevalence of microbial resistance has prompted numerous studies aimed at identifying new compounds for pharmaceutical and biotechnological applications. Extracts produced from the Streptomyces sp. SLA3 strain demonstrated antimicrobial potential, highlighting opportunities for further investigation (Santos et al., 2024).

The Streptomyces genus is widely recognized as one of the most prolific sources of natural antibiotics, notably including aminoglycosides, tetracyclines, sulfonamides, chloramphenicol, β-lactams, and macrolides (Goel et al., 2021). It is estimated that, since 1942, approximately 3,000 antibiotics have been discovered, with around 80% derived from species belonging to this genus (Bunbamrung et al., 2020; Mahmood and Kataoka, 2020). These data underscore the relevance of isolating the Streptomyces sp. SLA3 strain, given its potential for biosynthesizing compounds with antimicrobial activity.

Chemical characterization of the extract produced in this study revealed a predominance of the lipophilic phenolic compound 2,4-bis(1,1-dimethylethyl)phenol, along with long-chain saturated fatty acids, including myristic, palmitic, and stearic acids. These compounds confirm the lipophilic nature of the Streptomyces sp. SLA3 extract. The presence of these bioactive molecules can be associated with both the antimicrobial and antibiofilm activities observed, as numerous studies have reported that isolated phenolic compounds and fatty acids exhibit anticancer, anti-inflammatory, antimicrobial, and antibiofilm properties. Various assays indicate that lipophilic compounds can interfere with microbial membrane fluidity and modulate the expression of genes associated with biofilm production, providing a plausible basis for the antimicrobial activity observed against S. aureus in the SLA3 extract (Kim et al., 2021, 2022; Seenivasan et al., 2022; Sundar and Arunachalam, 2025).

The major compound, 2,4-bis(1,1-dimethylethyl)phenol, has been reported as a metabolic bioproduct in various animal, plant, and microbial species, including bacteria of the genus Streptomyces (Akshatha et al., 2016; Belghit et al., 2016; Zhao et al., 2020; Saikia et al., 2022). Zhao et al. (2020) highlighted the diverse bioactivities of this compound, including antioxidant, anti-inflammatory, insecticidal, nematicidal, antibacterial, antifungal, and antiviral effects. Beyond membrane interaction and gene modulation, 2,4-bis(1,1-dimethylethyl)phenol has been shown to influence the production of extracellular polymeric substances (EPS), interfering with bacterial intercellular communication (quorum sensing), facilitating disruption of pre-formed biofilms, and enhancing antimicrobial diffusion within microbial aggregates (Padmavathi et al., 2014; Padmavathi et al., 2015).

Thus, the presence of these bioactive compounds can be correlated with the antimicrobial activity observed for the Streptomyces sp. SLA3 extract. The metabolic extract exhibited antimicrobial activity against S. aureus, with MIC values ranging from 1,000-2,000 µg/mL. Several studies have highlighted the potential of Streptomyces strains in producing metabolites with potent antibacterial activity against S. aureus, including methicillin-resistant strains (MRSA). For instance, Jabila Mary et al. (2021) reported that Streptomyces sp. SBRK2, isolated from a marine sponge in India, produced 8-O-methyltetrangomycin, which displayed a 28 mm inhibition zone against MRSA and a MIC of 2 μg/mL. Similarly, Lin et al. (2021) isolated actinomycin D from Streptomyces parvulus, showing significant inhibitory activity against S. aureus with a MIC of 2 mg/mL. Bhandari et al. (2022) reported promising results with Streptomyces SB10, isolated from Nepali soil, producing metabolites such as brevianamide F and spiramycin, with inhibition zones of 30 mm and a MIC of 1.22 μg/mL. The combination of phenolic compounds and fatty acids may act synergistically, promoting membrane destabilization and interfering with bacterial metabolic processes.

It is also essential to evaluate the extract’s antibiofilm activity. Biofilms formed by S. aureus pose a significant challenge in infection management, as they persist on inert surfaces and biological tissues, particularly in hospital environments, and are closely associated with bacterial virulence. The assessment of biofilm formation revealed strain-dependent responses: while S. aureus CIBC 9722 exhibited significant inhibition of biofilm formation at subinhibitory concentrations, most tested strains showed increased biomass, indicating a complex extract-bacterium interaction.

This increase may be related to exposure to subinhibitory concentrations of the extract, a phenomenon widely described for S. aureus, in which levels below the MIC can stimulate biofilm production via adaptive mechanisms such as modulation of regulatory systems (agr/sarA), increased extracellular DNA release, and alterations in the extracellular matrix composition. Such behavior has been reported for various antimicrobials, including β-lactams, glycopeptides, fluoroquinolones, rifampin, mupirocin, and fosfomycin, typically at 1/2 to 1/8 MIC, resulting in increased biofilm biomass and stability (Luo et al., 2023; Elawady et al., 2024; Zeng et al., 2025).

Conversely, the eradication of pre-formed biofilms demonstrated greater consistency, with significant biomass reduction in several strains, suggesting that secondary metabolites act more effectively on already established biofilms than in their prevention. Singh and Dubey (2020) demonstrated, in biofilm interference assays against S. aureus ATCC 25923, up to 36.69% inhibition at 2×MIC; in assays evaluating pre-formed biofilms, the same strain exhibited 24.77% inhibition at 2×MIC. These findings highlight the potential of pre-formed biofilm eradication in mitigating antimicrobial resistance these pathogens.

Such assays provide a basis for developing new therapeutic formulations against pathogenic microorganisms. In addition to antimicrobial and antibiofilm activities, safety assessments are crucial. Toxicity evaluations demonstrated low hemolytic activity (EC₅₀ = 329.9 µg/mL) and moderate mortality in T. molitor larvae at concentrations up to 1000 µg/mL, indicating a favorable safety profile for potential therapeutic applications.

Integration of chemical and biological data suggests that phenolic compounds and fatty acids may be responsible for both antimicrobial activity and selective effects on biofilms, explaining the differing response patterns observed among strains and concentrations. From a biotechnological perspective, the results reinforce the potential of Streptomyces secondary metabolites as antimicrobial and antibiofilm sources, with low toxicity.

Acknowledgements

The authors thank Ceuma University and the Programa de Pós-Graduação em Biodiversidade e Biotecnologia da Rede BIONORTE for providing infrastructure and technical support essential for conducting this study. The authors also acknowledge financial support from Fundação de Amparo à Pesquisa do Estado do Maranhão (FAPEMA) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This research was funded by the Maranhão State Research Foundation (FAPEMA), under Edital FAPEMA 38/2022, Process ACT-FAPEMA 02510/2023.

Data Availability Statement

The entire dataset supporting the results of this study was published in the article itself.

References

• AKSHATHA, J.V., PRAKASH, H.S. and NALINI, M.S., 2016. Actinomycete endophytes from the ethno medicinal plants of Southern India: antioxidant activity and characterization studies. Journal of Biologically Active Products from Nature, vol. 6, no. 2, pp. 166-172. https://doi.org/10.1080/22311866.2016.1191971
  » https://doi.org/10.1080/22311866.2016.1191971
• AL-FAWARES, O., BASHABSHEH, R.H.F., NATSHEH, I.Y. and ABURAYYAN, W., 2024. Molecular investigation of Staphylococcus aureus isolated from inanimate surfaces in Jordanian hospitals. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 84, pp. e285397. https://doi.org/10.1590/1519-6984.285397 PMid:39907330.
  » https://doi.org/10.1590/1519-6984.285397
• AMARAL, S.S., SANTOS, D.C.P., PIRES, S.L., ALMEIDA, I.A., MOTA, A.G., ALMEIDA, B.L., SOUZA, G.S., MACHADO, D.J.S., ROSA, F.C., ALVES, M.S., ALIANÇA, A.S.S. and MIRANDA, R.C.M., 2023. Atividades biológicas de composos produzidos por Streptomyces sp. isolados do ambiente. In: M.R.C. SILVA, R.C.M. MIRANDA, E.H.C. RODRIGUES and D.F. SILVA, orgs. Indicadores de Qualidade Ambiental: uma perspectiva a partir da Zona de Transição Amazônica-Cerrado. 2. ed. São Luís: Pascal, vol. 2, pp. 29-41.
• AMORIM, E.A.F., CASTRO, E.J.M., DA SOUZA, S.V., ALVES, M.S., DIAS, L.R.L., MELO, M.H.F., DA SILVA, I.M.A., VILLIS, P.C.M., BONFIM, M.R.Q., FALCAI, A., SILVA, M.R.C., MONTEIRO-NETO, V., ALIANÇA, A., DA SILVA, L.C.N. and DE MIRANDA, R.C.M., 2020. Antimicrobial potential of Streptomyces ansochromogenes (PB3) isolated from a plant native to the Amazon against Pseudomonas aeruginosa. Frontiers in Microbiology, vol. 11, pp. 574693. https://doi.org/10.3389/fmicb.2020.574693 PMid:33162956.
  » https://doi.org/10.3389/fmicb.2020.574693
• BAUER, A.W., KIRBY, W.M., SHERRIS, J.C. and TURCK, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, vol. 40, no. 4, pp. 493-496. https://doi.org/10.1093/ajcp/45.4_ts.493 PMid:5325707.
  » https://doi.org/10.1093/ajcp/45.4_ts.493
• BELGHIT, S., DRICHE, E.H., BIJANI, C., ZITOUNI, A., SABAOU, N., BADJI, B. and MATHIEU, F., 2016. Activity of 2,4-Di-tert-butylphenol produced by a strain of Streptomyces mutabilis isolated from a Saharan soil against Candida albicans and other pathogenic fungi. Journal de Mycologie Médicale, vol. 26, no. 2, pp. 160-169. https://doi.org/10.1016/j.mycmed.2016.03.001 PMid:27107984.
  » https://doi.org/10.1016/j.mycmed.2016.03.001
• BHANDARI, S., BHATTARAI, B.R., ADHIKARI, A., ARYAL, B., SHRESTHA, A., ARYAL, N., LAMICHHANE, U., THAPA, R., THAPA, B.B., YADAV, R.P., KHADAYAT, K., ADHIKARI, A., REGMI, B.P. and PARAJULI, N., 2022. Characterization of Streptomyces species and validation of antimicrobial activity of their metabolites through molecular docking. Processes (Basel, Switzerland), vol. 10, no. 10, pp. 2149. https://doi.org/10.3390/pr10102149
  » https://doi.org/10.3390/pr10102149
• BUNBAMRUNG, N., INTARAUDOM, C., DRAMAE, A., THAWAI, C., TADTONG, S., AUNCHAROEN, P. and PITTAYAKHAJONWUT, P., 2020. Antibacterial, antitubercular, antimalarial and cytotoxic substances from the endophytic Streptomyces sp. TBRC7642. Phytochemistry, vol. 172, pp. 112275. https://doi.org/10.1016/j.phytochem.2020.112275 PMid:31986449.
  » https://doi.org/10.1016/j.phytochem.2020.112275
• CLINICAL AND LABORATORY STANDARDS INSTITUTE – CLSI, 2019. Performance standards for antimicrobial susceptibility testing 29th ed. Wayne, PA: CLSI. Supplement M100.
• DORNELAS, J.C.M., CARMO, P.H.F., LANA, U.G.P., LANA, M.A.G., PAIVA, C.A.O. and MARRIEL, I.E., 2023. Biocontrol potential of actinobacteria against Pantoea ananatis, the causal agent of maize white spot disease. Brazilian Journal of Biology, vol. 83, pp. e268015. https://doi.org/10.1590/1519-6984.268015 PMid:37283372.
  » https://doi.org/10.1590/1519-6984.268015
• ELAWADY, R., ABOULELA, A.G., GABALLAH, A., GHAZAL, A.A. and AMER, A.N., 2024. Antimicrobial Sub-MIC induces Staphylococcus aureus biofilm formation without affecting the bacterial count. BMC Infectious Diseases, vol. 24, no. 1, pp. 1065. https://doi.org/10.1186/s12879-024-09790-3 PMid:39342123.
  » https://doi.org/10.1186/s12879-024-09790-3
• GOEL, N., FATIMA, S.W., KUMAR, S., SINHA, R. and KHARE, S.K., 2021. Antimicrobial resistance in biofilms: exploring marine actinobacteria as a potential source of antibiotics and biofilm inhibitors. Biotechnology Reports (Amsterdam, Netherlands), vol. 30, pp. e00613. https://doi.org/10.1016/j.btre.2021.e00613 PMid:33996521.
  » https://doi.org/10.1016/j.btre.2021.e00613
• GOMES, D.L.R., PEIXOTO, R.S., BARBOSA, E.A.B., NAPOLEÃO, F., SABBADINI, P.S., DOS SANTOS, K.R.N., MATTOS-GUARALDI, A.L. and HIRATA, R., 2013. SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains. Journal of Medical Microbiology, vol. 62, no. Pt 5, pp. 754-760. https://doi.org/10.1099/jmm.0.052373-0 PMid:23449875.
  » https://doi.org/10.1099/jmm.0.052373-0
• JABILA-MARY, T.R., KANNAN, R.R., INIYAN, A.M., RAMACHANDRAN, D. and PRAKASH VINCENT, S.G., 2021. Cell wall distraction and biofilm inhibition of marine Streptomyces-derived angucycline in methicillin-resistant Staphylococcus aureus. Microbial Pathogenesis, vol. 150, pp. 104712. https://doi.org/10.1016/j.micpath.2020.104712 PMid:33359358.
  » https://doi.org/10.1016/j.micpath.2020.104712
• KIM, Y.G., LEE, J.H. and LEE, J., 2021. Antibiofilm activities of fatty acids including myristoleic acid against Cutibacterium acnes via reduced cell hydrophobicity. Phytomedicine : International Journal of Phytotherapy and Phytopharmacology, vol. 91, pp. 153710. https://doi.org/10.1016/j.phymed.2021.153710 PMid:34461422.
  » https://doi.org/10.1016/j.phymed.2021.153710
• KIM, Y.G., LEE, J.H., PARK, S., KIM, S. and LEE, J., 2022. Inhibition of polymicrobial biofilm formation by saw palmetto oil, lauric acid and myristic acid. Microbial Biotechnology, vol. 15, no. 2, pp. 590-602. https://doi.org/10.1111/1751-7915.13864 PMid:34156757.
  » https://doi.org/10.1111/1751-7915.13864
• LIN, Y., HUANG, L., ZHANG, X., YANG, J., CHEN, X., LI, F., LIU, J. and HUANG, R., 2021. Multi-omics analysis reveals anti-Staphylococcus aureus activity of actinomycin D originating from Streptomyces parvulus. International Journal of Molecular Sciences, vol. 22, no. 22, pp. 12231. https://doi.org/10.3390/ijms222212231 PMid:34830114.
  » https://doi.org/10.3390/ijms222212231
• LUO, Z.X., LI, Y., LIU, M.F. and ZHAO, R., 2023. Ciprofloxacin enhances the biofilm formation of Staphylococcus aureus via an agrC-dependent mechanism. Frontiers in Microbiology, vol. 14, pp. 1328947. https://doi.org/10.3389/fmicb.2023.1328947 PMid:38179460.
  » https://doi.org/10.3389/fmicb.2023.1328947
• MAHMOOD, A. and KATAOKA, R., 2020. Metabolite profiling reveals a complex response of plants to application of plant growth-promoting endophytic bacteria. Microbiological Research, vol. 234, pp. 126421. https://doi.org/10.1016/j.micres.2020.126421 PMid:32006789.
  » https://doi.org/10.1016/j.micres.2020.126421
• MATSUMOTO, A. and TAKAHASHI, Y., 2017. Endophytic actinomycetes: promising source of novel bioactive compounds. The Journal of Antibiotics, vol. 70, no. 5, pp. 514-519. https://doi.org/10.1038/ja.2017.20 PMid:28270688.
  » https://doi.org/10.1038/ja.2017.20
• PADMAVATHI, A.R., ABINAYA, B. and PANDIAN, S.K., 2014. Phenol, 2,4-bis(1,1-dimethylethyl) of marine bacterial origin inhibits quorum sensing mediated biofilm formation in the uropathogen Serratia marcescens. Biofouling, vol. 30, no. 9, pp. 1111-1122. https://doi.org/10.1080/08927014.2014.972386 PMid:25377484.
  » https://doi.org/10.1080/08927014.2014.972386
• PADMAVATHI, A.R., PERIYASAMY, M. and PANDIAN, S.K., 2015. Assessment of 2,4-Di-tert-butylphenol induced modifications in extracellular polymeric substances of Serratia marcescens. Bioresource Technology, vol. 188, pp. 185-189. https://doi.org/10.1016/j.biortech.2015.01.049 PMid:25641715.
  » https://doi.org/10.1016/j.biortech.2015.01.049
• QUINN, G.A., BANAT, A.M., ABDELHAMEED, A.M. and BANAT, I.M., 2020. Streptomyces from traditional medicine: sources of new innovations in antibiotic discovery. Journal of Medical Microbiology, vol. 69, no. 8, pp. 1040-1048. https://doi.org/10.1099/jmm.0.001232 PMid:32692643.
  » https://doi.org/10.1099/jmm.0.001232
• RAMOS, A.L.C.C., MENDES, D.D., SILVA, M.R., AUGUSTI, R., MELO, J.O.F., ARAÚJO, R.L.B. and LACERDA, I.C.A., 2020. Chemical profile of Eugenia brasiliensis (Grumixama) pulp by PS/MS paper spray and SPME-GC/MS solid-phase microextraction. Research, Social Development, vol. 9, no. 7, pp. E318974008.
• RAMOS, A.S., MARTINS, P.S.O., FIAUX, S.B. and LEITE, S.G.F., 2009. Solid-phase microextraction of 6-pentyl-α-pyrone produced by solid-state fermentation. Food Science and Technology, vol. 29, no. 3, pp. 523-528. https://doi.org/10.1590/S0101-20612009000300011
  » https://doi.org/10.1590/S0101-20612009000300011
• RASHID, N., SHAFEE M, IQBAL S, SAMAD A, KHAN, S.A., HASNI, M.S., REHMAN, Z.U., ULLAH, S., REHMAN, F.U., KHAN, G.I., AHMAD, S. and AKBAR, A., 2023. Contaminação de Staphylococcus aureus resistente à meticilina enterotoxigênica em peixes salgados de Gwadar Balochistan. Brazilian Journal of Biology, vol. 83, pp. e247701. https://doi.org/10.1590/1519-6984.247701
  » https://doi.org/10.1590/1519-6984.247701
• ROSA, F.C., MARTINS, P.S.O., FIAUX, S.B., LEITE, S.G.F., ALMEIDA, I.A., MOTA, A.G. and MIRANDA, R.C.M., 2023. Bioprospecting of secondary metabolites produced by endophytic actinomycete isolated from Aloe vera. Concilium, vol. 23, no. 22, pp. 227-239. https://doi.org/10.53660/CLM-2464-23T02
  » https://doi.org/10.53660/CLM-2464-23T02
• SAIKIA, J., MAZUMDAR, R. and THAKUR, D., 2022. Phylogenetic affiliation of endophytic actinobacteria associated with selected orchid species and their role in growth promotion and suppression of phytopathogens. Frontiers in Plant Science, vol. 13, pp. 1058867. https://doi.org/10.3389/fpls.2022.1058867 PMid:36570961.
  » https://doi.org/10.3389/fpls.2022.1058867
• SANTOS, D.C.P., GOUVEIA, S.S.A., PIRES, S.L., BELÉM, G.S.P., GUIMARÃES, S.V., AMARAL, S.S. and MIRANDA, R.C.M., 2024. Potencial antimicrobiano de metabólitos secundários de Streptomyces sp. isolados de solo impactado. Peer Review : Emerging Trends and Key Debates in Undergraduate Education, vol. 6, no. 5, pp. 234-245. https://doi.org/10.53660/PRW-1961-3622
  » https://doi.org/10.53660/PRW-1961-3622
• SATPATHY, S., SEN, S.K., PATTANAIK, S. and RAUT, S., 2016. Review on bacterial biofilm: an universal cause of contamination. Biocatalysis and Agricultural Biotechnology, vol. 7, pp. 56-66. https://doi.org/10.1016/j.bcab.2016.05.002
  » https://doi.org/10.1016/j.bcab.2016.05.002
• SEENIVASAN, A., MANIKKAM, R., KAARI, M., SAHU, A.K., SAID, M. and DASTAGER, S.G., 2022. 2, 4-Di-tert-butylphenol (2, 4-DTBP) purified from Streptomyces sp. KCA1 from Phyllanthus niruri: isolation, characterization, antibacterial and anticancer properties. Journal of King Saud University. Science, vol. 34, no. 5, pp. 102088. https://doi.org/10.1016/j.jksus.2022.102088
  » https://doi.org/10.1016/j.jksus.2022.102088
• SINGH, R. and DUBEY, A.K., 2020. Isolation and characterization of new endophytic actinobacterium Streptomyces californicus strain ADR1 as a promising source of anti-bacterial, anti-biofilm and antioxidant metabolites. Microorganisms, vol. 8, no. 6, pp. 929. https://doi.org/10.3390/microorganisms8060929 PMid:32575628.
  » https://doi.org/10.3390/microorganisms8060929
• SUNDAR, R.D.V. and ARUNACHALAM, S., 2025. 2,4-Di-terc-butilfenol do fungo endofítico Fusarium oxysporum atenua o crescimento de patógenos multirresistentes. Frontiers in Microbiology, vol. 16, pp. 1575021. https://doi.org/10.3389/fmicb.2025.1575021 PMid:40556898.
  » https://doi.org/10.3389/fmicb.2025.1575021
• TRISUWAN, K., RUKACHAISIRIKUL, V., SUKPONDMA, Y., PREEDANON, S., PHONGPAICHIT, S., RUNGJINDAMAI, N. and SAKAYAROJ, J., 2008. Epoxydans and pyrone from the marine-derived fungus Nigrospora sp. PSU-F5. Journal of Natural Products, vol. 71, no. 8, pp. 1323-1326. https://doi.org/10.1021/np8002595 PMid:18646829.
  » https://doi.org/10.1021/np8002595
• TYC, O., SONG, C., DICKSCHAT, J.S., VOS, M. and GARBEVA, P., 2017. The ecological role of volatile and soluble secondary metabolites produced by soil bacteria. Trends in Microbiology, vol. 25, no. 4, pp. 280-292. https://doi.org/10.1016/j.tim.2016.12.002 PMid:28038926.
  » https://doi.org/10.1016/j.tim.2016.12.002
• YANG, Z.G., SUN, H.X. and FANG, W.H., 2005. Haemolytic activities and adjuvant effect of Astragalus membranaceus saponins (AMS) on the immune responses to ovalbumin in mice. Vaccine, vol. 23, no. 44, pp. 5196-5203. https://doi.org/10.1016/j.vaccine.2005.06.016 PMid:16043270.
  » https://doi.org/10.1016/j.vaccine.2005.06.016
• ZENG, T., WANG, Y., ZHU, Q., XI, H., LIU, M., LIU, P., BAI, Y., YUAN, L., ZHAO, R., SHENG, Y.-Y. and DAI, Q., 2025. Sub-inhibitory concentrations of fosfomycin enhance Staphylococcus aureus biofilm formation by a sarA-dependent mechanism. Microbiology Spectrum, vol. 13, no. 9, pp. e0152125. https://doi.org/10.1128/spectrum.01521-25 PMid:40689791.
  » https://doi.org/10.1128/spectrum.01521-25
• ZHAO, F., WANG, P., LUCARDI, R.D., SU, Z. and LI, S., 2020. Natural sources and bioactivities of 2,4-Di-Tert-Butylphenol and its analogs. Toxins, vol. 12, no. 1, pp. 35. https://doi.org/10.3390/toxins12010035 PMid:31935944.
  » https://doi.org/10.3390/toxins12010035
• ZGODA, J.R. and PORTER, J.R., 2001. A convenient microdilution method for screening natural products against bacteria and fungi. Pharmaceutical Biology, vol. 39, no. 3, pp. 221-225. https://doi.org/10.1076/phbi.39.3.221.5934
  » https://doi.org/10.1076/phbi.39.3.221.5934

Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  03 Aug 2026
• Date of issue
  2026

History

• Received
  29 Nov 2025
• Accepted
  29 Jan 2026