Evaluation of antimicrobial activity of extract of actinobacteria isolated from impacted soil against strains of resistant Staphylococcus aureus

Avaliação da atividade antimicrobiana do extrato de actinobactérias isoladas de solo impactado contra cepas resistentes de Staphylococcus aureus

Abstract

This study investigated the antibacterial potential of secondary metabolites from actinobacteria isolated from landfill-impacted soil in Pinheiro-MA against resistant Staphylococcus aureus. The extract produced inhibition halos up to 34 mm and showed a minimum inhibitory concentration of 500 µg/mL for S. aureus ICH 4808. In Tenebrio molitor, larval survival remained 100% except at 1,000 µg/mL. The extract reduced biofilm formation by about 80%, highlighting its promise for antimicrobial bioproduct development.

Keywords:
Streptomyces sp.; secundary metabolic; resistence; bacteria; ESKAPE

Resumo

Este estudo investigou o potencial antibacteriano de metabólitos secundários de actinobactérias isoladas de solo impactado por aterro sanitário em Pinheiro-MA contra Staphylococcus aureus resistente. O extrato produziu halos de inibição de até 34 mm e apresentou concentração inibitória mínima (CIM) de 500 µg/mL para S. aureus ICH 4808. Em Tenebrio molitor, a sobrevivência larval manteve-se em 100%, exceto na concentração de 1,000 µg/mL. O extrato reduziu a formação de biofilme em cerca de 80%, evidenciando seu potencial promissor para o desenvolvimento de bioprodutos antimicrobianos.

Palavras-chave:
Streptomyces sp.; metabólito secundário; resistência; bactéria; ESKAPE

1. Introduction

Several soil microorganisms, including fungi and bacteria, produce enzymes that degrade organic compounds, thriving in environments rich in nutrients and shaped by physicochemical changes, microbial competition, and adaptive responses (Cárceles Rodríguez et al., 2022). Extremophilic microorganisms from harsh environments such as arctic regions and deserts exhibit unique adaptations that enable survival under extreme conditions (Rampelotto, 2024). Improper solid waste disposal also poses environmental and public health risks by facilitating the spread of pathogenic microorganisms through air and water, contaminating ecosystems and nearby populations (Chagas et al., 2023).

Antimicrobial resistance has become one of the major global health concerns due to the misuse of antibiotics, leading to the rise of “superbugs” capable of resisting conventional treatments (Levine, 2006; WHO Collaborating Centre for Drug Statistics Methodology, 2021; Choucair et al., 2021). Continuous antibiotic exposure creates selective pressure that promotes multidrug resistance, particularly among hospital-associated pathogens from the ESKAPE group—Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.—responsible for severe nosocomial infections (Vale de Macedo et al., 2021).

Actinobacteria, especially Streptomyces spp., are known for producing structurally diverse secondary metabolites derived from metabolic intermediates such as acetyl-CoA and amino acids (Kim et al., 2020; Krysenko, 2023). These compounds include anthramycin-type antibiotics with strong antimicrobial, antifungal, and antitumor activities (Bhattarai et al., 2022). Given the global spread and adaptability of S. aureus, which causes infections ranging from mild skin lesions to severe systemic diseases (Newstead et al., 2020; Cassat and Thomsen, 2021), this study evaluates the antibacterial potential of secondary metabolites from Streptomyces strains isolated from solid waste-contaminated soils, emphasizing their promise as candidates for biotechnological and pharmaceutical innovation.

2. Methodology

2.1. Microorganisms used

Among the microorganisms used, the tests initially included 10 strains of Staphylococcus aureus. All strains were isolated from clinical blood cultures (Isolado Clínico Hemocultura – ICH) and are available in the culture collection of the Laboratory of Electrochemistry and Biotechnology at CEUMA University.

2.2. Soil collection, isolation and identification of actinobacterias

Soil samples were collected from a solid-waste–impacted landfill in Pinheiro, Maranhão, Brazil (2º31'12.7''S 45º07'15.2''W) (Figure 1). Actinobacteria isolation followed Clark (1965): 1 g of soil was serially diluted (10⁻¹–10⁻⁴) in saline, and 100 µL aliquots were plated on Potato Dextrose Agar (PDA) and incubated at 28 °C for 7 days. Colonies exhibiting actinobacterial morphology were purified on PDA, preserved at 4 °C, and identified by micromorphological characteristics according to Shirling and Gottlieb (1966).

Figure 1
Dump site where the soil was collected, located in Pinheiro, Maranhão, Brazil. Source: Satellite Image: Google Satellite – Google; Data: IBGE (2021).

2.3. Preparation of the extract

Secondary metabolites were produced by submerged fermentation following Amorim et al. (2020), with actinobacterial colonies incubated in potato dextrose broth at 28 °C and 180 rpm for 14 days. Metabolites were extracted using the ethyl acetate liquid–liquid method (1:1) described by Trisuwan et al. (2008), and the organic phase was concentrated by rotary evaporation. The crude extract was resuspended in 1% DMSO at 10,000 µg/mL to prepare the stock solution.

2.4. Evaluation of antimicrobial activity

Antimicrobial activity was assessed using the crude extract diluted in 1% DMSO (10,000 µg/mL) against clinically relevant S. aureus strains. Agar diffusion followed standard Kirby–Bauer methodology (Bauer, 1966; CLSI, 2020) on Müller-Hinton Agar, with chloramphenicol, DMSO, and sterile extract as controls. MIC values were determined by broth microdilution using resazurin after 48–72 h of incubation. In vivo efficacy was evaluated with the Tenebrio molitor larvae model (Souza et al., 2015), applying MIC-based doses and monitoring survival and behavioral changes to estimate LC₅₀.

2.5. Evaluation of antivirulence activity - biofilm assays

The biofilm-forming capacity of S. aureus isolates was assessed in TSB using the modified crystal violet assay of O’Toole (2011), with optical-density quantification following Ferro et al. (2016). Biofilm inhibition was evaluated by incubating bacterial suspensions (1×10⁷ CFU/mL) with extract concentrations as described by Wai-Kei Tsang et al. (2012). Pre-formed biofilms were similarly exposed to the extracts for 24 h, then fixed, stained, and measured at 570 nm.

3. Results

3.1. Isolation and identification of actinobacteria

Purified colonies exhibited small, rounded morphology, predominantly white with some black surfaces, and burnt-yellow pigmentation in the medium. Colonies displayed raised, rough textures with aerial mycelium. Microscopic analysis revealed clustered micrococcus-like structures and short blue-stained hyphae, characteristic of Streptomyces spp.

3.2. Antimicrobial activity

The lyophilized extract from the soil actinomycete yielded 33 mg/mL and showed antimicrobial activity in agar diffusion (Table 1) and MIC assays. At 10,000 µg/mL, inhibition halos were formed against nine Staphylococcus strains, with no effect on S. aureus ATCC 10, suggesting the need for higher concentrations. These results indicate secretion of inhibitory metabolites by the actinomycete. In the MIC assay, the lowest MIC was 500 µg/mL for S. aureus ICH 4808, while strains ICH 03501 and ICH 4201 required 1,000 µg/mL. No inhibition was observed for ICH 9722, indicating resistance. MIC testing was not performed for S. aureus ICH 10.

Table 1
Mean diameters of inhibition zones (mm) produced by the Streptomyces extract and chloramphenicol (control) against the tested pathogens in liquid medium.

Microorganisms tested	Halo mean (mm) and SD
	Extract	Control
S. aureus ICH 10	-	-
S. aureus ICH 03501	19 ± 1	36.7 ± 0.1
S. aureus ICH 4201	19 ± 1	35.0 ± 1
S. aureus ICH 4808	18 ± 1	33.4 ± 0.46
S. aureus ICH 9722	21 ± 1	34.3 ± 1.33

• Legend: Standard Deviation (SD); Isolated Clinical Blood Culture (ICH); (-) = Absence of halo. Source: Author himself.

3.3. Infection model with Tenebrio molitor

Larvae survival after 10 days was 100%, except at 1,000 µg/mL, where survival dropped to 60% on day seven, indicating low toxicity and safety of the metabolite at lower concentrations (Figure 2).

Figure 2
Analysis of data obtained from the survival curve with Tenebrio molitor after 10. Source: Authors.

3.4. Biofilm assays

Biofilm inhibition tests were restricted to S. aureus due to extract toxicity at 1,000 µg/mL. All isolates were strong biofilm producers (Table 2). The Streptomyces metabolite caused marked inhibition at the MIC, reducing biofilm formation by 61% and 60% in S. aureus ICH 03501 and ICH 4808, respectively (Table 3). At ½ and ¼ MIC, inhibition persisted for these strains, whereas ICH 4201 showed a transient increase at ½ MIC followed by reduction at ¼ MIC (Table 3). For pre-formed biofilms, the metabolite maintained inhibitory activity, achieving 58% reduction in ICH 03501 at MIC and moderate decreases in ICH 4201 and ICH 4808 at subinhibitory levels (Table 4) (Xie et al., 2023).

Table 2
Formation and intensity of in vitro biofilm production by Staphylococcus aureus species.

Microorganisms test	Intensity of biofilm production
S. aureus ICH 03501	Produtor moderado
S. aureus ICH 4201	Produtor forte
S. aureus ICH 4808	Produtor forte

• Legend = Clinical Isolate Hemoculture (ICH). Source: Authors.

Table 3
Interference of the Streptomyces extract on biofilm formation.

MICROORGANISMS	EXTRACT INTERFERENCE
	MIC (µg/mL)	Reduced biofilm formation in %
S.aureus ICH 03501	1,000 µg/mL	61%
	500 µg/mL	19%
	250 µg/mL	10%
S.aureus ICH 4201
	1,000 µg/mL	52%
	500 µg/mL	76%
S. aureus ICH 4808	250 µg/mL	37%
	500 µg/mL	60%
	250 µg/mL	37%
	125 µg/mL	20%

• Legend = Clinical Isolate Hemoculture (ICH). Source: Authors.

Table 4
Interference of the Streptomyces on Pre-formed Biofilm.

MICROORGANISMS	EXTRACT INTERFERENCE
	MIC (µg/Ml)	Reduced biofilm formation in %
S. aureus ICH 03501	1000 µg/mL	58%
	500 µg/mL	65%
	250 µg/mL	67%
S. aureus ICH 4201	1000 µg/mL	61%
	500 µg/mL	73%
	250 µg/mL	68%
S. aureus ICH 4808	500 µg/mL	61%
	250 µg/mL	79%
	125 µg/mL	88%

• Legend = Clinical Isolate Hemoculture (ICH). Source: Authors.

3.5. Chemical characterization of the extract

The detected compounds—hexadecanoic acid, octadecanoic acid, 4-(3-methyl-2-butenyl)-1H-indole, hedycaryol, and 2-methylisoborneol—showed chromatographic peaks proportional to their abundance. The dominant fatty acids, which presented the largest peaks, likely account for most of the activity by disrupting the S. aureus membrane. The minor constituents (prenylated indole, hedycaryol, and 2-MIB) may contribute complementary antivirulence and cell-destabilizing effects. Together, major and minor metabolites appear to act synergistically to produce the antimicrobial effect (Figure 3).

Figure 3
Chromatogram indicating the compound fragments from the chemical extraction of the secondary metabolite Streptomyces sp. Source: Authors.

4. Discussions

Actinomycetes thrive in diverse habitats, producing secondary metabolites with pharmaceutical potential due to their adaptability and plant interactions (José et al., 2021). The indiscriminate use of medications—driven by sociocultural and economic factors, poor prescription practices, and self-medication—intensifies antimicrobial resistance (Batista et al., 2021; Brito and Trevisan, 2021). Natural antimicrobial compounds, often from soil microorganisms, inhibit or kill bacteria depending on dosage (Sheikh et al., 2022). Phytochemistry explores these bioactive substances from natural sources (Pereira, 2022), emphasizing the relevance of microbial extracts. Impacted soils, particularly those contaminated by waste, host actinomycetes such as Streptomyces, which produce adaptive metabolites (Ataíde et al., 2020). Extracts from Streptomyces isolated from solid waste sites showed antibacterial activity against resistant strains, aligning with findings from Streptomyces sp. MPO11 (Liberal, 2018; Fuzeti, 2023). Waste-contaminated soils favor actinobacterial diversity (Selvarajan et al., 2022) and play ecological roles in soil recovery (Tong et al. 2015; Araújo Leal et al., 2021; Fernandes et al., 2021). The extract tested reduced Staphylococcus aureus biofilm formation, indicating potential for controlling resistant infections. Conserving soil biodiversity is crucial, as microbial metabolites contribute to ecosystem balance and human health (Guerra et al., 2022; Chagas et al., 2023; ). The Streptomyces extract presented metabolites from different classes with strong antimicrobial plausibility against Staphylococcus aureus. Fatty acids palmitate and stearic acid act in membrane disruption, a mechanism well established by Casillas-Vargas et al. (2021) and highly effective against Gram-positive bacteria. The prenylated indole 4-(3-Methyl-2-butenyl)-1H-indole reinforces the effect by interfering with DNA/RNA synthesis and modulating quorum sensing and biofilm, as described by Yan et al. (2021). The sesquiterpene hedycaryol contributes as an additional lipophilic disruptor (Ogundajo et al., 2021), while 2-MIB, despite its low activity, confirms the typical Streptomyces profile. Taken together, these compounds suggest a multifactorial and possibly synergistic action, although the lack of fractionation and MIC/MBC assays limits experimental confirmation of this potential, indicating the need for further studies.

5. Conclusion

This study highlights the therapeutic potential of Streptomyces sp., demonstrating antimicrobial efficacy against Staphylococcus aureus. The findings reinforce the urgency of discovering new agents to combat multidrug-resistant bacteria and raise public awareness about antibiotic misuse.

Acknowledgements

The authors thank Ceuma University (UNICEUMA), the Postgraduate Program in Biodiversity and Biotechnology (BIONORTE Network), and the Electrochemistry and Biotechnology Laboratory for providing infrastructure and technical support essential for conducting this study. The authors also acknowledge financial support from Maranhão State Research Foundation (FAPEMA), (Edital 38/2022; Process ACT-FAPEMA 02510/2023) and Coordination for the Improvement of Higher Education Personnel (CAPES).

Data Availability Statement

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

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

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

History

• Received
  01 Dec 2025
• Accepted
  16 Mar 2026