Trends Sci. 2025; 22(11): 10533

The term multidrug-resistant (MDR) bacteria refer to bacterial strains resistant to at least 3 different classes of antibiotics [1]. The increasing resistance of pathogenic bacteria to various antibiotics has become a critical issue in the healthcare sector. The misuse and overuse of antibiotics, along with missed doses, are primary factors contributing to antibiotic resistance [2]. This resistance enables sublethal bacteria to synthesize enzymes that degrade antibiotics, modify protein components targeted by antibiotics, and alter cell membrane permeability, thereby creating a barrier that prevents antibiotics from reaching their target cells.

The rising prevalence of MDR bacteria in infection cases has been reported in numerous countries. Researchers have identified MDR bacteria in samples collected from patients with urinary tract infections in hospitals [3,4]. Additionally, MDR bacteria have been detected in patients with solid organ transplant infections and wound infections [5-8].

The exploration of natural bioactive compounds is crucial in addressing the challenges posed by MDR pathogenic bacteria. Streptomyces, one of the largest genera in the phylum Actinobacteria, has significant potential as a producer of natural bioactive compounds [9]. This genus consists of Gram-positive filamentous bacteria characterized by a high G + C nitrogenous base content. The bioactive compounds produced by Streptomyces exhibit antibacterial activity distinct from conventional antibiotics, making them promising candidates for the development of new antibacterial agents to combat MDR pathogenic bacteria. Furthermore, Streptomyces is responsible for producing the highest number of antibacterial bioactive compounds, with nearly 60% of the antibacterial compounds sourced from microbes are obtained from Streptomyces [9,10].

The diversity of Streptomyces bioactive compounds is particularly intriguing due to their unique chemical structures and broad applications in medicine. Moreover, these bioactive compounds generally have fewer side effects, promoting extensive research into the identification of promising active molecules from Streptomyces in various environments. For instance, Streptomyces sp. Al-Dhabi-90, isolated from a marine environment, has been reported to produce 3-methylpyridazine, an antimicrobial agent effective against MDR bacteria [11]. Similarly, Maiti et al. [12] successfully isolated Streptomyces sp. from soil, which produces picolinamycin, a compound capable of inhibiting multiple MDR pathogenic bacteria. Furthermore, Streptomyces sp. PA5.6, isolated from forest soil, was also found to produce bioactive compounds such as benzebenzeneacetic acid, 4-hydroxy, and benzeneacetamide, all of which can inhibit the growth of various MDR bacterial strains [13]. S. malachitospinus, isolated from Hopea ferrea endophytes, has also demonstrated inhibitory effects against S. mutans [14].

Beyond marine and soil environments and plant endophytes, the rhizosphere of mangrove plants is another unique habitat for discovering bioactive compound-producing Streptomyces. In the mangrove ecosystem, Streptomyces can adapt to extreme conditions such as high salinity and low oxygen levels by producing bioactive compounds with potential applications in drug development [15]. The rhizosphere of the Baros Mangrove Forest in Yogyakarta, Indonesia, presents a particularly distinctive microenvironment for exploring various Streptomyces species capable of producing novel bioactive compounds [16]. Furthermore, the unique physicochemical conditions of the rhizosphere offer opportunities to discover Streptomyces species capable of producing novel bioactive compounds to effectively inhibit the growth of MDR bacteria [17,18]. Notably, Rhizopora apiculata exhibits the highest species diversity within the Baros Mangrove Forest, which spans 1.46 ha [16]. To date, no studies have been conducted on Streptomyces from the rhizosphere of R. apiculata in mangrove environments as potential producers of bioactive compounds that inhibit MDR bacteria. Therefore, this study aims to screen Streptomyces species from the rhizosphere of R. apiculata in the Baros Mangrove Forest, Yogyakarta, for their potential as new antibacterial agents against MDR pathogens. Additionally, the specific bioactive compounds produced by selected Streptomyces strains will be identified.

Materials and methods

Sample collection

Samples of the rhizosphere of R. apiculata were collected from the Baros Mangrove Forest in Yogyakarta, Indonesia (08°00’28.6 S”S110°16’ 59.4”E). The samples were placed in sterile plastic bags, securely sealed, and stored at 4 °C for further analysis.

Isolation of Streptomyces from the rhizosphere of R. apiculata in the Baros Mangrove Forest, Yogyakarta, Indonesia

For isolation, 5 g of rhizosphere samples were heated in an oven at 70 °C for 30 min. The samples were then serially diluted using 0.85% physiological saline to achieve a final concentration of 10⁻⁵. A 100 µL aliquot of the diluted sample suspension was inoculated into starch casein nitrate agar (SCNA) medium using the pour plate method, with 100 µL of nystatin (100 µL/100 mL) added as an antifungal agent. Following an incubation period of 7 - 14 days at 30 °C, the resulting bacterial colonies were purified using the streak plate method on malt extract agar [19].

Screening of Streptomyces isolates for bioactive compound production

Bioactive compound production by Streptomyces isolates was assessed using the perpendicular streak method. Seven-day-old Streptomyces cultures were streaked centrally onto Mueller–Hinton agar plates and incubated at 30 °C for 5 days. The MDR Pathogenic test bacteria (E. coli, P. aeruginosa, S. aureus and B. subtilis) were then streaked perpendicularly to the Streptomyces growth and incubated at 30 °C for an additional 2 days. Antibacterial activity was indicated by clear zones of inhibition surrounding the test strains [20]. Isolates exhibiting the broadest spectrum and largest inhibition zones were selected for further polyphasic identification.

Polyphasic identification of the selected Streptomyces isolates

Selected Streptomyces isolates were identified through a polyphasic approach integrating phenotypic and genotypic analyses. Phenotypic characterization included assessments of cultural, morphological, biochemical, and physiological traits. Cultural properties, such as aerial and substrate mycelium coloration, growth type, and diffusible pigment production were examined on 6 media types after 7 days of incubation at 30 °C. The 6 media types used were Nutrient Agar (NA), Starch Casein Agar, Starch Casein Nitrate Agar (SCNA), Tryptone Yeast Extract (TYE), Malt Extract Agar (MEA), and Inorganic Salt Agar (ISA). Morphological traits, including colony morphology, hyphal branching, and spore characteristics (chain formation, shape, and surface ornamentation), were examined using scanning electron microscopy (SEM; JSM-6510 LA) [21-23].

Biochemical profiling involved assays for catalase activity, starch hydrolysis, milk coagulation, and peptonisation. The ability to utilize various carbon (e.g., glucose, fructose, lactose and sucrose) and nitrogen sources (e.g., L-arginine, yeast extract, peptone and ammonium salts) was also tested. Physiological responses were evaluated across a range of pH (4 -13), temperatures (16, 30 and 37 °C), and NaCl concentrations (0 - 9 % w/v) [23].

Genotypic identification was conducted via 16S rDNA gene sequencing. Genomic DNA was extracted using the Zymo-Research Quick-DNA Fungal/Bacterial Miniprep Kit. PCR amplification was performed with universal primers 27F (5′‐AGAGTTTGATCMTGGCTCAG‐3′) and 1492R (5′‐TACGGYTACCTTGTTACGACTT‐3′) using a Bio-Rad T100 Thermal Cycler [24]. The 25 μL PCR reaction mix contained 1 μL DNA template, 12.5 μL 2× MyTaq Red Mix, 1 μL of each primer (0.5 μM), and 9.5 μL ddH₂O. Thermal cycling conditions were: initial denaturation at 96 °C for 1 min; 30 cycles of denaturation at 96 °C for 45 s, annealing at 55.6 °C for 1 min, and extension at 72 °C for 2 min; followed by a final extension at 72 °C for 7 min. Amplified products were verified via 0.8 % agarose gel electrophoresis with SYBR Safe staining, purified with the Zymoclean™ Gel DNA Recovery Kit, and sequenced bidirectionally. Sequences were edited using GeneStudio and aligned using ClustalW. BLASTn analysis was performed by comparing the 16S rDNA gene sequence from strain ACSAN21-05 with the NCBI database (https://blast.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed using the Maximum Likelihood method in MEGA11 with 1,000 bootstrap replicates.

Production and extraction of bioactive compounds

Streptomyces sp. ACSAN21-05 was initially cultured on MEA at 30 °C for 7 days, and 1 g of the resulting cell pellet was inoculated into 100 mL starch casein broth. The culture was incubated for 10 days at 30 °C with continuous agitation at 120 rpm in a water bath shaker. Following incubation, the culture was centrifuged at 4,000 rpm for 35 min (Gemmy PLC Series 3), and the supernatant containing secreted bioactive compounds was collected. Extraction was performed with ethyl acetate at a 1:1 (v/v) ratio, followed by overnight incubation at 30 °C with shaking at 120 rpm. The organic phase was separated and concentrated using a rotary evaporator at 45 °C until a paste-like residue was obtained. The crude extract was further dried at 55 °C and stored for subsequent bioactivity assays [25].

Antimicrobial activity testing and Minimum Inhibitory Concentration (MIC) of bioactive compound extracts

The antimicrobial activity of crude extracts from Streptomyces sp. ACSAN21-05 was assessed using the well diffusion method [11]. The MDR strains of E. coli, P. aeruginosa, S. aureus, and B. subtilis were inoculated into nutrient agar (NA) plates using the pour plate technique. Bacterial suspensions were standardized to 0.5 McFarland turbidity (∼10⁸ CFU/mL). Wells were created using a sterile cork borer, and 50 µg/mL of the crude extract was added to each well. Plates were incubated at 37 °C for 24 h, and inhibition zones were measured to evaluate antibacterial activity.

Minimum inhibitory concentration (MIC) values were determined using the broth microdilution method in 96-well microplates [11]. The crude extract was dissolved in 1.5% dimethyl sulfoxide (DMSO) to prepare 4 concentrations: 6.25, 12.5, 25 and 50 µg/mL. Test strains were cultured in nutrient broth to 0.5 McFarland density and combined with the extract in equal volumes (100 µL each) per well. Plates were incubated at 37 °C for 24 h. Bacterial growth was quantified by measuring optical density at 600 nm using a spectrophotometer. Azithromycin (50 µg/mL) served as a positive control.

Profiles of bioactive compounds of Streptomyces sp. ACSAN21-05

The chemical composition of the volatile bioactive compounds produced by Streptomyces sp. ACSAN21-05 was analyzed using gas chromatography-mass spectrometry (GC-MS; GCMS-QP2010S, Shimadzu). A 2 µL sample of the ethyl acetate extract was injected into an EC-5 capillary column. The oven temperature was programmed to increase from 60 to 305 °C, with the injector set at 300 °C. Helium was used as the carrier gas, maintaining a constant flow rate of 2 mL/min. The mass spectrometry scan began at 5.20 min and ended at 80 min. Compound identification was carried out by comparing the obtained mass spectra to entries in available reference MS databases [26].

Data analysis

All experiments were performed in triplicate, and results are reported as mean ± standard deviation. Statistical significance of differences in inhibition zone diameters and MIC values was evaluated using 1-way analysis of variance (ANOVA), followed by Duncan’s multiple range test. Analyses were conducted using SPSS software (version 25.0), with significance defined at p < 0.05.

Results and discussion

Streptomyces isolates producing bioactive compounds

A total of 15 Streptomyces isolates were obtained from the rhizosphere of R. apiculata in the Baros Mangrove Forest, Yogyakarta, Indonesia (Table 1). Five of these isolates demonstrated the ability to produce bioactive compounds, as evidenced by the formation of clear inhibition zones in the screening test. The mean diameters of the inhibition zones varied among isolates. Notably, Streptomyces ACSAN21-05 exhibited the most significant bioactive compound production, effectively inhibiting both Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus and B. subtilis) pathogenic bacteria. The inhibition zones for these strains exceeded 40 mm in diameter, suggesting that the bioactive compounds produced by Streptomyces ACSAN21-05 possess broad-spectrum antimicrobial activity against both Gram-negative and Gram-positive pathogens.

Table 1 Streptomyces isolates producing bioactive compounds using the perpendicular streak method.

Note: EC: E. coli, PA: P. aeruginosa, SA: S. aureus, BS: B. subtilis

Polyphasic identification of selected Streptomyces isolates

The identification of the selected Streptomyces sp. ACSAN21-05 isolate was performed through a polyphasic approach, integrating both phenotypic and genotypic analyses. Phenotypic characterization, based on cultural traits, is summarized in Table 2 and Figure 1. The strain demonstrated vigorous growth across 5 distinct media, with the formation of both aerial and substrate mycelia in varying colours, including gray, white-gray, and light gray. Notably, the isolate produced a distinctive golden-yellow pigment when cultured on TYE and MEA, highlighting its potential for secondary metabolite production.

Table 2 Cultural characteristics of Streptomyces sp. ACSAN21-05 on 5 different media.

Figure 1 Growth of Streptomyces sp. ACSAN21-05 on various media (A) NA; (B) SCNA; (C) TYE; (D) MEA; and (E) ISA.

The morphological features of Streptomyces sp. ACSAN21-05 are summarized in Table 3. Colonies of this strain were round, measuring 6 mm in diameter, with flat edges, convex elevation, and powdery surfaces due to the production of spore-like structures. Gram staining revealed that the cells were Gram-positive. The spore chain type was classified as polysporous, with each chain containing over 50 spores, a typical characteristic of the Streptomyces genus. The spores exhibited irregular, rugose surface ornamentation, indicating the presence of surface wrinkles, and were rod-shaped with a distinct central notch. Scanning electron microscopy (SEM) further confirmed the presence of recti flexible aerial hyphae, displaying straight or flexible spore chains, some of which were arranged in fascicles (Figure 2).

Table 3 Morphological characteristics of Streptomyces ACSAN21-05.

Figure 2 Morphology of the aerial mycelium of Streptomyces sp. ACSAN21-05. (A) 1,000× magnification; (B) 10,000× magnification.

The phenotypic characterization of Streptomyces sp. ACSAN21-05, based on its biochemical and physiological properties, is detailed in Table 4. Biochemical analysis revealed a positive catalase reaction, starch hydrolysis, milk coagulation (positive coagulase), and the conversion of peptone into casein (positive peptonization). The strain demonstrated the ability to utilize various carbon sources, including dextrose, fructose, galactose, glucose, maltose, mannitol, xylose, lactose, sorbitol, and sucrose. It also grew on several nitrogen sources, such as yeast extract, peptone, L-arginine, L-tyrosine, KNO₃, (NH₄)₂SO₄, and (NH₄) H₂PO₄. Physiologically, Streptomyces sp. ACSAN21-05 exhibited growth across a pH range of 4 - 8, temperatures from 30 to 37 °C, and NaCl concentrations of 0 - 9%. These phenotypic traits were consistent with the profile described in Bergey’s Manual of Systematic Bacteriology, confirming its identification as Streptomyces anulatus ACSAN21-05 [27].

BLASTn analysis of the 16S rDNA gene sequence from Streptomyces sp. ACSAN21-05, compared with the NCBI database, revealed a 98.78% identity match with S. anulatus (Table 5). Phylogenetic analysis further supported this finding, classifying Streptomyces sp. ACSAN21-05 as S. anulatus (Figure 3). Thus, the polyphasic identification approach confirmed the consistency between phenotypic and genotypic data, solidifying the identification of Streptomyces sp. ACSAN21-05 as S. anulatus.

Antimicrobial activity of the crude extract of bioactive compounds from Streptomyces anulatus ACSAN21-05

The crude extract of bioactive compounds from Streptomyces anulatus ACSAN21-05 demonstrated significant antimicrobial activity (p < 0.05) against 4 multidrug-resistant (MDR) pathogenic bacteria, as assessed by the inhibition zone formation (Table 6). The extract exhibited the strongest inhibitory effect against MDR E. coli, followed by MDR S. aureus, MDR P. aeruginosa, and MDR B. subtilis, with inhibition zone diameters of 17, 16, 15 and 14 mm, respectively. These results surpass those reported by Djebbah et al. [28], where S. anulatus ACSAN21-05 produced bioactive compounds with larger inhibition zones and stronger antimicrobial activity against the tested MDR pathogens.

Minimum Inhibitory Concentration (MIC) of bioactive compound extract of Streptomyces anulatus ACSAN21-05

The Minimum Inhibitory Concentration (MIC) results for the crude bioactive compound extract from Streptomyces sp. ACSAN21-05, compared to streptomycin as a standard antibiotic, are shown in Table 7. MDR E. coli exhibited the lowest MIC of 12.5 µg/mL, followed by MDR S. aureus at 25 µg/mL. The MIC values for both MDR P. aeruginosa and MDR B. subtilis were 50 µg/mL. Notably, the MIC values of the bioactive compounds from Streptomyces sp. ACSAN21-05 were lower than those of streptomycin against several pathogens. These findings suggest that the bioactive compounds produced by Streptomyces sp. ACSAN21-05 hold potential as novel antimicrobial agents.

Table 4 Biochemical and physiological characteristics of Streptomyces sp. ACSAN21-05.

Table 5 BLASTn results of the 16S rDNA gene sequence of Streptomyces sp. ACSAN21-05.

Figure 3 Phylogenetic tree of Streptomyces sp. ACSAN21-05 based on the 16S rDNA gene sequence, constructed using the Neighbor-Joining method with 1,000 bootstrap replicates

Table 6 Antibacterial activity of the crude extract of bioactive compounds from Streptomyces anulatus ACSAN21-05 against 4 MDR bacterial pathogens.

Table 7 Minimum inhibitory concentration (MIC) of crude ethyl acetate extract of bioactive compounds from Streptomyces anulatus ACSAN21-05.

Metabolite profile of the broth extract from Streptomyces anulatus ACSAN21-05

GC-MS analysis of the crude extract from ethyl acetate extraction revealed 68 peaks, representing compounds in the extract at varying concentrations (Figure 4). The numbers on the peaks correspond to their respective retention times in minutes. Mass spectrometry (MS) analysis identified the structures and characteristics of the compounds eluted at different retention times. Among the 68 volatile compounds detected, 22 exhibited antibacterial activity, as summarized in Table 8.

Figure 4 GC-MS chromatogram of the ethyl acetate extract from Streptomyces anulatus ACSAN21-05.

Table 8 Metabolite profile of the of the ethyl acetate extract from Streptomyces anulatus ACSAN21-05.