Research Article | | Volume 14 Special Issue 2 (July to August, 2025) | Pages 85 - 95

Isolation and Characterization of Marine Actinomycetes from Specific Red Sea Algae: Evaluation of Their Antimicrobial and Antioxidant Activities

1
Department of Biology, Faculty of Science, King Abdulaziz University, Kingdom of Saudi Arabia
Under a Creative Commons license
Open Access
Received
Aug. 4, 2025
Revised
Sept. 30, 2025
Accepted
Sept. 27, 2025
Published
Sept. 5, 2025

Abstract

Bacteria associated with algae play a crucial role in the ecological balance of marine ecosystems. Actinomycetes have many advantageous characteristics that increase the bioavailability of vital nutrients in soil and regulate the growth of microbial pathogens. This study aimed to identify the culturable actinomycetes, associated with some algal samples from the Red Sea and investigate their antimicrobial and antioxidant activities. The actinomycetes were isolated from four algal species collected from Yanbu cityon starch nitrate agarmedium supplemented with 10% NaCl and tested for their antimicrobial activity against a panel of pathogenic multidrug-resistant bacteria and for their antioxidant potential using appropriate assays. The collected Algae were identified as Ulva lactuca and Codium tomentosum (green algae), Padina gymnospora (brown algae) and Hypnea valentia (red algae)The results demonstrated that out of 4 different algal samples collected from the Red Sea, 15 filamentous bacterial isolates were obtained, purified and screened for antibacterial activities. Five isolates showed detectable antibacterial activities using the agar plug diffusion assay. Excellent antibacterial activities were recorded by the isolate RAD3 against Staphylococcus aureusStreptococcus pyogenes and Enterococcus faecalis in addition to Escherichia coliAt the same time, it exhibited lower antimicrobial activity against Pseudomonas aeruginosa, Salmonella typhi, Shigella sonnei and Serratia marcescens. The Minimal Inhibitory Concentrations (MICs) of the RAD3 extract were ranged from 50-100 µg/ml for the tested bacterial pathogens. No antibacterial activity was recorded against Klebsiella pneumonia and Acinetobacter baumannii. The isolate RAD3was the most active in bacterial inhibition and prevented E. faecalis biofilm formation by 63% and exopolymeric substances (EPS) by 47%. The ethyl acetate extracts of the five selected bacteria showed promising antioxidant potential by scavenging DPPH (2,2-diphenyl-1-picrylhydrazyl) method. Out of the 5 chosen bacteria, RAD3 showed 66.37% free radical scavenging with EC50 = 29.0 µg/ml and the results were compared to the positive control and the synthetic antioxidant Butylated Hydroxytoluene (BHT) which showed 96% free radical scavenging with EC50 = 19.7 µg/ml. The 5 chosen bacterial isolates were morphologically characterized and genetically identified by the analysis of 16S rDNA as species belonging to the genus Streptomyces. In conclusion, these findings highlight the potential of bacteria associated with the Red Sea algae as a source of novel antimicrobial and antioxidant compounds for food and medicine industries.

Keywords
Red Sea Streptomyces, antimicrobial agents, Biofilm inhibition, DPPH

INTRODUCTION

The marine environment encompasses 70% of the Earth's surface and comprises highly productive zones that contain various subsystems supporting nearly 80% of the world's biota, including microorganisms, animals, algae, sponges and protozoa [1-3]. Its vast phylogenetic diversity exceeds the terrestrial environment due to location, temperature and salinity variations [4]. There are widespread symbiotic relationships between algae and bacteria, considered an ancient survival strategy that provides microorganisms with advantages for proliferation, such as access to nutrient-rich habitats and increased environmental stability. Additionally, microbial aggregates on the algal surface offer greater protection against external stresses [5]. The isolates of the genus Endozoicomonas exhibit highly diverse and adaptable symbiotic relationships with a wide array of marine hosts, such as sponges, fish and corals and play a role in protein and carbohydrate transport and cycling. Furthermore, they control high bacterial growth inside their hosts through competition and exclusion methods [6].

 

Marine bacteria are a diverse group of microorganisms that can live in close association with various types of algae. They play essential roles in the ecology and physiology of their hosts through processes such as nutrient cycling, symbiosis and disease resistance. Secondary metabolites released by different types of macroalgae can selectively attract and enhance the growth of these specific bacteria [7]. In a study from Finland, 17 bacterial species were isolated from four algae cultures: Chlorella pyrenoidosa, Scenedesmus obliquusIsochrysis sp. and Nitzschia microcephala. The bacterial community of Isochrysis sp. differs significantly from the other tested algae [8,9]. Bacteria associated with brown algae have emerged as a promising source of bioactive metabolites. In a study on Panjang Island, Jepara, twenty-three marine bacterial strains were isolated from Sargassum using marine agar and they showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis. The most active bacterial strain was identified as Bacillus subtilis [10]. From Red Sea sediments, 27 strains of actinomycetes were isolated and the most active isolate was identified as Streptomyces sp., which showed antimicrobial and anticancer activities with no cytotoxic effect, likely due to the presence of many anthraquinone compounds [11]. Also, bacteria from seaweed surfaces and invertebrates produce many antibacterial compounds that inhibit the most dangerous resistant bacteria, MRSA and vancomycin-resistant Enterococcus [12,13]. Moreover, marine Streptomyces from India's Bengal coast inhibit the growth of multidrug-resistant (MDR) bacteria which became a global threat and a matter of concern [14]. Similarly, isolated Streptomyces sp. MK388207 is from the Gulf of Suez and it has a strong antimicrobial agent against some pathogens, such as Candida albican [15]. Moreover, out of 21 crude extracts of marine bacterial strains associated with sponges, Rhodococcus erythropolis showed excellent antibacterial activities against MRSA [16] while marine Streptomyces sp. Al-Dhabi-100, isolated from Jazan region of Saudi Arabia, showed growth inhibition of Enterococcus faecalis, Bacillus subtilis, S. aureus, S. epidermidis and Klebsiella pneumoniae, with MIC ranging from 32.5-250 µg/ml. Additionally, the strain showed anti-tubercular and antioxidant potentials [17]. These findings suggest the therapeutic potential of marine-derived bacterial extracts against bacterial infections.

 

Different contaminants from certain hotspot sources like wastewater, sludge and natural fertilizer scan potentially spread, which enhances the emergence of new antimicrobial-resistant bacterial strains that contribute annually to 700,000 deaths worldwide and are likely to increase to 10 million in the year 2050 [18,19]. These bacteria have developed biochemical and physiological mechanisms, including the bioactive compounds with distinctive structural features accumulation, serving various functions such as reproduction, communication and defense against predation, infection and competition [20]. This is why, it is essential to search for new biologically active compounds from distinct sources to obtain novel antimicrobial agents. Marine water needs more precious studies to discover new species with excellent marine metabolites with structurally unique structures and action [21,22]. The Red Sea, with its extreme salinity and temperature, hosts unique microbial communities that have evolved to produce diverse bioactive compounds. Actinomycetes, especially those associated with algae, are prolific sources of antimicrobial and antioxidant metabolites with therapeutic potential. Thus the study was aimed to isolate and characterize the marine actinomycetes from Red Sea algae, highlighting their significance as an untapped resource for novel drug discovery and strategies against antimicrobial resistance and oxidative stress-related diseases [23-27].

METHODS

Algal Sample Collection and Actinobacterial Isolation

Algae were chosen as a source for actinomycete isolation because their nutrient-rich surfaces provide micro-niches that support diverse microbial communities capable of producing bioactive metabolites. Red algae, in particular, were prioritized as they exhibited distinct colony morphologies-colored, powdery and filamentous growth-characteristic of actinomycetes, making them strong candidates for antimicrobial and antioxidant screening. Building on this rationale, isolating marine actinomycetes from algae was a multi-step process that required careful sterile techniques and specialized media to obtain pure cultures for further study and potential biotechnological applications.

 

In this research, algal samples were collected from their natural habitat in sterile bags and washed several times with sterile water to avoid contamination, identified and classified. Algae were collected during winter 2022 by a local diver from a 3-4 m depth of Yanbu shores, Saudi Arabia and transferred to the laboratory in an ice box. To remove sand and necrotic parts, algal samples were washed with seawater followed by distilled water [28] and then identified at the Biology Department, Faculty of Science, King Abdulaziz University, according to color and morphological features.

 

All algal samples were processed to isolate actinomycetes using two selective media: Zobell Marine Agar (ZMA2216; Himedia, India) and modified starch nitrate agar supplemented with 10% NaCl [29]. Zobell Marine Agar is widely used for the isolation of marine bacteria, while starch nitrate agar provides nutrients that favor actinomycete growth and inhibit most other true bacteria. Sterilized surfaces of the collected algae were cut into 1 cm² squares and inoculated directly onto the agar surface of both media. The plates were incubated at 25°C for four days, after which individual colonies were selected for further purification by streaking, based on colony morphology. Colonies displaying characteristic actinomycete features-colored, powdery appearance and filamentous growth-were purified, examined microscopically for mycelial structures and preserved on slants of the same medium or in glycerol stocks at -80°C for future use [30]. Following successful isolation, the purified actinomycete cultures were subjected to phytochemical screening to evaluate their capacity for secondary metabolite production. Standard qualitative tests for alkaloids, flavonoids, tannins and saponins were performed using reagents such as Mayer’s reagent, ferric chloride and foam tests, enabling the detection of key bioactive compounds with potential antimicrobial and antioxidant activity.

 

Actinobacterial Characterization

Various methods, such as microscopy, biochemical tests and molecular techniques (like DNA sequencing), are used to identify the isolated actinomycetes to the genus or species level and characterize their properties. Out of 15 actinobacteria, five were examined and identified using morphological examination on Oatmeal agar at 25°C and biochemical tests. Gram stain detected after examination with a light microscope (CX21FS1 Olympus, China), the aerial and substrate mycelia colors and some biochemical tests like oxidase and catalase were carried out by conventional methods used in the microbiology laboratories [31].

 

Molecular Identification

According to the instructions of the manufacturer (QIAamp DNA Mini Kit, Qiagen, Germany), the DNA of the selected colonies was extracted, purified and preserved at -20°C for further analysis. Its purity was confirmed using the NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) [32]. For each DNA bacterial extract,16S rRNA gene sequences were amplified using two internal Universal Primers 27F (5'-AGA GTT TGA TCM TGG CTC AG-3') and 511 R (5’-GCG GCT GCTGGC ACR KAG T -3') and PCR products was obtained, purified (QIA quick PCR purification kit) and sequenced (Big Dye Terminator). Nucleotides blast of NCBI and phylogenetic analysis were performed.

 

The Tested Bacterial Pathogens

The pathogenic Gram-positive and negative bacteria, Acinetobacter baumannii, Escherichia coli, Klebsiella oxitoca, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Shigella sonnei, Enterococcus faecalis, Staphylococcus aureus (MRSA) and Streptococcus pyogenes were collected from KAU hospitals on plate agar. All isolates were examined using Gram stain and preserved on agar slants of nutrient agar at 4°C until used.

 

Bacterial Extract Preparations

The bacterial isolates were cultured in 500 ml flasks containing 100 ml of broth medium and incubated under shaking at 120 rpm for 7 days, at 25°C. After the fermentation period, the cells were separated by centrifuging at 5000 rpm for 10 min. The supernatants were mixed with an equal amount of ethyl acetate and agitated for 12 hours at room temperature. The ethyl acetate extract was evaporated under reduced pressure at 50°C by a Rotary evaporator (HS-2005S-N, Hahnvapor, Hahnshin Scientific, Korea). The collected dried extracts were saved in dark glass bottles and stored at -20°C for antimicrobial, antioxidant and phytochemical analysis [33].

 

The Antimicrobial Activity of the Isolated Actinobacteria

The isolates were screened for their antimicrobial activity using an agar plug diffusion assay. All actinomycete isolates (15) were grown on SNA medium at 25°C for 5 days. After incubation, the medium was cut aseptically with a sterile corkborer (6 mm) and the obtained discs were deposited on the opposed side on the Mueller Hinton agarplate, inoculated with 0.1 mL of suspensions of the tested pathogenic bacteria and all plates were incubated for 24 hours at 37°C. The substances diffuse from the plug to the agar. Then, the antimicrobial activity of the isolates was detected by the appearance of an inhibition zone (mm) around the agar plug. Moreover, a disc (6 mm diameter) fromRAD3, grown on SNA medium at 25°C for 5 days was transferred to 250 ml conical flasks containing 48 ml SN broth medium. The flasks were incubated in an incubator shaker (100 rpm) at 25°C for 5 days and the cultures were centrifuged at 4,000 rpm for 15 min at 4°C. The supernatant was collected, extracted with the same volume of ethyl acetate (v/v), dried and re-suspended in 2 ml DMSO and the antibacterial activity of the extract was detected using Agar well diffusion method and paper disc diffusion methods against MRSA and E. faecalis [34-36]. The mean diameter of the produced inhibition zone was determined. Broth microdilution assays were used to detect the minimal inhibitory concentration of the tested actinobacteria against a panel of pathogenic bacteria [36].

 

Effect of the Extract of Actinobacteria on Biofilm and Exopolysaccharide Formation by E. faecalis

All collected actinobacterial isolates were screened for inhibition of biofilm and exopolysaccharide formation using Crystal Violet Staining method [37,38], respectively as described before [39].

 

Antioxidant (DPPH Free Radical Scavenging) Activity

The five selected Actinobacteria were grown in the SNA medium at 25°C for 4 days (pre-culture) and 2 ml of the pre-culture (6×106 CFU/ml) was transferred to 250 ml conical flask containing 48 ml of fresh sterile SNB medium and the flasks incubated at 100 rpm and 25°C for 7 days. Cells were removed and the supernatant was used to determine the Antioxidant (free radical scavenging) activity [28]. Different concentrations of the actinobacteria extract were prepared from a stock solution of 10 mg/ml in methanol. In a 96-well plate containing 100 µl of 0.1 mM DPPH (Sigma-Aldrich, USA) in methanol and 100 µl of the tested bacterial extract (500, 400, 200, 100, 50 and 25 µg/ml). The plate was incubated for 30 min. at 20°C in a dark place [40]. Ascorbic acid and butylated hydroxytoluene (BHT) (Sigma-Aldrich, USA) were used as positive controls while DPPH solution (200 µl) was considered as negative control. The absorbance at 515 nm was measured using a Micro-titer plate reader (HT Synergy, USA) and the percentage scavenge activity was calculated [41]:

 

Where, Ac: Absorbance of control (DPPH), As: Absorbance of the sample.

 

The effective concentration of the extract produced a 50% reduction of DPPH activity (EC50) which was detected by drawing the relationship between inhibition percentage and extract concentrations.

 

Preliminary Phytochemical Analysis

The dry extracts (0.1 g) were dissolved in 5% dimethyl sulfoxide (DMSO) and analyzed for the presence of saponins, flavonoids, anthocyanins, betacyanins, steroids and tannins [42,43].

 

Statistical Analysis

All experiments were carried out in triplicate and data are expressed as Mean±standard deviation (SD). Antimicrobial activity data (zone of inhibition) were analyzed using one-way ANOVA followed by Tukey’s post-hoc test to compare differences between isolates. Antioxidant activity assays across different concentrations were evaluated using two-way ANOVA to assess both isolate- and concentration-dependent effects. A p-value <0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 22 (IBM Corp., Armonk, NY, USA)/GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA).

RESULTS

Isolation of Different Actinobacteria from Algal Samples

The following information is about the collection and identification of marine macroalgae from the coastal area of the Red Sea of Yanbu, Saudi Arabia (Figure 1). Four types of marine macroalgae were identified: two species of Chlorophyte (Green Algae), one species of Phaeophyte (Brown Algae) and one species of Rhodophyte (Red Algae). The specific algae identified were Ulva lactuca and Codium tomentosum from green algae, Padina gymnospora from brown algae and Hypnea valentia from red algae as shown in Figure 2. Two different media were used to isolate associated actinobacteria with the algae: Zobell Marine agar and SNA with 10% NaCl. The Zobell Marine agar was unsuitable for actinobacteria isolation and give weak growth and undefined colonies of actinobacteria, while the SNA with 10% NaCl was excellent for actinobacterial isolation as the colonies were good separated and colored. Fifteen different actinobacteria were isolated, purified and studied from SNA with 10% NaCl while no isolate was recovered from Zobell Marine agar. Seven isolates were from Ulva, three from Codium, three from Padina and two from Hypnea (Figure 3). The isolates were named RAD1 to RAD15.

 

Figure 1: The study area of the algal collection, Yanbu city, Saudi Arabia

 

Figure 2(a-d): The green algae, (a) Ulva lactuca, (b) Codium tomentosum, the brown algae, (c) Padina gymnospora and the red algae and (d) Hypnea Valentia

 

Figure 3: Percentage of isolated actinobacteria from Ulva lactucaCodium tomentosumPadina gymnospora and Hypnea Valentia

*Significant bacterial counts compared to control (Ulva)

 

Screening of the Isolated Actinobacteria for Antagonistic Activities

Antimicrobial activities of the 15 bacterial isolates were detected using agar disc diffusion assay and 9 bacterial pathogens. Moderate activity was recorded against E. coli as test organism by isolate RAD1, RAD3 and RAD14 while no activity was recorded by the other isolates (Figure 4a). Of the 15 isolates, 5 (33%) showed potent antimicrobial activity against more than one tested pathogens. Approximately 60% of the isolates showed inhibitory activity against Streptococcus pyogenes and 50% showed inhibitory activity against Staphylococcus aureus. Only 10% inhibited Acinetobacter baumannii and 19-20% inhibited Klebsiella oxitoca and Escherichia coli which was used as control bacteria (Figure 4b).

 

Figure 4: Screening of the 15 isolated actinobacteria for (a) Antimicrobial activities against E. coli using agar well diffusion assay and (b) The antimicrobial activities of the actinobacteria (15 isolates) against different bacterial pathogens

*Significant antibacterial activity compared to control (E. coli)

 

Some isolates showed antimicrobial activities against all tested pathogens, with one isolate (RAD3) showing the most potent antimicrobial activity (Table 1). The bacterial index was calculated for each actinobacterial isolate and the highest index was recorded for isolate RAD3, followed by RAD7. The lowest minimum inhibitory concentration (MIC) was recorded for S. aureusS. pyogenes and Enterococcus faecalis and it was ranged from 50 µg/ml while for Pseudomonas aeruginosaShigella sonneiSerratia marcescensE. coli and Salmonella typhi, it was ranged from 75-100 µg/ml. The active material of isolate RAD3 was extracted with ethyl acetate, showing excellent antimicrobial activity against MRSA and E. faecalis using two different methods, paper disc diffusion assay and agar well diffusion assay (Figure 5).

 

Table 1: The antimicrobial activities of five selected actinobacteria against different bacterial pathogens using paper disc diffusion assay

Tested bacteria

Antimicrobial activity of five selected actinomycetes

Ampicillin (Control )

MIC (µl/ml)

RAD1

RAD3

RAD7

RAD9

RAD14

RAD3 extract

Ampicillin (Control)

Acinetobacter baumannii

ND

ND

11±2.9

ND

ND

ND

ND

ND

Escherichia coli

12±2.0

13±1.4

ND

ND

14±1.0

20±1.1

75.0±25.0*

7.5±1.5

Klebsiella oxitoca

ND

ND

10±0.8

11±0.5

13±0.6

21±10

ND

7.5±1.0

Pseudomonas aeruginosa

10±2.4

13±2.0

11±0.9

11±0.5

13±0.4

29±2.0

75.0±25.0*

7.5±1.0

Salmonella typhi

11±0.6

13±0.8

10±1.0

11±0.4

11±0.6

22±1.6

100±25.0*

7.5±1.0

Serratia marcescens

10±2.0

16±0.8

14±0.33

16±1.1

13±0.8

30±1.4

75.0±25.0*

5.0±0.5

Shigella sonnei

11±2.0

13±0.3

10±2.2

14±0.7

11±0.8

26±0.4

100.0±25.0*

7.5±0.5

Enterococcus faecalis

17±1.0

21±0.29

14±0.57

10±0.3

18±1.0

30±0.4

50.0±2.5

5.0±1.5

Staphylococcus aureus (MRSA)

19±2.1

22±1.45

11±0.49

14±1.4

14±2.4

40±0.6

50.0±2.5*

5.0±1.5

Streptococcus pyogenes

17±1.9

23±2.7

11±0.54

13±1.2

15±2.5

37±0.6

50.0±2.5*

5.0±1.5

Bacterial index**

13.95*

17.20*

12.75*

14.8*

16.8*

30.7

ND

LSD

1123

229

1158

720

543

619

11290

411

 

Figure 5(a-c): The antimicrobial activity of the crude extract of the isolate RAD3 using paper disc diffusion assay and against (a) MRSA and (b) E. faecalis and using agar well diffusion assay against (c) E. faecalis

 

Actinobacterial Extracts Inhibited of Biofilm Formation and EPS Secretion of the Bacterial Pathogens

Additionally, the actinobacteria extracts inhibited biofilm formation to different degrees. The isolate RAD3 extract was the most active in bacterial growth inhibition and prevented biofilm formation by 63% and exopolysaccharide (EPS) by 47% while the lowest activities were recorded by isolate RAD1 extract (Table 2).

 

Table 2: The inhibition of biofilm and exopolysaccharide formation using E. faecalis as test strain and the antioxidant activity detected by DPPH and compared to the control (ascorbic acid and BHT) of the selected actinobacteria extracts

Actinobacteria extract

Biofilm inhibition (%)

EPS inhibition (%)

DPPH reducing %

RAD1

19*a

23*a

55.08±0.51*a

RAD3

63*b

47*d

66.38±0.72*b

RAD7

39*c

27*c

60.16±0.52*c

RAD9

30*c

24*a

50.10±0.32*a

RAD14

28*c

25*a

58.02±0.12*c

Negative control (no extract)

0

0

0

BHT

ND

ND

96.0±1.19d

Ascorbic acid

ND

ND

100

*Significant results compared to negative control, The results are the mean of three replicates, Values within a column followed by different alphabets are significantly different at p≤0.05, ND: Not detected

 

The Antioxidant Activities of the Actinobacterial Extracts

Also, the antioxidant activities of the selected actinobacteria were detected by DPPH free radical scavenging activity and the results were significantly different compared to the control, ascorbic acid (p<0.05). The highest scavenging percentage was recorded for RAD3 extract, which demonstrated notable antioxidant activity, the lowest was by extract RAD 9 and extracts of isolate RAD1, RAD7 and RAD14 recorded moderate activities. The percentage of reduced DPPH radical by bacterial ethyl acetate extracts at a concentration of 1mg/ml is shown in Table 2. The most potent scavenger among bacterial species was RAD3 extract which recorded 66% activity of the standard antioxidant. EC50 of the actinobacteria was detected and compared to controls (Figure 6). RAD 3 extract has EC50 = (29.14 µg/ml) relatively higher than the synthetic antioxidant BHT EC50 = (17.22 µg/ml).

 

Figure 6: Effective concentration (µg/ml) of the actinobacteria extracts, reduced DPPH radicals by 50% (EC50 values)

 

The Primary Analysis of the Actinobacterial Extracts

The primary analysis revealed the presence of flavonoids, anthocyanins, betacyanins and steroids but no saponins or tannins, among the extract of the isolate RAD 3, as shown in Table 3.

 

Table 3: Preliminary phytochemical analysis of ethyl acetate extract of the isolate RAD3

Bacterial extract

Phytochemicals

Saponins

Flavonoids

Anthocyanin

Betacyanin

Tannins

Steroids

Isolate RAD3

-

+

+

+

-

+

+: The presence of the material, -: The absent of the material

 

Characterization and Identification of the Selected Actinomycetes

The growth of the selected actinobacteria (RAD1, RAD7, RAD9 and RAD14) on oatmeal agar is illustrated in Figure 7 while the growth, Gram stain and shape of aerial and substrate mycelia, examined under a scanning electron microscope of the isolate RAD3, are summarized in Figure 8. It's evident that all isolates displayed good growth on oatmeal agar and belonged to the gray series, except isolate RAD1, which belonged to the white series. Some morphological, biochemical and physiological characteristics of the selected actinobacteria are presented in Table 4. The spores are in chains that are straight, spiral or closed spiral shapes with smooth or rough surfaces and the aerial and substrate mycelia were well-developed with different colors. Soluble pigments of different colors were detected for all strains and no production of catalase and urease enzymes was observed. The actinobacteria showed no visible change on blood agar, such as staining or clearing around the bacterial colony (gamma hemolysis), as they produced no hemolytic enzymes. Additionally, the selected actinobacteria were identified through molecular analysis as species belonging to the genus Streptomyces (Table 5). Isolate RAD1 was isolated from Ulva while RAD3 and RAD7 were from Codium. Similarly, RAD 9 and RAD14 were from Padina and Hypnea, respectively. All isolates, RAD1, RAD3, RAD7, RAD9 and RAD14 were identified as a filamentous bacteria of the genus Streptomyces. The phylogenetic tree analysis of the obtained bacteria is shown in Figure 9 and the isolates were identified as S. filamentosus RAD1, S. glaucescens RAD3, S. chilikensis RAD7, S. pseudogriseolus RAD9 and Streptomyces sp. RAD14. The similarity levels ranged from 95-97%, while the percentages of G+C were high, ranging from 60-70%.

 

Table 4: Some morphological, biochemical and physiological characteristics of the selected actinobacteria grown on Oat meal agar at 25°C

Isolate

Growth

Spore chain

Spore surface

Color

Aerial mycelia

Substrate mycelia

Soluble Pigment

Catalase

Urease

Hemolysis

RAD1

+

Straight

Smooth

White to gray

White

Dark brown

Pale brown

-ve

-ve

γ

RAD3

++

Straight

Smooth

Gray

Reddish gray

Reddish brown

Pale brown

-ve

-ve

γ

RAD7

++

Closed spiral

Rough

Gray

Pale gray

Dark gray

Reddish gray

-ve

-ve

γ

RAD9

++

Spiral

Rough

Gray

Gray

Dark gray

Yellowish gray

-ve

-ve

γ

RAD14

++

Spiral

Smooth

Gray

Dark gray

Black

Dark gray

-ve

-ve

γ

γ: Gamma hemolysis, -ve: Negative result, +: Moderate growth, ++: High growth

 

Table 5: Molecular identification of actinomycetes associated with algal samples based on 16S rRNA gene sequence analysis

Isolate No.

Source

Close relative in GenBank

Closest species

Identity (%)

G+C (%)

RAD1

Ulva lactuca

NR_115665.1

Streptomyces filamentosus

95

60

RAD3

Codium tomentosum

NR_118246.1

Streptomyces glaucescens

95

67

RAD7

Codium tomentosum

CP009438.1

Streptomyces chilikensis

97

70

RAD9

Padina gymnospora

EU598259.1

Streptomyces pseudogriseolus

95

67

RAD14

Hypnea valentia

KF991642.1

Streptomyces sp. XJSS-67

97

66

 

 

Figure 7: The growth of the most active antibacterial producer actinobacteria on starch nitrate agar after 10 days of growth at 25°C

 

Figure 8(a-c): The isolate RAD 3 on (a) Oatmeal agar, (b) Examined under a light microscope after Gram staining x1000 and (c) Examined under a scanning electron microscope

 

Figure 9: The phylogenetic tree of the obtained actinobacteria on 16S rRNA sequences

DISCUSSION

The Red Sea is one of the most important and unique marine environments, hosting diverse habitats such as sea-grass, macroalgae, sponges and mangroves with rich microbial communities24. Four algae were collected during winter to obtain special bacterial isolates with excellent antimicrobial activity. The absence of these activity was usually recorded during summer and autumn due to the unfavorable and dry conditions that prevailed during these successive seasons in seawater and sediments [44,45]. The Red Sea is known for its rich biodiversity, its high salinity and unique physical and chemical conditions making it a valuable source of epiphytic bacteria, which have been defined as non-parasitically populations and can survive, live and multiply on the surface of various organs of plants, algae and animals. These epiphytic bacteria occupy ecological niches that could be occupied by pathogens and can produce bioactive compounds for pharmaceutical industries [46]. Epiphytic actinobacteria are known for their filamentous growth, broad antagonistic effects against pathogens and ability to produce bioactive compounds with various biological activities, such as antimicrobial, anticancer and immunosuppressive properties [47]. However, the isolation of actinomycetes from algae in the Red Sea is not well studied. Out of fifteen actinobacterial isolates associated with the four collected algae, five were selected for their antimicrobial activities and identified based on their morphology and sequences of the 16 S rRNA. DNA was extracted and amplified for molecular identification of the strains to the species level. The 16S rRNA sequence was compared to the GenBank database. Similarly, various strains of actinobacteria, such as S. chilikensisS. glaucescens and S. pseudogriseolus, have been isolated from different marine sediment in India and S. pseudogriseolus was found to produce several bioactive compounds with antimicrobial activity [48]. Understanding and isolating these bacteria is crucial, especially in light of the growing concern about antibiotic-resistant bacteria, which cause a significant number of deaths annually and are projected to cause even more in the future [18]. Therefore, the search for new biologically active compounds from various sources is essential to develop novel antimicrobial agents. Different marine bacteria play a pivotal role as primary colonizers on the surfaces of organisms within the marine environment and have been isolated from a diverse range of biological and ecological niches, including seaweeds, marine invertebrates, sediments and water and produce antimicrobial agents effective against multidrug-resistant bacteria [49]. Similarly, five marine bacteria with inhibitory effects against E. coliS. aureus and Listeria monocytogenes were isolated by Chbel et al. [50]. The relationship between bacteria and their algae is considered an ancient survival strategy that provides microorganisms with advantages for proliferation, such as access to nutrient-rich habitats and increased environmental stability. Microbial aggregates on the algal surface offer higher protection against external stresses, play a role in host-associated protein and carbohydrate transport and cycling and control high pathogen growth inside their host through competition and exclusion methods [5,6].

 

Secondary metabolites released by different types of macroalgae can selectively attract and enhance the growth of specific bacteria [10]. Four bacterial isolates from red algae and among them, Lysinibacillus odyssey exhibited broad-spectrum antibacterial activity against E. coliKlebsiella pneumoniaeShigella strains [50]. Similarly, 15 actinomycetes were isolated from 4 different sponge samples collected from the Red Sea and Streptomyces sp. Sp9 extract showed potent antitumor activity, due to the presence of heliomycin and tetracenomycin D [51].

 

The antioxidants are natural phytochemicals present in plants and macroalgae. The discovery of new species with significant antimicrobial and antioxidant activities in marine water is recommended because these microorganisms may be new species and have developed mechanisms to synthesize bioactive compounds with distinctive structures and features that help them defend against predators and competition [52-55]. In this study, the five selected isolates exhibited great potential as antioxidants, with RAD3 showing the most promising results. As far as we know, this could be the first study on the antioxidant activity of associated actinobacteria with algae in Saudi Arabia. It was known that Actinobacteria produce some pigments with significant antioxidant activity against DPPH radicals [56]. Similar to our results, the bacteria associated with the brown algae revealed ≥60% DPPH scavenging activity [30,56] and can act as a potential probiotic. The presence of Steroids, flavonoids and tannins in most bacteria extracts may explain the antioxidant activities, in addition to others such as polysaccharides, fatty acids and proteins [57-59] and many studies indicated a positive correlation between the antioxidant activity and the total phenolic compounds [60,61].

 

This study had few limitations. Sampling was restricted to a few algal species and locations in the Red Sea, which may not reflect the full actinobacterial diversity. Only culturable isolates were examined, potentially overlooking non-culturable strains with bioactive potential. Antimicrobial and antioxidant activities were tested in vitro without in vivo validation and the chemical structures of the active compounds were not characterized. Additionally, advanced analyses such as metabolomics or biosynthetic gene cluster identification were not performed, limiting mechanistic insights.

 

Further investigations are recommended to uncover the potential hidden in the Red Sea shores. Finally, the identified isolates belonged to the genus Streptomyces, family Streptomycetaceae and order Actinomycetales of the class Schizomycetes. The bacteria of this genus are mainly found in soil and occasionally isolated from marine sources and showed antimicrobial and antioxidant activities [62-64].

CONCLUSIONS

This study highlighted the untapped bioactive potential of Red Sea algal-associated actinobacteria, with Streptomyces glaucescens emerging as a particularly promising isolate. Identified through morphological and molecular methods, its extract exhibited strong antibacterial activity, effectively inhibited biofilm formation and EPS production and showed notable antioxidant effects. These properties position it as a valuable candidate for the development of novel therapeutic agents targeting infectious diseases and oxidative stress-related disorders. To fully realize its potential, future research should focus on characterizing the bioactive compounds responsible, elucidating their mechanisms of action and assessing their applications in medicine, pharmaceuticals and related industries.

 

Acknowledgement

The authors gratefully acknowledge King Abdulaziz University, Department of Biological Science, Faculty of Science, Saudi Arabia.

 

Conflicts of Interest

The author reports no conflict of interest.

REFERENCES

  1. Webster, N.S. and M.W. Taylor. "Marine sponges and their microbial symbionts: Love and other relationships." Environmental Microbiology, vol. 14, no. 2, 2012, pp. 335-346.
  2. Al-Saif, S.S.A. et al. "Antibacterial substances from marine algae isolated from Jeddah coast of Red Sea, Saudi Arabia." Saudi Journal of Biological Sciences, vol. 21, no. 1, 2014, pp. 57-64. https://doi.org/10.1016/j.sjbs.2013.06.001.
  3. Thushari, G.G.N. and J.D.M. Senevirathna. "Plastic pollution in the marine environment." Heliyon, vol. 6, no. 8, 2020, e04709.
  4. Datta, D. et al. "Bioactive compounds from marine invertebrates for potential medicines: An overview." International Letters of Natural Sciences, vol. 7, 2015, pp. 42-61.
  5. Lo Giudice, A. and C. Rizzo. "Bacteria associated with benthic invertebrates from extreme marine environments: Promising but underexplored sources of biotechnologically relevant molecules." Marine Drugs, vol. 20, no. 10, 2022, p. 617.
  6. Neave, M.J. et al. "Diversity and function of prevalent symbiotic marine bacteria in the genus Endozoicomonas." Applied Microbiology and Biotechnology, vol. 100, 2016, pp. 8315-8324.
  7. Wang, J. et al. "Diversity of epiphytic bacterial communities on male and female Sargassum thunbergii." AMB Express, vol. 12, no. 1, 2022, p. 97.
  8. Alaidroos, B. et al. "Evaluation of the biological activities of two macro-algae collected from the Red Sea of Jeddah." European Online Journal of Natural and Social Sciences, vol. 12, 2022, pp. 551-559.
  9. Schwenk, D. et al. "Algae-bacteria association inferred by 16S rDNA similarity in established microalgae cultures." MicrobiologyOpen, vol. 3, no. 3, 2014, pp. 356-368.
  10. Susilowati, R. et al. "Isolation and characterization of bacteria associated with brown algae Sargassum spp. from Panjang Island and their antibacterial activities." Procedia Environmental Sciences, vol. 23, 2015, pp. 240-246.
  11. Abdelfattah, M.S. et al. "Isolation and characterization of marine-derived actinomycetes with cytotoxic activity from the Red Sea coast." Asian Pacific Journal of Tropical Biomedicine, vol. 6, no. 8, 2016, pp. 651-657.
  12. Lemos, M.L. et al. "Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds." Microbial Ecology, 1985, pp. 149-163.
  13. Armstrong, E. et al. "The symbiotic role of marine microbes on living surfaces." Hydrobiologia, vol. 461, 2001, pp. 37-40.
  14. Saurav, K. and K. Kannabiran. "Chromium heavy metal resistance activity of marine Streptomyces VITSVK5 spp. (GQ848482)." Pharmacologyonline, vol. 3, 2009, pp. 603-613.
  15. Hamed, M.M. et al. "Antimicrobial activity of marine actinomycetes and the optimization of culture conditions for the production of antimicrobial agent(s)." Journal of Pure and Applied Microbiology, vol. 13, 2019, pp. 2177-2188.
  16. Anteneh, Y.S. et al. "Antimicrobial activities of marine sponge-associated bacteria." Microorganisms, vol. 9, no. 1, January 2021, p. 171. doi:10.3390/microorganisms9010171.
  17. Al-Dhabi, N.A. et al. "Metabolite profiling of Streptomyces sp. El baz-100 isolated from the marine environment in Saudi Arabia with antibacterial, anti-tubercular and antioxidant potentials." Journal of King Saud University-Science, vol. 32, no. 2, 2020, pp. 1628-1633.
  18. Salam, M.A. et al. "Antimicrobial resistance: A growing serious threat for global public health." Healthcare (Basel), vol. 11, no. 13, July 2023, p. 1946. Doi:10.3390/health care11131946.
  19. Indraningrat, A.A. et al. "Bioprospecting sponge-associated microbes for antimicrobial compounds." Marine Drugs, vol. 14, no. 5, May 2016, p. 87. Doi:10.3390/md14050087.
  20. Doshi, G.M. et al. “Novel antibiotics from marine sources.” International Journal of Pharmaceutical Sciences and Nanotechnology, vol. 4, no. 3, 2011, pp. 1446-1461.
  21. Mancini, I. et al. “Recent synthesis of marine natural products with antibacterial activities.” Bentham Science Publishers, 2007.
  22. Hassanali, K. and R. Mahon. “Encouraging proactive governance of marine biological diversity of areas beyond national jurisdiction through strategic environmental assessment (SEA).” Marine Policy, vol. 136, 2022.
  23. Ameen, F. et al. “Marine fungi showing multifunctional activity against human pathogenic microbes and cancer.” PLOS One, vol. 17, no. 11, 2022, e0276926.
  24. Bernardi, J. et al. “Screening for antioxidant capacity of tropical reef seaweeds: Prospection for new natural antioxidants.” Tropical Oceanography, vol. 45, no. 2, 2017, pp. 16-30.
  25. Priyankaa, P. et al. “Prospecting exopolysaccharides produced by selected bacteria associated with marine organisms for biotechnological applications.” Chinese Journal of Polymer Science, vol. 33, no. 2, 2015, pp. 236-244.
  26. Rout, S. and A. Kumar. “A review on the potentiality of marine seaweeds.” World Journal of Pharmacy and Pharmaceutical Sciences, vol. 4, no. 10, 2015, pp. 458-476.
  27. Ahmed, E.F. et al. “A comparative biochemical study on two marine endophytes, Bacterium SRCnm and Bacillus sp. JS, isolated from Red Sea algae.” Pakistan Journal of Pharmaceutical Sciences, vol. 29, no. 1, 2016, pp. 17-26.
  28. Ishakani, A.H. et al. “Indian journal of science and technology IndJST.” Indian Journal of Science and Technology, vol. 9, no. 4, 2016. https://doi.org/10.17485/ijst/2016/v9i4/78665.
  29. Aly, M.M. et al. “Physiological response of Zea mays to NaCl stress with respect to Azotobacter chroococcum and Streptomyces niveus.” Pakistan Journal of Biological Sciences, vol. 6, no. 24, 2003, pp. 2073-2080.
  30. Horta, A. et al. “Antioxidant and antimicrobial potential of the Bifurcaria bifurcata epiphytic bacteria.” Marine Drugs, vol. 12, no. 3, 2014, pp. 1676-1689. https://doi.org/10.3390/md 12031676.
  31. Fawole, M. and B. Oso. Characterization of bacteria: Laboratory manual of microbiology. 4th ed., Spectrum Book Ltd., 2004.
  32. Gaget, V. et al. “DNA extraction from benthic cyanobacteria: Comparative assessment and optimization.” Journal of Applied Microbiology, vol. 122, no. 1, 2017, pp. 294-304. https://doi.org/10.1111/jam.13332.
  33. Yoghiapiscessa, D. et al. “Antimicrobial and antioxidant activities of bacterial extracts from marine bacteria associated with sponge Stylotella sp.” American Journal of Biochemistry and Biotechnology, vol. 12, no. 1, 2016, pp. 36-46. https://doi.org/10.3844/ajbbsp.2016.36.46.
  34. Naikpatil, S.V. and J.L. Rathod. “Selective isolation and antimicrobial activity of rare actinomycetes from mangrove sediment of Karwar.” Journal of Ecobiotechnology, vol. 3, 2011, pp. 48-53.
  35. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests, approved standard. 7th ed., CLSI document M02-A11, Clinical and Laboratory Standards Institute, 2012.
  36. Balouiri, M. et al. “Methods for in vitro evaluating antimicrobial activity: A review.” Journal of Pharmaceutical Analysis, vol. 6, no. 2, 2016, pp. 71-79. https://doi.org/10.10 16/j.jpha.2015.11.005.
  37. Christensen, G.D. et al. “Methods for studying microbial colonization of plastics.” Methods in Enzymology, vol. 253, 1995, pp. 477-500.
  38. Dubois, M. et al. “Colorimetric method for determination of sugars and related substances.” Analytical Chemistry, vol. 28, no. 3, 1956, pp. 350-356.
  39. Jastaniah, S.D. et al. “New strategies for inhibition of Listeria monocytogenes and Klebsiella pneumoniae biofilm formation and persistence.” Journal of Contemporary Medical Sciences, vol. 8, no. 6, 2022, pp. 400-407.
  40. Rinaldi, F. et al. "Neem Oil Nanoemulsions: Characterization and Antioxidant Activity." Journal of Enzyme Inhibition and Medicinal Chemistry, vol. 32, no. 1, 2017, pp. 1265-1273.
  41. Valko, M. et al. "Free Radicals and Antioxidants in Normal Physiological Functions and Human Disease." International Journal of Biochemistry and Cell Biology, vol. 39, 2007, pp. 44-84.
  42. Fadeyi, M.O. et al. "Epidermal and Phytochemical Studies in the Genus Boerhavia (Nyctaginaceae) in Nigeria." International Journal of Crude Drug Research, vol. 27, no. 3, 1989, pp. 178-184. https://doi.org/10.3109/138802089090 53960
  43. Varadharajan, V. et al. "Physicochemical, Phytochemical Screening and Profiling of Secondary Metabolites of Annona Squamosa Leaf Extract." Journal of Pharmaceutical Research, vol. 1, no. 4, 2012, pp. 1143-1164.
  44. Chakraborty, B. et al. "Streptomyces filamentosus Strain KS17 Isolated from Microbiologically Unexplored Marine Ecosystems Exhibited a Broad Spectrum of Antimicrobial Activity against Human Pathogens." Process Biochemistry, vol. 117, 2022, pp. 42-52. https://doi.org/10.1016/j.procbio. 2022.03.010
  45. El-Bestawy, E.A. et al. "Distribution and Biodiversity of Antimicrobial Agents Producing Marine Bacteria along Alexandria Coasts." Journal of Applied Sciences Research, vol. 6, no. 12, 2010, pp. 2199-2211.
  46. Srinivasan, R. et al. "Marine Bacterial Secondary Metabolites: A Treasure House for Structurally Unique and Effective Antimicrobial Compounds." Marine Drugs, vol. 19, no. 10, 2021, p. 530.
  47. Khallaf, H. et al. "Isolation and Molecular Characterization of Some Plant Growth Promoting Actinomycetes from Cultivated Soil for Management of Some Pathogenic Microbes." Agriculture and Biology Research, vol. 39, no. 1, 2023, pp. 412-420.
  48. Ray, L., et al. "Streptomyces chilikensis sp. nov., a Halophilic Streptomycete Isolated from Brackish Water Sediment." International Journal of Systematic and Evolutionary Microbiology, vol. 63, pt. 8, 2013, pp. 2757-2764. https://doi. org/10.1099/ijs.0.046284-0
  49. Qureshi, K.A. et al. "Bio-Characterizations of Some Marine Bacterial Strains Isolated from Mangrove Sediment Samples of Four Major Cities of Saudi Arabia." Journal of Environmental Biology, vol. 41, no. 5, 2020, pp. 1003-1012.
  50. Chbel, A., et al. "Isolation, Molecular Identification and Antibacterial Potential of Marine Bacteria from Deep Atlantic Ocean of Morocco." Avicenna Journal of Medical Biotechnology, vol. 14, no. 3, 2022, pp. 206-215.
  51. Abdelfattah, M.S. et al. "Heliomycin and Tetracinomycin D: Anthraquinone Derivatives with Histone Deacetylase Inhibitory Activity from Marine Sponge-Associated Streptomyces sp. SP9." 3 Biotech, vol. 8, no. 6, 2018, p. 282. https://doi.org/10.1007/s13205-018-1304-1.
  52. El-Kurdi, N. et al. "Anti-Quorum Sensing Activity of Some Marine Bacteria Isolated from Different Marine Resources in Egypt." Biotechnology Letters, vol. 43, 2021, pp. 455-468.
  53. Sarkar, G. and K. Suthindhiran. "Diversity and Biotechnological Potential of Marine Actinomycetes from India." Indian Journal of Microbiology, vol. 62, 2022, pp. 475-493. https://doi.org/10.1007/s12088-022-01024-x
  54. Selvaraj, S. et al. "Studies on Phytochemical Screening, Antioxidant Activity and Extraction of Active Compound (Swertiamarin) from Leaf Extract of Enicostemma Littorale." Asian Journal of Pharmaceutical and Clinical Research, vol. 7, no. 4, 2014, pp. 240-244.
  55. Oh, J.Y. et al. "Potential Applications of Radioprotective Phytochemicals from Marine Algae." Algae, vol. 31, no. 4, 2016, pp. 403-414. https://doi.org/10.4490/algae.2016.31.12.1
  56. El-Newary, S.A. et al. "Production, Characterization and Biological Activities of Acidic Exopolysaccharide from Marine Bacillus amyloliquefaciens 3MS 2017." Asian Pacific Journal of Tropical Medicine, vol. 10, no. 7, 2017, pp. 652-662. https://doi.org/10.1016/j.apjtm.2017.07.005
  57. Al-Enazi, N.M. et al. "Antimicrobial, Antioxidant and Anticancer Activities of Laurencia CatarinensisLaurencia Majuscula and Padina Pavonica Extracts." Saudi Pharmaceutical Journal, vol. 26, no. 1, 2018, pp. 44-52. https:// doi.org/10.1016/j.jsps.2017.11.001
  58. Alassali, A. et al. "Advanced Techniques in Biology and Medicine." Advanced Techniques in Biology and Medicine, vol. 4, no. 1, 2016. http://dx.doi.org/10.4172/2379-1764.1000 163.
  59. Vidal-Novoa, A. et al. "Anti-Atherogenic Properties Associated with the Antioxidant Activity from the Hydrophilic Extracts of Halimeda Incrassata (Chlorophyta, Bryopsidales)." African Journal of Agricultural Research, vol. 12, no. 4, 2017, pp. 208-220. https://doi.org/10.5897/AJAR 2014.9328
  60. Agatonovic-Kustrin, S. et al. "Assessment of Antioxidant Activity in Victorian Marine Algal Extracts Using High Performance Thin-Layer Chromatography and Multivariate Analysis." Journal of Chromatography A, vol. 1468, 2016, pp. 228-235. https://doi.org/10.1016/j.chroma.2016.09.041
  61. Surget, G. et al. "Marine Green Macroalgae: A Source of Natural Compounds with Mineralogenic and Antioxidant Activities." Journal of Applied Phycology, vol. 29, 2016, pp. 575-584. https://doi.org/10.1007/s10811-016-0968-3
  62. Quinn, G.A. et al. "Streptomyces from Traditional Medicine: Sources of New Innovations in Antibiotic Discovery." Journal of Medical Microbiology, vol. 69, 2020, pp. 1040-1048.
  63. Rammali, S. et al. "Antimicrobial and Antioxidant Activities of Streptomyces Species from Soils of Three Different Cold Sites in the Fez-Meknes Region, Morocco." Scientific Reports, vol. 12, 2022, p. 17233. https://doi.org/10.1038/s415 98-022-21644-z
  64. Ghanem, N.B. et al. "Isolation and Enumeration of Marine Actinomycetes from Seawater and Sediments in Alexandria." Journal of General and Applied Microbiology, vol. 46, 2000, pp. 105-111.
Recommended Articles
Case Report

Interstitial Lung Disease in Dermatomyositis and Polymyositis in Patients from Saudi Arabia: A Single Tertiary Centre Retrospective Report

...
Published: 05/09/2025
pdf Download PDF
Research Article

Comparative Evaluation of Verbal, Printed, and Virtual Reality Approaches for Post-Operative Dental Care Instructions: Perspectives of Patients and Dental Students

...
Published: 05/09/2025
pdf Download PDF
Case Report

Clinical profile of Behcet’s Disease patients from Saudi Arabia: A Single Tertiary Centre, Retrospective Report

...
Published: 05/09/2025
pdf Download PDF
Research Article

The Impact of Oral Health Status on Daily Performances Using the Child-OIDP Index Among 12-14-Year-Old Children in Residential Schools of Bangalore

...
Published: 05/09/2025
pdf Download PDF
Copyright © Journal of Pioneering Medical Sciences until unless otherwise.