A novel Streptomyces zaomyceticus metabolite exhibits antifungal activity against Candida albicans in African catfish

Abstract

Fungal infection, particularly that caused by Candida albicans, pose emerging challenges in aquaculture, leading to significant economic and health losses in cultured fishes. However, limited research has explored eco-friendly antifungal alternatives for Clarias gariepinus, a species of high commercial value and susceptibility to fungal pathogens. This study aimed to isolate and identify marine-derived Actinobacteria capable of producing potent antifungal compounds effective against C. albicans. An initial screening of 30 Actinobacteria isolates obtained from thirty marine samples collected from Marsa Alam, Egypt was conducted. Among these, Streptomyces zaomyceticus (PQ395286) was identified as a producer of lucidin (1,3-diethoxy-1,1,3,3-tetramethyl lucidin), a novel bioactive metabolite with strong antifungal activity. In vitro assays demonstrated that lucidin inhibited C. albicans (ATCC 10221) with a minimum inhibitory concentration (MIC) of 0.5 μg/mL as confirmed by scanning electron microscopy. To evaluate its in vivo efficacy, C. gariepinus were randomly divided into four groups (three replicates each, 10 fish per replicate). Group 1 served as the negative control (no infection or treatment); Group 2 received lucidin exposure at 100 μg/L through water administration; Group 3 was intraperitoneally infected with C. albicans (3.6 × 10⁴ CFU/fish); and Group 4 was both infected and treated with lucidin. Lucidin treatment markedly alleviated infection-induced alterations in hepatorenal biomarkers, nonspecific immunity, and antioxidant status. It significantly enhanced nitric oxide (NO) production, lysozyme (LYZ) activity, and overall antioxidant capacity. Histological observations confirmed that lucidin effectively restored the normal architecture of the liver, kidney, and spleen. Collectively, these findings highlight lucidin as a promising natural antifungal agent that can effectively mitigate C. albicans infection in C. gariepinus, thereby contributing to the development of sustainable antifungal strategies in aquaculture.

Introduction

Microorganisms are prolific producers of diverse metabolites with significant biological activities. These metabolites are generally classified into primary metabolites, which are essential for cell growth and metabolic processes (such as amino acids, nucleotides, and organic acids) [1] and secondary metabolites, which are synthesized during the stationary growth phase and often possess ecological or pharmacological functions [2]. Among these, bioactive compounds constitute a specialized category of secondary metabolites that exhibit distinct biological effects, including antimicrobial, antifungal, antiviral, and anticancer activities [3–5].

The extensive and, in some cases, inappropriate use of conventional antibiotics has contributed to the emergence of multidrug-resistant (MDR) pathogens, posing serious challenges to public health, veterinary medicine, and aquaculture [6–8]. Consequently, there is growing scientific interest in exploring novel antimicrobial agents of natural origin that can serve as safe, eco-friendly alternatives to synthetic drugs [9–11].

Actinobacteria, particularly the genus Streptomyces, are well recognized as a major source of bioactive secondary metabolites. These Gram-positive, high-G + C microorganisms are responsible for producing nearly 75% of known antibiotics, including vancomycin, erythromycin, and gentamicin [12–15]. In addition to antibacterial agents, Streptomyces spp. synthesize numerous metabolites exhibiting antifungal, anticancer, and anti-inflammatory properties [16–18]. Recent studies have also highlighted their potential as probiotics in aquaculture due to their ability to inhibit pathogenic microorganisms and enhance host immunity [19, 20].

Advanced analytical techniques such as gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS), and nuclear magnetic resonance (NMR) spectroscopy have facilitated the extraction and structural elucidation of microbial metabolites [21–23]. Among these, GC–MS is widely used for detecting low-molecular-weight bioactive compounds and identifying antimicrobial agents derived from Streptomyces species [24–26].

Lucidin, an anthraquinone derivative produced by S. zaomyceticus PQ395286, has been reported to possess diverse bioactivities, including antiprotozoal effects [27]. However, its antifungal potential has not yet been investigated, particularly in the context of aquaculture. Given the urgent need for alternative antifungal strategies, exploring lucidin’s bioactivity against fish-pathogenic fungi offers a promising research avenue. It is important to note that, while this study demonstrates lucidin’s antifungal efficacy against C. albicans, it does not claim that resistance to lucidin will never develop. Rather, lucidin may act through mechanisms different from those of conventional antifungal drugs, potentially reducing the risk or delaying the onset of resistance.

In aquaculture, opportunistic fungal pathogens such as C. albicans are increasingly recognized as significant threats, causing skin lesions, internal organ damage, and high mortality rates in economically important species such as tilapia, zebrafish, and African catfish [28]. The scaleless skin and high stress sensitivity of C. gariepinus make it particularly vulnerable to fungal infections, often resulting in hepatic and renal impairment and weakened immune responses. As C. gariepinus represents a cornerstone of Egypt’s aquaculture industry [29], the development of sustainable and effective antifungal interventions is essential.

The marine environment represents a rich and largely untapped reservoir of biologically active natural products. Since the mid-1980s, more than four thousand marine-derived bioactive compounds have been identified from a broad spectrum of organisms, including invertebrates, marine plants, and their associated or sediment-dwelling microorganisms. These metabolites display diverse pharmacological properties such as antibacterial, antiviral, antifungal, antiprotozoal, antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective effects [30, 31].

Among these, antifungal metabolites have attracted increasing scientific attention. Reports indicate that the majority of marine antifungal compounds originate from sponges and bacteria, many of which exist in close symbiotic relationships. Nevertheless, other microbial sources, particularly marine fungi and free-living bacteria, remain insufficiently explored despite their potential to yield novel antifungal secondary metabolites [32]. This research direction highlights the growing importance of lucidin as promising candidates for the discovery and development of new antifungal agent against C. albicans in C. gariepinus, while offering an updated overview of their chemical diversity and biological potential.

Therefore, the present study aimed to isolate, purify, and characterize the bioactive compound lucidin from S. zaomyceticus PQ395286 and to evaluate its in vitro and in vivo antifungal efficacy against C. albicans infection in C. gariepinus. Furthermore, the study investigated lucidin’s impact on immunological, antioxidant, and histopathological parameters, providing the first evidence of its potential as a natural antifungal agent for aquaculture applications.

Material and methods

Sample collection from the sea

A total of thirty marine samples—comprising ten sediment samples and twenty seawater samples were collected from ten distinct locations along the Red Sea coast at depths ranging from 30 to 1000 cm [26]. Sediment samples were collected aseptically using a sterile stainless-steel corer, while seawater samples were obtained using pre-sterilized Niskin bottles. Immediately after collection, temperature, pH, and salinity were measured on-site using a thermometer, a calibrated portable pH meter, and a salinity meter, respectively. All samples were transferred into sterile polyethylene containers, properly labeled, and transported to the laboratory under refrigerated conditions (4 °C) for subsequent microbiological analyses.

Isolation of marine actinobacteria

Actinobacteria were isolated from both sediment and seawater samples collected from ten Red Sea locations. Each sample was serially diluted in sterile seawater, and 0.1 mL aliquots of appropriate dilutions were spread onto Starch Casein Agar (SCA) and ISP2 medium, both supplemented with cycloheximide (50 µg/mL) and nalidixic acid (20 µg/mL) to inhibit fungal and Gram-negative bacterial growth, respectively [33, 34]. The plates were incubated at 28 ± 2 °C for 7–14 days. Distinct, dry, chalky colonies typical of actinobacteria were picked and repeatedly sub-cultured to obtain pure isolates. A total of 30 morphologically distinct Actinobacteria isolates were obtained (10 from sediment samples and 20 from seawater samples). Daily observations of the SCA plates revealed the growth of actinomycetes, which were easily identified by their characteristic chalky to leathery appearance. Spore development and morphology were examined using a light microscope to confirm their filamentous nature. Individual colonies were harvested and subcultured on SCA [35]. The colonies exhibited unique traits, such as differences in shape, colour, and the amount of diffusible pigment. To achieve optimal sporulation, isolates were incubated on SCA slants at 28 °C for 5–14 days. According to [36] et al. (2021), all isolates were maintained on ISP2 slants at 4 °C for short-term preservation and in 20% glycerol at −20 °C for long-term storage. These 30 isolates were further subjected to primary antifungal screening against C. albicans (ATCC 10221) using the agar well diffusion method to identify potential bioactive strains.

Pathogenic fungi

Multi-drug-resistant pathogenic fungi, including C. albicans (ATCC 10221), were kindly provided by the Microbiology Department, Faculty of Science, Ain Shams University. The isolates were stored at −20°C in Sabouraud dextrose broth (Oxoid, USA) or brain heart infusion broth supplemented with 20% (v/v) glycerol for future use [37].

Screening tests for antimicrobial activity

Primary screening of the laboratory-isolated MDR C. albicans against the isolated Actinomycetes was performed using the overlay method and agar well diffusion assay [36, 38]. The overlay approach involved spot-inoculating Sabouraud's agar plates with a 7-day-old Actinomycetes culture and incubating them for 7 days at 28 ± 2 °C. After incubation, 3 ml of soft agar was overlaid on the plates, which were then inoculated with a freshly prepared suspension of the test fungus (approximately 10⁸ CFU/ml) and incubated for 24 h at 37 °C. Growth inhibition zones around the Actinomycetes colonies indicated antifungal activity. For the agar well diffusion method, Sabouraud dextrose agar (SDA) (Sigma-Aldrich) plates were spread with 0.1 ml of the test fungal strain. Wells were made using sterilised 1000 µl microtips, and 100 µl of crude extract from each isolated Actinomycete was added. The plates were then incubated for 24 h at 37 °C. The growth inhibition zone was measured in millimetres.

Bioassay of actinobacterial culture supernatants

The yeast was cultivated on Sabouraud's agar for 24 h at 35°C. The inoculum was prepared in a sterile saline solution (0.85%) and adjusted to match the turbidity of a 0.5 McFarland standard at 530 nm (Spectrophotometer SP-830 plus, Nangang, Taipei, Taiwan) to achieve 10⁶ colony-forming units (CFU)/mL. It was then diluted with Sabouraud's broth at a 1:1000 dilution. The diffusion method is a common technique for assessing the antifungal activity of plant or microbial extracts. The agar plate surface was inoculated by spreading a volume of the Streptomyces sp. inoculum, similar to the disk-diffusion method. A 6–8 mm diameter well was aseptically punched with a sterile cork borer or tip, and a volume (20–100 µL) of the antifungal extract solution at the required concentration was added to the well; a fluconazole disc (150 µg) served as the control. The agar plates were then incubated under appropriate conditions depending on the test microbe. The antimicrobial ingredient diffuses throughout the agar media, inhibiting the growth of the tested Candida ATCC 10221 [39–41].

Macro and microscopic features

The cultural properties of pure actinomycetes colonies on SCA media, including elevation, surface, and aerial and substrate mycelium colour and pigment synthesis, were recorded according to Bergey's Manual of Determinative Bacteriology [42]. A cover slip culture method was used to examine the microscopic features of the isolates. Observations included the morphology of the substrate and aerial mycelia, and the formation of coil and chain-shaped spores, rectiflexibiles, unbranched and branched chains, retinaculum-apertum, and spiral spores [27].

Genomic DNA extraction from Streptomyces

Genomic DNA was extracted using a slightly modified version of the methodology described by [43] et al. (2004). Strain SCA3-4 was grown in YE liquid medium (pH 7.4) at 28 °C for three days on a rotary shaker (150 rpm). A 1.0 ml culture aliquot was centrifuged at 9,000 rpm for 30 s. The pellets were resuspended in 480 µl of ethylenediaminetetraacetic acid (EDTA). Lysozyme (120 µL) was added, and the mixture was incubated for 50 min at 37°C. After centrifugation for 2 min at 12,000 rpm, the pellets were resuspended in 600 µL of lysis solution and incubated at 80 °C for 5 min. After cooling, 1.8 µL of RNase A was added to the sample, which was incubated for 15 min at 37°C. Then, 200 µL of phenol/chloroform (1:1) was added, and the mixture was left on ice for five minutes. Following a 5-min centrifugation at 13,000 rpm and 4 °C, the supernatant (about 600 µL) was transferred to a sterile 1.5 ml microcentrifuge tube and mixed with an equal volume of isopropanol. After the supernatant was removed, the pellet was washed with 0.5 ml of 70% ethanol and centrifuged for one minute at 12,000 rpm. The pellets were air-dried and then resuspended in 100 µl of TE buffer (a pH 7.7 Tris–HCl buffer containing 1.0 mM EDTA). Genomic DNA was assessed using 1% agarose gel electrophoresis and stored at −20°C. Evaluating the purity of genomic DNA by using short, 1 × TBE buffer contain 0.5 µg/mL of ethidium bromide was used to prepare 1% (w/v) agarose gel. DNA samples were loaded into the wells with a DNA ladder after being combined with loading dye. DNA integrity was verified by visualising the bands under UV transillumination after electrophoresis was performed at 80–100 V for roughly 30–40 min. After that, the extracted DNA was kept at −20°C until it was needed [44].

Extract preparation

After 1, 2, 3, and 7 days of cultivation, the broths were centrifuged at 9,000 rpm for 10 min to separate the mycelium from the supernatant. The mycelium was extracted twice with 10 mL of MeOH/Acetone (1:1) for 10 min each, separated by a 10-min centrifugation stage at 9,000 rpm. The mycelial extracts of S. zaomyceticus (PQ395286) were combined. The supernatant was extracted twice with 20 mL of EtOAc at 190 rpm for 30 min. Thus, one extract from the mycelium and one from the supernatant (culture broth) were obtained from each sample. Sterile culture media were used as controls (blanks for each culture media). After extraction, samples were vacuum-dried in an Eppendorf® Concentrator Plus system, and the resultant material was diluted to 1.0 mg/mL using HPLC-grade MeOH [45].

Analysis, purification, and identification of active ingredients

The extract was first tested using silica gel thin-layer chromatography (TLC) (Sigma-Aldrich, Germany). TLC was performed using a variety of solvent systems with different polarities, such as chloroform:ethyl acetate (3:9) and toluene:ethyl acetate:formic acid (7:3:0.2), for Streptomyces metabolites to select the solvent system that provided the highest resolution [46]. The Streptomyces metabolite extracts were applied via capillary tubes to pre-coated TLC plates, which were then developed in a TLC chamber with the appropriate mobile phase. A 10 µL aliquot of the extract solutions was applied to the TLC plate. After air-drying, the developed TLC plates were examined in a UV TLC viewer at both 254 and 366 nm. The rate of flow (Rf) values for the observed spots were computed [46]. Each band was scraped off separately, extracted using methanol, and placed in a different vial. The antifungal properties of each band were then examined using an agar-well diffusion test. After several purifications, 6 mg of the antifungal active ingredient was dried in a freeze-drying concentrator (SPD121P, Thermo Fisher, USA).

Nuclear magnetic resonance (NMR) spectroscopy

Chloroform was the solvent used to acquire the spectra. The main purpose of nuclear magnetic resonance (NMR) spectroscopy is to accurately determine the structural and quantitative characteristics of synthesized metabolites. NMR technique, including 1D-¹H, 1D-¹H ¹H J-resolved, 1H-1H NOESY, 1H-1H COSY, 1H-1H TOCSY, 1H used to achieve this. The data were compared to those of similar compounds produced by Streptomyces [47].

LC–MS/MS analyses

Liquid chromatography–mass spectrometry analyses were performed using an HPLC (Shimadzu®) connected to a mass spectrometer ESI-IT (Amazon SL, Bruker Daltonics) equipped with an ion-trap analyser and an electrospray ionisation source. The chromatographic separation used a C18 column (Phenomenex® Luna, 5 µm, 4.6 × 250 mm) with solvents A (0.1% formic acid in H₂O) and B (0.1% formic acid in MeOH), starting at 5% B and increasing to 100% B over 30 min, then holding at 100% B for 5 min. The method used a column temperature of 40 °C, a flow rate of 1.0 mL min⁻¹, and an injection volume of 15 µL. The mass spectrometer operated in positive mode with a capillary voltage of 3500 V and a mass range of 100 to 1500 atomic mass units (amu). The untargeted mode [48] fragmentation at MS2 level using a ramp of collision energy from 50 up to 75 eV) was used with an end plate offset of 500 V, nebuliser pressure at 60 psi, dry gas at 10 L min⁻¹, and dry temperature at 320°C.

Gas chromatography-mass spectrometry (GC–MS) examination of crude extracts

Crude extracts were analysed using GC–MS in accordance with the methods previously detailed by Kitson et al. [49] For GC–MS analysis, crude extracts were dissolved in spectroscopy-grade methanol and filtered through a 0.2-µm filter. The analysis used a Thermo Fisher Scientific Trace GC equipped with a DSQ II MS and a DB-5MS capillary column (30.0 m × 0.25 mm × 0.25 µm). Helium was used as the carrier gas at a flow rate of 1 mL per minute, with the injector temperature at 250°C. The column temperature was programmed to start at 60 °C and increase by 5 °C per minute to 100°C. After 5 min of isothermal holding, it was ramped up to 250 °C at 10°C/min, held for 35 min, elevated to 280 °C at 8°C/min, and then held for 25 min. The mass spectrometer operated in electron ionisation mode at 70 eV, continuously scanning from 50 to 650 atomic mass units. The mass spectra were compared to the National Institute of Standards and Technology (NIST, United States) library to identify the constituents.

Minimum inhibitory concentration (MIC) of organic extract fractions

The MIC is the lowest concentration of an antimicrobial agent that completely inhibits visible growth of the organism in the microdilution wells. A pure culture of a specific yeast, grown overnight and diluted to a concentration of 1 × 10⁵ to 1 × 10⁶ CFU/mL in Tryptic soy broth, was used. polystyrene microtitre plates 96-well (Costar, Corning Inc., USA) were used for the broth microdilution technique. Vacuum-dried ethyl acetate or diethyl ether metabolites of Streptomyces were dissolved in tween 20 (1 gm/1 ml) and diluted (concentration range: 0.125 μg/ml to 512 μg/ml) in either RPMI 1640 medium (Thermo ScientificTM Oxoid, Ltd., Basingstoke, Hampshire, United Kingdom) for testing fungal isolates.

The 96-well microtitre plate was then incubated for 24 to 48 h at 37 °C after each well was injected with 100 μL of fungal suspension (5 × 10⁶ CFU/ml). There were both positive and negative controls. The minimum inhibitory concentration (MIC) is the lowest concentration that prevents the development of bacteria or fungi [50].

In vitro application of antimicrobial activity by scanning electron microscopy

Scanning electron microscopy (SEM) studies were carried out in compliance with the defined protocol to determine the morphological changes of C. albicans treated with identified metabolite [51]. After the C. albicans strain reached the logarithmic stage in new MH liquid medium, the discovered metabolite was added at a final drug dosage of 2 MIC, and the combination was agitated for 8 h at 28 °C. The precipitation from the lower layer was collected and preserved at 4 ◦C with 2.5% glutaraldehyde for SEM (SU8100, Hitachi, Ltd., Japan) evaluation following centrifugation at 4 ◦C for 10 min at 5000 rpm and three sterile PBS washes [52].

Fungal isolate

A pathogenic isolate of C. albicans was obtained from naturally infected C. gariepinus in the Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Egypt, and confirmed to be harmful to C. gariepinus. This isolate was identified using standard biochemical tests and the VITEK® 2 system (BioM’erieux, Marcy l’Etoile, France). C. albicans was cultivated on Sabouraud dextrose medium (Hi-media, India) and incubated for two days at 24 °C. Selected colonies were suspended in sterile PBS (phosphate-buffered saline).

In VIVO application

Fish and culture conditions

In this experiment, apparently healthy African catfish (N = 120, average body weight 183.40 ± 0.40 g) were obtained from the Al-Abbassa fish hatchery. The study was conducted under ethical approval number (ZU-IACUC/1/F/234/2024); the study was applied in Aquatic Animal Medicine Department at Zagazig University, Egypt. Fish were randomly placed in 100-L glass aquariums with dechlorinated tap water. Fish were acclimatized for two weeks with continuous aeration provided by electric air compressors with air stones. The water in the aquaria was partially cleaned and replaced each day. The laboratory maintained a controlled photoperiod (12 h of light and 12 h of dark) with consistent temperature (28.3 ± 1.1 °C), dissolved oxygen (6.18 ± 0.4 mg/L), pH (6.9 ± 0.1), and total ammonia (0.035 ± 0.01 mg/L) levels [53, 54].

According to [55], (2012), the basal diet (Table 1) was formulated in Fish Research Centre, Zagazig University, to provide fish with the required nutrients. The ingredients were mechanically mixed using a beef mincer with a 1.5 mm diameter before being pelletized. These pellets were air-dried with frequent rotation to ensure uniform drying, and then stored at 4 °C until use. Fish were fed at 3% of their body weight twice daily (8:00 AM and 2:00 PM), and feed amounts were adjusted biweekly based on fish weight.

Table 1.

The components of basal diet and proximate composition (% on a dry weight basis)

Ingredients	%
Fish meal 70.7% CP	23
Fish oil	2
Yellow corn	10
Soybean meal 49% CP	33
Wheat flour	10
Corn gluten 67% CP	4
Wheat bran	15
Premixa	3
Calculated chemical analysis (%)
Crude lipids	6.17
Crude fiber	4.69
Crude protein	40.12
NFE b	41.09
Ash	7.92
Methionine	0.81
Lysine	2.45
GE MJ/kg c	19.85

^aPremix: each 1 kg of premix contain: Vit A 550000 IU, Vit E 11000 mg, Vit D 110000 IU, Vit C 50 g, Vit K 484 mg, Vit B1 440 mg, Vit B2 660 mg, Vit B3 13,200 mg, Vit B5 1100 mg, Vit B6 1045 mg, Vit B9 55 mg, Biotin 6.6 mg, iron 6.6 g, Choline 110,000 mg, copper 330 mg, Zn 6.6 g, Se 44 mg, Mn 1320 mg, Iodine 110 mg. NEF, Nitrogen free extract; GE, Gross energy

^bNEF = 100 − (crude protein + crude lipids + ash + crude fiber).

^cGross energy (GE) was calculated as 23.6 kJ/g protein, 39.5 kJ/g lipid, and 17.0 kJ/g NFE (NRC, 2011)

Challenge test

The lethal dose (LD50) of C. albicans was estimated following the technique outlined by Zayed et al. [38]. Five groups (20 fish per group; 10 fish per replication) and one control were created from 110 fish. Ten fish were placed in each 100 L aquarium with adequate aeration. Prior to the experimental infection, the fish were fasted for twenty-four hours. The five groups were injected intraperitoneally with 0.2 mL of different C. albicans dilutions (10⁵–10⁹ CFU/mL). The control group was injected with 0.2 mL of sterile saline. Throughout the 96-h trial, no food was given to the fish and 25% of the aquarium water was exchanged daily. The fish mortality percentage was recorded at 24, 48, 72, and 96 h post-challenge, and the 96 h LD50 was determined using a Probit model analysis (SPSS 21). The LD50 was estimated to be 1.5 × 10⁸ CFU/mL for C. albicans. A dose of 4.6 × 10⁷ CFU/mL was selected for the experimental trial. The injected fish were monitored daily for fifteen days. Clinical signs and mortalities were recorded. The selected inoculum (4.6 × 10⁷ CFU/mL; 0.2 mL/fish) represented approximately 30% of the estimated LD50 (1.5 × 10⁸ CFU/mL), which provided a consistent infection and measurable clinical signs without excessive mortality, allowing for a reliable evaluation of lucidin's efficacy.

Determination of the therapeutic concentration of lucidin

Fish (N = 90 fish; 10 fish/group) were exposed to nine different concentrations of lucidin (0, 25, 50, 75, 100, 125, 150, 175, and 200 μg/L) for 10 days to determine the trial concentration according to [56] et al. (2022). Each group was kept in a 100 L well-aerated aquarium. The fish were fed a commercial diet (30% protein; at a rate of 2% of total biomass) twice daily (8:00 a.m. and 3:00 p.m.). A 25% water exchange was performed daily by siphoning to remove excretory wastes. Daily clinical observations and the mortality rate were recorded. Based on preliminary range-finding exposure (25–200 μg/L), concentrations between 25 and 150 μg/L were safe. Therefore, 100 μg/L was chosen as an effective and non-toxic therapeutic level. During the therapeutic trial, aquarium water was completely replaced every two days to maintain water quality. After each renewal, lucidin was reintroduced at the same concentration to maintain a consistent therapeutic level.

Experimental design

Fish were equally distributed into four experimental groups, each with three replicates (aquaria). Ten fish were allocated to each replicate (n = 10 per aquarium; total n = 30 per group). Each aquarium (100 L capacity) was continuously aerated, and 25% of the water was exchanged daily to maintain water quality. The experimental period lasted for 15 days.

• Group 1 (G1, Negative Control): Fish neither received lucidin nor were infected with C. albicans.

• Group 2 (G2, Lucidin only): Fish were exposed to 100 μg/L lucidin in the aquarium water.

• Group 3 (G3, Positive Control): Fish were intraperitoneally (I/P) injected with 0.2 mL of C. albicans suspension (4.6 × 10⁷ CFU/mL).

• Group 4 (G4, C. albicans + Lucidin): Fish were injected I/P with C. albicans (0.2 mL of 4.6 × 10⁷ CFU/mL) and simultaneously treated with 100 μg/L lucidin in the water.

At the end of the experimental period, five fish from each replicate (n = 15 per group) were randomly sampled for biochemical, immunological, and histopathological analyses. Data were expressed as mean ± SD, and statistical significance was determined at p < 0.05 using one-way ANOVA followed by Duncan’s multiple range test.

Sampling procedures

Five fish from each aquarium were randomly selected and anesthetized using 95 mg/L clove oil (Oleum, Egypt) within 3 min [57]. Blood samples were taken from the caudal blood vessels using sterile syringes without anticoagulant and centrifuged at 1075 × g for 20 min for serum separation. The serum samples were stored at −20°C in a deep freezer until use. Additionally, tissue samples (n = 3) from the liver, kidney, and spleen were obtained for histological analysis.

Hematological and biochemical analysis

Hematological analysis was performed according to Ser et al. [58]. Serum indicators for kidney damage, urea and creatinine, were evaluated according to the methods of Coulombe and Favreau [59] and Larsen [60], respectively.

Serum antioxidant and nonspecific immune parameters

Antioxidative biomarkers, including total antioxidant capacity (TAC) and catalase (CAT), were measured according to Aebi [61] and Melekh et al. [62]. The activity of superoxide dismutase (SOD) was estimated using a method described by Nishikimi et al. [63] and Wink et al. [64]. Reduced glutathione (GSH) concentration was detected using the method of Beutler et al. [65]. Lysozyme (LYZ) activity was measured by turbidimetric assay [66]. Nitric oxide (NO) levels were measured according to the method described by Wink et al. [67].

Histopathological evaluation

At the end of the experimental trial (15 days), samples of the liver, kidney, and spleen were fixed in 10% buffered neutral formalin solution for 48 h, dehydrated through a graded ethanol series (70, 80, 95, 95 and 100%), cleared in xylene, and embedded in paraffin. Sections of 5-micron thickness were cut using a microtome (Leica RM 2155, England). The sections were stained with hematoxylin and eosin and routinely examined under a microscope [67]. Images for each section were captured using a Leica® microscope and an AmScope® digital microscope [68].

Statistical analysis

Data were expressed as mean ± standard deviation (SD). The data were statistically analyzed using one-way analysis of variance (ANOVA) with SPSS software version 14 (SPSS Inc., Chicago, IL, USA). Duncan’s multiple range test was applied to detect statistical differences between groups at a significance level of p < 0.05.

Results

Isolation of Streptomyces from collected samples

The marine samples, collected from the mangrove ecosystem, yielded eight actinomycetes isolates. Every isolate displayed colonial morphology typical of Streptomyces. The antifungal activity of all isolates was examined. One isolate, designated strain RAS24, was selected for further characterization and bioactivity assessment due to its pronounced activity.

Antifungal action of actinomycetes on Candida albicans (ATCC 10221)

The dual culture plate assay revealed significant variation in the antifungal activity of the twenty distinct actinomycetes isolates against C. albicans. Only one isolate demonstrated consistent and strong antifungal activity. The supernatant of S. RAS24 was applied at varying doses to assess inhibition. The growth of the yeast strain decreased as the concentration increased, suggesting a concentration-dependent effect. The minimal inhibitory concentration that suppresses the growth of Candida ATCC 10221. by 50% (MIC50) and 70–90% (MIC70₋90) was determined. Metabolites from S. RAS24 inhibited the growth of C. albicans, resulting in reduced MIC50 and MIC70₋90 values (Table 2). Compared to fluconazole, the MIC50 and MIC 50–90 values for the Streptomyces metabolites were lower (Table 2).

Table 2.

Fluconazole MIC50 and MIC70-90 values and the S. RAS24Q supernatant in comparison to Candida strain

Inhibitor	Disc zone dimater (mm) C. albicans ATCC 10221
S. RAS24Q	31 ± 0.2
Control	28 ± 0.3

Biochemical and morphological description of strain ras24

Strain RAS24 is Gram-positive, aerobic, and filamentous, with rectiflexibilis spore chains and a smooth spore surface, as shown in Fig. 1. The strain developed a brown diffusible pigment on ISP2 medium, although it grew well on all tested solid media with varying colony colors. It produced acetoin, hydrogen sulfide, and the enzymes arginine dihydrolase and urease, and hydrolyzed starch. Of all the carbon sources investigated, the strain utilized only glycerol, glucose, and starch. It grew at temperatures between 20 and 40 °C and tolerated up to 4% sodium chloride, as shown in Table 3 and Fig. 1.

Fig. 1 .

Scanning electron micrograph of RAS24 strain showing rectiflixibilis spore chain and smooth spore surface

Table 3.

The RAS24 strain's morphological, biochemical, and physiological traits

Characteristic	Result
Gram	+
Spore chain	rectiflixibilis
Spore surface	Smooth
Diffusible pigment	brown
Cellulase	-
Arabinose	+
Lysine	+
Glucose	+
Maltose	+
Lactose	−
Starch	+

Following amplification from genomic DNA, the isolates were identified at the genus level using 16S rDNA gene sequences. Analysis of all sequences using the RDP database clearly indicated that the isolate belongs to S. zaomyceticus PQ395286. The phylogenetic relationships of the isolate to type strains were determined using the Maximum Likelihood method in MEGA 7 software, based on a nucleotide BLAST search, as shown in the phylogenetic tree in Fig. 2.

Fig. 2.

Maximum Likelihood analysis of molecular phylogenetics. The Maximum Likelihood approach, which is based on the Tamura-Nei model, was used to estimate the evolutionary history [43]. Branch lengths are expressed in terms of the number of substitutions per site, and the tree is depicted to scale. In MEGA7, evolutionary analyses were performed [43]

Further elucidation of the anti-fungal compounds produced by S. zaomyceticus PQ395286 by NMR

When the NMR analysis was conducted in deuterated methanol, we identified the compound as 8-O-methyltetrangomycin (1). However, when measured in deuterated chloroform, the sample fully converted to 8-O-methyltetrangulol (2), as shown in Fig. 3. Evidently, residual hydrochloric acid in the deuterated chloroform catalysed the conversion of 1 to 2. Both compounds displayed typical signals for a methoxyanthraquinone system. The distinct oxidation state of the six-membered ring D was revealed by the two methylene carbons (C-2 and C-4) in 8-O-methyltetrangomycin, whereas two olefinic methine signals were found for C-2 and C-4 in 8-O-methyltetrangulol. Signal assignment was confirmed by COSY, HSQC, and HMBC experiments. The structure elucidation was validated by comparing the spectral data of 1 and 2 with values reported in the literature, as shown in Fig. 3.

Fig. 3.

S. zaomyceticus PQ395286 produces the Lucidin chemical compound by NMR.S

LC–MS profiles of crude extracts from isolate RAS24

We used high-resolution LC–MS to examine extracts from S. zaomyceticus PQ395286 (strain RAS24), which showed strong antifungal activity that varied depending on the fermentation mode (solid or liquid media). Both targeted and untargeted analyses were performed. Initially, potential sum formulae were predicted and the mass spectra of all major compounds were interpreted. The anticipated chemical formulas and accurate masses were then compared against the Dictionary of Natural Products (DNP). Subsequently, extracted ion chromatograms were created to search for metabolites predicted from bioinformatic analysis, specifically 1,3-diethoxy-1,1,3,3-tetramethyl lucidin, matching ions [M + H]⁺, [M + 2H]²⁺, and [M-H]⁻.

GC-mass profiles of the semi-purified metabolite from isolate RAS24

The TLC-purified compound was analysed using GC–MS. Compounds were identified based on their retention time, molecular weight, and mass spectral fragmentation. Lucidin, a recently identified antifungal metabolite isolated from S. zaomyceticus, was confirmed as the active compound. As seen in Fig. 4, the abundance of the material in the active band corresponds to the peak in this region.

Fig. 4.

zaomyceticus PQ395286 produces Lucidin chemical compound by GC—mass

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

This study examined the fungistatic and fungicidal efficacy of synthesized lucidin against C. albicans. The MIC and MBC values demonstrated that lucidin can inhibit 31 mm and 35 mm.respectively fungal growth at low concentrations The significant inhibitory effect of lucidin on microbial cells is shown in Table 2 and Fig. 5.

Fig. 5.

Photograph of the antifungal susceptibility testing of Lucidin (50 μg/ml), (70 μg/ml), and (100 μg/ml) demonstrates the zone of inhibition on Muellar Hinton agar

Antifungal activity screening of lucidin

Lucidin demonstrated antifungal action against C. albicans with an MIC value of 0.25 μg/ml. Figure (6) illustrates the morphological changes in C. albicans following lucidin treatment. The untreated control group appeared as non-motile, oval, spherical cells that tended to form chains (Fig. 6, A). Treatment with lucidin significantly reduced the number of fungal cells and caused severe surface irregularities and loss of structural integrity (Fig. 6, B).

Fig. 6.

Scanning electron microscopy (SEM) micrograph of C. albicans culture after Lucidin. The adverse control group was (A). B was the group that had 2MIC treatment

Hepatorenal function indicators

After 15 days, fish infected with C. albicans (G3) showed significantly higher mean serum concentrations of ALT, AST, urea, and creatinine (p < 0.05) compared to the control (Table 4). In G4, where infected fish were treated with lucidin, a significant (p < 0.05) decrease in these liver and kidney damage markers were observed, suggesting a restorative effect.

Table 4.

Effect of C. albicans and\or lucidin on the hepatorenal function of African catfish after 15 days

Parameters	G1	G2	G3	G4
ALT(U/L)	4.19 ± 0.17b	3.03 ± 0.18b	12.59 ± 0.42a	7.91 ± 0.46c
AST(U/L)	32.44 ± 0.39d	40.22 ± 0.55c	163.50 ± 1.61a	102.86 ± 2.84c
Creatinine (mg/dl)	0.081 ± 0.004b	0.088 ± 0.002b	0.640 ± 0.030a	0.140 ± 0.020b
Urea (mg/dl)	3.44 ± 0.12b	3.98 ± 0.12bc	8.79 ± 0.27a	5.09 ± 0.23b

^{a, b, c, d} Means with different superscripts differed significantly (P < 0.05) (n = 30)

G1: (control negative group): Fish-neither received C. albicans nor treatment, G2 Control positive group treated by lucidin only in water, G3 Control positive group infected with C. albicans without treatment, G4 Infected with C. albicans and treated by lucidin, ALT Alanine aminotransferase, AST Aspartate aminotransferase

Antioxidant and immune parameters

After 15 days, a significant decline (p < 0.05) in serum antioxidant and immune parameters was observed in G3 (Table 5). The addition of lucidin to the water in G4 significantly (p < 0.05) increased antioxidant activity, lysozyme (LYZ) activity, and nitric oxide (NO) levels compared to G3. Fish in G2 (lucidin only) also showed a significant (p < 0.05) enhancement in these parameters compared to the control (Table 5).

Table 5.

Effect of C. albicans and/or lucidin on the antioxidant and immune parameters of African catfish after 15 days

Parameters	G1	G2	G3	G4
TAC (μmol/ml)	2.41 ± 0.29b	3.18 ± 0.46a	0.92 ± 0.02c	2.06 ± 0.15b
CAT(U/ml)	3.05 ± 0..11a	2.67 ± 0..19a	0.52 ± 0.05c	1.77 ± 0.14b
SOD (U/ml)	29.31 ± 0.58b	33.51 ± 1.40a	15.41 ± 1.61d	22.48 ± 1.30c
GSH (μmol/ml)	33.17 ± 1.16b	38.45 ± 1.97a	14.17 ± 0.87c	32.34 ± 1.29b
LYZ (μmol/ml)	2.99 ± 0.10b	3.68 ± 0.37a	0.58 ± 0.05d	2.07 ± 0.22c
NO (μmol/ml)	19.32 ± 0.39b	23.87 ± 1.87a	7.37 ± 0.80c	18.21 ± 1.4b

^{a, b, c, d} Means with different superscripts differed significantly (P < 0.05) (n = 30). G1: (control negative group): Fish-neither received C. albicans nor treatment

G2 Control positive group treated by lucidin only in water, G3 Control positive group infected with C. albicans without treatment, G4 Infected with C. albicans and treated by lucidin, TAC Total antioxidant capacity, SOD Superoxide dismutase, CAT Catalase activity, GSH Reduced glutathione, LYZ Lysozyme activity, NO Nitric oxide

Clinical signs and postmortem lesions

Fish infected with C. albicans (G3) exhibited clinical signs including lethargy, erratic swimming, loss of body reflexes, and anorexia. Upon dissection, affected fish showed fin rot, erythema, skin depigmentation, and congestion, inflammation, and hemorrhages in internal organs (Fig. 7). The severity of these clinical signs and postmortem lesions was markedly reduced in G4 after lucidin treatment. Fish in all lucidin-supplemented groups had lower cumulative mortality rates compared to the infected control group.

Fig. 7.

Photograph of C. gariepinus of G3 (infected with C. albicans without treatment) showed: A and C. Loss of skin pigmentation and emaciation (black arrow) with slight erythema (white arrow). B Congestion of liver, kidney, spleen, and gall bladder (white arrow). B Congestion of liver and spleen (red arrow) with fibrous tissue inflammation of internal organs (black arrow). D Photograph of C. gariepinus of G4 (infected with C. albicans and treated with lucidin) showed relieve of the congestion and inflammation of internal organs

Histopathological findings

The histopathological findings are shown in Fig. (8, 9 and 10). The liver (Fig. 8A) of the control group (G1) showed normal hepatic architecture. G3 (infected control) revealed degenerative changes and perivascular coagulative necrosis of hepatocytes (Fig. 8B). G4 (infected and treated) showed amelioration of hepatic structure, with only moderate vacuolation in some hepatocytes (Fig. 8C). The kidney of the control group exhibited normal histology of glomeruli and renal tubules (Fig. 9D). G3 showed necrosis in some renal tubules and glomerular atrophy (Fig. 8E). G4 exhibited minor shrinkage of some glomerular tufts but otherwise apparently normal renal tubular epithelium (Fig. 9F). The spleen of the control group exhibited normal white and red pulp structures (Fig. 10G). G3 showed marked depletion of lymphoid elements in the white pulp (Fig. 10H). G4 showed re-formed lymphoid elements in the majority of the spleen tissue (Fig. 10I).

Fig. 8.

Photomicrographs of H&E-stained sections of the liver (Figure A-C) from African catfish showing: A Normal histological architectures of hepatic cells, sinusoids and central veins were demonstrated in "control group" of liver. B G3" control infected group" revealed degenerative changes in most hepatic tissue beside presence of perivascular co-agulative necrosis of hepatocytes that represented by homogenous eosinophilic cytoplasm with pyknotic nuclei. C However, ameliorations in histological structure of hepatic tissue beside vacuolation in moderate number of hepatocytes were seen in G4 "treated group by lucidin" (Scale bar 20 μm)

Fig. 9.

Photomicrographs of H&E-stained sections of the liver (Figure D-F) from African catfish showing: D Control group of kidney exhibited normal histology of glomeruli, renal tubules, and hematopoietic series with number of melanomacrophages. E Fish in G3, showed necrotic some renal tubules, and atrophied glomerular tufts. F However, exhibited shrinkage of some glomerular tufts and apparently normal renal tubular epithelium were seen in G4. (Scale bar 20 μm)

Fig. 10.

Photomicrographs of H&E-stained sections of the liver (Figure G-I) from African catfish showing: G Control group of spleen exhibited normal histological structures of white pulp with melanomacrophages around ellipsoids arterioles beside normal red pulp. H G3 showed markedly depletion of lymphoid elements of white pulp around the ellipsoids arterioles. I However, G4 showed re-formed lymphoid elements in the majority of spleen tissue. (Scale bar 20 μm)

Discussion

This study provides the first evidence that lucidin, a secondary metabolite produced by S. zaomyceticus PQ395286, exhibits potent antifungal activity against C. albicans infection in C. gariepinus. The rising prevalence of fungal pathogens in aquaculture underscores the need for effective and environmentally safe alternatives to conventional antifungal agents [69]. Our findings highlight lucidin as a promising natural compound with both antifungal and immunoprotective properties.

The results confirm that S. zaomyceticus produces bioactive secondary metabolites capable of inhibiting C. albicans, which is consistent with the well-known antimicrobial potential of actinobacteria [70]. The structural identification of lucidin through GC–MS and NMR [71, 72]. Although the experimental dose was below the in vitro MIC, lucidin remained effective IN VIVO due to continuous exposure, potential tissue accumulation, and the host’s immune contribution as explained by Toutain et al. [69]; Abdel-Rhman et al. [73]; Pereira et al. [74]. Its known immunostimulatory and antioxidant properties likely enhanced antifungal efficacy, as supported by the observed morphological damage to C. albicans cells (Fig. 6).

Scanning electron microscopy revealed pronounced membrane disruption and deformation of C. albicans cells following exposure to lucidin, suggesting that its antifungal effect is mediated through membrane destabilization. This mechanism aligns with previous reports describing anthraquinone derivatives-such as emodin and chrysophanol-as potent agents that impair fungal cell wall integrity and interfere with ergosterol synthesis [71, 75]. Lucidin treatment markedly reduced clinical signs, postmortem lesions, and mortality in infected fish, confirming its strong in vivo antifungal efficacy [75].

Beyond its direct antifungal activity, lucidin significantly enhanced immune responses (lysozyme and nitric oxide levels) and antioxidant defenses (SOD, CAT, and GPx activities) in infected fish. These findings indicate that lucidin not only suppresses fungal proliferation but also strengthens host immunity and mitigates oxidative stress associated with infection. Comparable immunostimulatory effects have been reported for other natural compounds, including curcumin, allicin, and quercetin derivatives [66], which support host resilience against opportunistic pathogens.

The enhanced hepatorenal function and histopathological observations further demonstrated lucidin’s protective influence on hepatic and renal tissues, with treated fish showing markedly reduced cellular degeneration and vascular congestion compared to infected controls [28]. The dual antifungal and host-protective actions observed in this study position lucidin as a valuable candidate for sustainable fish health management.

Nevertheless, the present study has certain limitations. It was conducted on a single fish species under controlled laboratory conditions and over a relatively short experimental period. Comprehensive toxicity studies, environmental risk assessments, and field-scale validations are required to confirm the practical applicability of lucidin in aquaculture. Future molecular and transcriptomic investigations may also clarify the pathways underlying its immunomodulatory and antioxidant effects [76].

In conclusion, lucidin represents a natural, eco-friendly compound with demonstrated antifungal efficacy and host-protective properties against C. albicans infection in C. gariepinus. Its dual-action potential makes it a promising alternative to synthetic antifungal agents. Further studies integrating lucidin into feed or water treatment systems may pave the way for its use as a sustainable therapeutic and prophylactic agent in aquaculture.

Conclusion

This study successfully identified and evaluated a new S. zaomyceticus metabolite, lucidin, which exhibits antifungal properties against C. albicans infection in C. gariepinus. Treatment with lucidin at 100 μg/L significantly improved fish health indicators, increased survival rates, and resulted in lower cumulative mortality in all lucidin-supplemented groups compared to the infected controls. Lucidin exposure effectively restored hepatorenal function, enhanced innate immune responses (increased lysozyme activity and nitric oxide production), and boosted antioxidant enzyme levels (SOD, CAT, GPx). Histological examination confirmed remarkable tissue recovery in the liver, kidney, and spleen of infected fish. These findings confirm that lucidin possesses potent antifungal and immunoprotective effects and could serve as a promising natural alternative to chemical antifungals in aquaculture. Its efficacy, water solubility, and biological compatibility highlight its potential for integration into feed formulations or bath treatments to control fungal infections in fish farms. Future studies should focus on field-scale validation, long-term safety and residue analysis, comparative trials with commercial antifungal drugs, and the optimization of lucidin delivery systems for industrial application. Overall, this research provides the first in vivo evidence of lucidin’s antifungal potential in fish and establishes a foundation for its development as a sustainable, eco-friendly antifungal solution for aquaculture.

Authors’ contributions

Rewan Abdelaziz: Writing – review & editing, Supervision, Methodology, Investigation, Data curation, Conceptualization, Validation, and Investigation. Saeedah M. Almutairi, Mohamed T. Yassin: Writing – review & editing, Supervision, Funding acquisition. Mohamed R. AbdelGawwad, Rania Ali:Writing – review & editing, Validation, Funding acquisition. Rania Ali, Mohamed M. Ammar, Sheeren Abdelaziz:Writing – review & editing, Visualization.

Funding

Open access funding provided by The Science, Technology & Innovation. Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank. (EKB). Open access funding provided by the Science, Technology & Innovation. Funding Authority (STDF), Egypt in cooperation with the Egyptian Knowledge. Bank (EKB).

Data availability

Availability of data and materials supporting the findings of this study are available upon reasonable request from the corresponding author.

Declarations

Ethics approval and consent to participate

The experiment was carried out in compliance with the rules established by the local committee for the care of experimental animals. The experimental protocol was approved by the Ethics of the Institutional Animal Care and Use Committee of Zagazig University, Egypt. The experimental procedures were conducted by following the NIH general guidelines for the Care and Use of Laboratory Animals in scientific investigations (ZU-IACUC/1/F/234/2024). The fish were transported in plastic bags to the Wet Lab at Zagazig University's Department of Aquatic Animal Medicine in Egypt from Al-Abbassa Fish Hatchery in Sharkia Governorate, Egyptian permission from owners.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.