Evaluating the antibacterial properties of deep-sea sponges Dactylospongia elegants, Stelletta fibrosa, and Haliclona manglaris from the Jordanian Gulf of Aqaba

Sponge collection

Deep-sea sponge specimens were collected in July 2022 from the Jordanian Gulf of Aqaba using manned submersibles equipped with robotic arms during the OceanXplorer expedition under permit number (9,795), issued by Aqaba special economic zone authority. Ecological parameters were recorded at each collection site. The samples included Sponge 1, collected at a depth of 260 m (temperature: 21.17 °C, salinity: 40.7 PSU, dissolved oxygen: 5.96 mg/L); Sponge 2, collected at 290 m (temperature: 21.1 °C, salinity: 40.7 PSU, dissolved oxygen: 5.92 mg/L); and Sponge 3, collected at 140 m (temperature: 22.5 °C, salinity: 40.66 PSU, dissolved oxygen: 6.21 mg/L). All samples were preserved in 70% ethanol at −4 °C and placed in the Laboratory for Molecular and Microbial Ecology (LaMME), The University of Jordan, Amman. Figure 1 illustrates the location of the sample collection, with an image of the sample.

Identification of sponge samples

To determine the species of sponge, 25 mg of sponge tissue was divided into tiny fragments. The DNeasy Blood and Tissue Kit from Qiagen (Manchester, UK) was then used to extract the DNA from sponge tissue (Harper et al., 2023). The 28S ribosomal RNA gene served as the molecular marker (Yang et al., 2017), with the primers listed in Table 1. This marker is commonly employed in sponge barcoding for species identification (Timmers et al., 2022).

Gene amplification was performed using the polymerase chain reaction (PCR), veriti™ 96-Well Thermal Cycler (Applied Biosystems, Waltham, MA, USA) with the Enti-link PCR master mix from ELK Biotechnology Co. (Wuhan, China). The PCR was conducted under the following conditions: 3 min of initial denaturation at 94 °C, 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C (depending on the gene) for 30 s, 83 s of extension at 72 °C, and a final 10 min of extension at 72 °C. A 1% agarose gel from Cleaver Scientific (Warwickshire, UK) was used to verify the PCR results, which were then examined using a gel documentation system. After that, the PCR products were shipped to Macrogen, South Korea, for sequencing. Finally, we aligned the acquired sequences with sponge species using Molecular Evolutionary Genetics Analysis (MEGA) software by the Center for Evolutionary Medicine and Informatics, Arizona State University, Tempe, AZ, USA. and the Basic Local Alignment Search Tool (BLAST) database from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). A phylogenetic tree was constructed using the neighbor-joining method and the aligned 28S rRNA sequences of closely related sponges obtained from the GenBank database collections (Cristianawati et al., 2019).

Ethanolic extraction

After collecting the sponges, the specimens were immersed in ethanol/water solution (70% v/v) and kept at −20 °C for storage. Ethanol was used to extract both hydrophilic and hydrophobic components in optimum yield (Bashari et al., 2019). The samples were then blended and heated at 55–60 °C for 2 h while being soaked in ethanol, followed by dark resting overnight. The samples were filtered afterward, and the concentrated rinse was rotary evaporated; the paste extract was lyophilized to yield the powder.

Preparation of extract

To make the stock solution, 25 mg of extract powder was dissolved in one ml of dimethyl sulfoxide (DMSO) from Scharlau (Barcelona, Spain) to reach a concentration of 25 mg/ml. This liquid was stored in a Falcon tube after being vortexed and filtered with a 0.45 µm nylon syringe filter. After that, sterile distilled water was used to dilute the stock solution to different concentrations (5, 10, 15, and 20 mg/mL).

Antimicrobial activity

To cover both Gram-positive and Gram-negative bacteria, six bacterial strains were chosen. Among the Gram-positive bacteria were Bacillus pumilus (isolate), Staphylococcus aureus American Type Culture Collection (ATCC 29213), and Staphylococcus epidermidis (ATCC 51625). Among the Gram-negative bacteria were Escherichia coli (ATCC 25922) and Klebsiella aerogenes (isolate). Methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 1026) is a multidrug-resistant bacterium (MDR) showing resistance to β-lactams, macrolides, fluoroquinolones, and often clindamycin (Chambers & De Leo, 2009). It was subjected to additional testing after the extract showed antibacterial efficacy against Staphylococcus aureus.

The antibacterial activity of the three sponge extracts was assessed using the agar well diffusion method. The Muller-Hinton broth (MHB) from Oxoid (Basingstoke, UK) was first mixed with the bacterial strain, and it was then incubated overnight at 37 °C (Magaldi et al., 2004). Following incubation, the density of the culture was modified to satisfy the McFarland turbidity criteria of 0.5 (Salameh et al., 2024).

Using sterile cotton swabs, the test organisms were cultured on Muller-Hinton agar plates, followed by a 10-minute drying period. The agar plates were then prepared with eight mm-diameter wells made with a sterile cork borer. Following that, various concentrations of the sponge extracts (5, 10, 15, and 20 mg/ml) were added to these wells using micropipettes. To allow for pre-diffusion, the culture plates were set on the bench for an hour before being incubated for 24 h at 37 °C (Ibrahim et al., 2022a; Ibrahim et al., 2022b).

Following an 18-hour incubation period, the diameter of the inhibition zones (measured in millimeters) around the wells was used to determine the antibacterial activity (Geetha & Roy, 2013). For all microorganisms, 10 µg gentamycin (Yassine, Ammar & Shabbar, 2013) was utilized as the positive control, and MRSA-specific 30 µg vancomycin (Hannan et al., 2008) was used. Additionally, 80% DMSO was used as the negative control, which was the highest solvent concentration utilized in the extract dilutions. This ensured that any antimicrobial activity observed was due to the extract itself and not the solvent. To reduce the possibility of experimental errors, each experiment was run three times.

The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC)

The minimal inhibitory concentration (MIC) procedure, done as in a (Balouiri, Sadiki & Ibnsouda, 2016) protocol with some adjustments, involves the preparation of various extract concentrations using sterile MHB. These concentrations were then added to a 96-well plate in a specific order: for Sponge 2 concentrations: 5, 4, 3, 2, 1.0.5, 0.25, 0.125 mg/ml for all bacterial strains, while Sponge 3 concentrations: 20, 19, 18, 17, 16, 15, 14, 13, 12 mg/ml for S. aureus and 15, 14, 13, 12, 11, 10, 9, 8, 7 mg/ml for S. epidermidis. The selection of these concentrations depended on the results obtained from the well diffusion agar method. Negative controls included 80% DMSO and sterile MHB, while positive control comprised a bacterial suspension.

The minimal bactericidal concentration (MBC) was established by transferring and evenly spreading the treated culture broth from the wells with concentrations equal to or greater than the MIC onto agar plates. The MBC was defined as the lowest concentration of the fraction needed to entirely eradicate the test microorganism, indicated by no growth observed on the agar plate following incubation at 37 °C for 24 h (Majali et al., 2019).

Sponge extract chemical composition using LC-MS-MS

Liquid chromatography with tandem mass spectrometry (LC-MS-MS) was performed by Smart Labs Group (Amman, Jordan) using Shimadzu LC (Kyoto, Japan) for the most active sponge extract. It consisted of CBM-20A (control bus module), CTO-30A (column oven), LC-30AD (liquid chromatograph), SIL-30AC (autosampler), and LCMS-8030 (liquid chromatograph mass spectrometer - Triple Quad MS). 50 mg of the sponge extract was extracted with methanol (MeOH). Then, the crude extract was cleaned using a solid phase extraction column, in which the 100% MeOH fraction was retained. A working concentration of 2.5 mg/mL of the crude extract was used. LC-MS spectra consisted of retention time (minutes), accurate molecular ions (>five ppm accuracy), and MS-MS daughter ions. The LC method used for the analysis spanned 15 min with a gradient of 100% H₂O (with 0.1% formic acid) to 95% acetonitrile (ACN).

Statistical analysis

All statistical analyses were conducted using GraphPad Prism™ version 10.3.0 (GraphPad Software Inc., San Diego, CA, USA). Two-way ANOVA was used to analyze differences between multiple groups, followed by Tukey’s test for post-hoc comparisons. Results were expressed as the mean ± standard error of the mean (SEM), with statistical significance defined as p ≤ 0.05.

Sponge DNA barcoding

The 28S rRNA gene was successfully amplified from three sponge samples via PCR, on agarose gel electrophoresis, yielding bands ranging from 1,000 to 1,300 base pairs (Supplementary 1). Our results confirm high-quality DNA amplification for sequencing.

The 28S rRNA sequences from the three specimens showed similarities with other Spongillidae species when analyzed using BLAST. The sequences were matched to Spongillidae GenBank sequences that already existed. The following are the findings of the BLAST analysis for the three sponge samples’ 28S rRNA sequence alignment with GenBank. Sponge 1 was determined to be Stelletta fibrosa, which is a member of the genus Stelletta, class Demospongiae, order Tetractinellida, and family Ancorinidae. The sponge 2 was found to be Dactylospongia elegans, which is also a member of the class Demospongiae but belongs to the family Thorectidae, order Dictyoceratida, and genus Dactylospongia. Sponge 3 was found to be Haliclona manglaris, which is a member of the family Chalinidae, genus Haliclona, and order Haplosclerida under the class Demospongiae. The phylogenetic tree constructed using the Neighbor-joining method with 1000 bootstrap support indicates high 28s rRNA sequence similarities with previously mentioned species (Fig. 2).

Antimicrobial activity

The extracts’ antibacterial activity at various concentrations was evaluated using the agar well diffusion technique, and the diameter of the inhibition zone was measured in millimeters (mm). The results are depicted in Table 2 and Supplementary 2.

S. fibrosa (Sponge 1) demonstrated no positive results against the selected bacteria. In contrast, D. elegans (Sponge 2) exhibited significant activity against Gram-positive bacteria (S. aureus, B. pumilus, S. epidermidis, and MRSA). However, H. manglaris (Sponge 3) showed only modest activity against two bacterial strains (S. aureus and S. epidermidis).

Positive controls, gentamycin (10 µg) and vancomycin (30 µg), showed clear inhibitory zones against all tested microorganisms, validating the technique. In contrast, the negative control (80% DMSO) exhibited no inhibition, Table 2, showing that the observed antibacterial activity was primarily due to sponge extracts.

Figure 3 presents the (MIC) and (MBC) values for the extracts from D. elegans (Sponge 2) and H. manglaris (Sponge 3), both of which exhibited antibacterial activity against the tested bacteria. Statistical analysis revealed significant differences (p < 0.05) between the two sponge extracts for each bacterial strain. At lower concentrations, D. elegans (Sponge 2) yielded promising results, especially against B. pumilus, with a MIC of 0.125 mg/mL and an MBC of 0.5 mg/mL. In contrast, H. manglaris (Sponge 3) showed notable effectiveness against S. aureus, with both its MIC and MBC values reaching eight mg/mL.

LC-MS-MS

Through LC-MS-MS analysis (Supplementary 3), the ethanol extract of D. elegans (sponge 2) was characterized as in Table 3. The prominent compounds identified were a high proportion (over 7%) of gallic acid, caffeic acid, dactyloquinone, and bolinaquinone. Other significant metabolites, including indole-3-carbaldehyde, hyatellaquinone, linoleic acid, chromazonarol, ilimaquinone, isospongiaquinone, mamanuthaquinone, cyclospongiaquinone, pelorol, ergosterol, manoalide, and lectin, are also present at more than 2% in the extract.