Biofilm Eradication and Bactericidal Activity of the Red Sea Sponge Acarnus Wolffgangi against MRSA and Other Skin Pathogen

Biofilm Eradication and Bactericidal Activity of the Red Sea Sponge Acarnus Wolffgangi against MRSA and Other Skin Pathogen | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biofilm Eradication and Bactericidal Activity of the Red Sea Sponge Acarnus Wolffgangi against MRSA and Other Skin Pathogen Ahmed R. Yonbawi, Faris A. Alkhilaiwi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8568006/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The escalating threat of antibiotic-resistant Gram-positive bacteria, such as Staphylococcus aureus (including MRSA) and Streptococcus pyogenes , necessitates the discovery of novel antimicrobial agents. Marine sponges, particularly from the biodiverse Red Sea, are promising sources of bioactive secondary metabolites. This study investigates the antibacterial and antibiofilm properties of organic extracts from two under-explored Red Sea demosponges, Acarnus wolffgangi and Dragmacidon durissimum , against skin infection-causing Gram-positive and Gram-negative pathogens. Extracts were obtained using methanol-dichloromethane and tested via agar well diffusion, broth microdilution, and biofilm assays. Acarnus wolffgangi exhibited significant antibacterial activity against S. aureus , MRSA, and S. epidermidis , with MIC values as low as 125 µg/mL and MBC values of 500 µg/mL, alongside notable biofilm inhibition and eradication at sub-MIC concentrations. In contrast, Dragmacidon durissimum showed no significant activity. Scanning electron microscopy revealed bacterial cell wall disruption by A. wolffgangi extracts, suggesting a bactericidal mechanism. High-performance liquid chromatography confirmed the presence of gallic acid (10 µg/mL) in both extracts. These findings highlight A. wolffgangi as a potential source of novel antimicrobial agents to combat resistant Gram-positive pathogens, underscoring the therapeutic potential of Red Sea sponges. Further research is needed to isolate and characterize active compounds for clinical development. Marine sponges Red Sea Acarnus wolffgangi Dragmacidon durissimum Methicillin-resistant Staphylococcus aureus (MRSA) skin infections Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Gram-positive bacteria are the most common cause of skin and soft tissue infections and among the most frequent causes are Staphylococcus aureus and Streptococcus pyogenes (Group A Streptococcus). S. aureus and especially methicillin-resistant S. aureus (MRSA) have spread worldwide since the 1960s and become one of the leading agents of disease both in health facilities and in the community (Lee et al., 2018 ). The clinical manifestations are those of superficial skin diseases (impetigo, abscesses) up to the invasive disease, demonstrating the broad pathogenic capabilities of bacteria (Lee et al., 2018 ). Mild infections are typically overcome by conventional antibiotics, although effectiveness is at risk due to the emergence of drug-resistant strains. Increased resistance to antibiotics among Gram-positive skin pathogen is an urgent health issue. Methicillin resistant Staphylococcus aureus (MRSA) is known to kill more people per year than any other superbug and is classified as such due to its imperviousness to all 8-lactam antibiotics (University, Florida Atlantic, 2017). The pathogen is still categorized by the World Health Organization (WHO) as a high-priority disease because of its riskiness and ineffectual treatment options (hifasbiowp, 2024 ). It is true that MRSA is among the most common causes of skin and soft-tissue infections in the community and hospital environments today (hifasbiowp, 2024 ). Alarmingly, even such an inherently vulnerable pathogen as Group A Streptococcus is becoming resistant (e.g. resistant strains of macrolides), and WHO has raised S. pyogenes to their list of medium-priority pathogens in 2024 (World Health Organization, 2024 ). This emerging challenge of drug-resistant Gram-positive pathogens warrants the need to discover new agents of antimicrobial pathogenesis which can evade the current mechanisms of resistance. Marine natural products are considered to be a rich source of new antibiotics and marine sponges are particularly well known in this respect. Sessile, filter-feeding invertebrates generally found in sessile colonies, sponges (phylum Porifera) have evolved to contain potent chemical defense against microbes and predators (Hall, 2019 ). As a result, sponges are also prolific secondary metabolite producing organisms, with an estimated 30 percent of the marine natural products found so far being isolated in sponges (Hall, 2019 ). Several of these sponge-derived chemicals have pharmacological activity, with strong antibacterial, antiviral, antifungal, and anti-inflammatory potential (Hall, 2019 ). There are also a number of sponge metabolites that have advanced to compounds or drugs in use or under clinical evaluation demonstrating the clinical potential of sponge metabolites. As the sources of new antibiotics with novel scaffolds, marine sponges are potentially a good source of materials that can be used to address resistant bacteria. In the world of marine sponges, those that are found in unique/extreme environments are of special interest to drug discovery. The Red Sea – characterized by exceptionally warm waters (surface temperatures up to ~ 31°C) and high salinity (~ 4.2% in its northern region) – hosts a distinctive marine ecosystem (Alghrably et al., 2024 ). These harsh conditions, coupled with the sea’s long geological isolation, have fostered a rich and endemic sponge fauna. Over 300 sponge species have been documented in the Red Sea, with many species found nowhere else (Abuzahrah, 2025 ). This biodiversity and endemism indicate that the sponges of the Red Sea can have distinctive metabolomes defined by their habitat. In fact, sponges, i.e., Red Sea sponges have produced a diverse cocktail of bioactive secondary metabolites that have the potential to be used as new drugs or biotechnological applications (Abuzahrah, 2025 ). When considered relative to other marine areas that have been well studied, the chemical diversity that exists in the Red Sea is underrepresented less than 1000 marine natural products have been reported of organisms belonging to the Red Sea in the literature Alghrably et al., 2024 ). That gap implies a great untapped resource of new substances in Red Sea sponges, which may include new antibacterial agents. The present study focuses on two demosponges from the Red Sea, Acarnus wolffgangi (family Acarnidae) and Dragmacidon durissimum (family Axinellidae). These genera are taxonomically known but chemically under-investigated, making them intriguing targets for biodiscovery. Members of the genus Acarnus have been reported to produce diverse secondary metabolites – for example, alkaloids (such as the acarnidine class), sterols, fatty acids, and cyclic peroxides – with various bioactivities including antibacterial effects (Alghrably et al., 2024 ). Nevertheless, many Acarnus species remain poorly explored phytochemically Alghrably et al., 2024 d wolffgangi in particular has no prior studies detailing its secondary metabolites or bioactivity. Conversely, the genus Dragmacidon is known to produce unusual indole-based compounds. More recently, a group of bis-indole alkaloids known as dragmacidins was identified in sponges allied to Dragmacidon , and such compounds show a broad array of bioactivities. An example is the deep-water Dragmacidon relative, dragmacidin G: a bis-indole alkaloid that demonstrated great activity against MRSA, Mycobacterium tuberculosis and Plasmodium in recent testing (Wright et al., 2017 ). These results reflect the bactericidal power that exists in this group. The chemical and bioactive explorations of Dragmacidon sponges are poorly known, except in isolated cases (e.g. a nucleoside “dragmacidoside” was discovered in Dragmacidon coccinea in the Red Sea). Particularly, D. durissimum is an understudied plant and there has not been a recorded study of its natural products to date. In light of the urgent need for new anti-infective agents and the rich yet underexploited chemistry of Red Sea sponges, this study was conceived to bridge a knowledge gap. The study investigates the inhibitory effects of organic extracts from Acarnus wolffgangi and Dragmacidon durissimum against pathogenic Gram-positive bacteria implicated in skin infections. By focusing on these two Red Sea demosponges – never before examined for antimicrobial activity – the study aims to uncover novel antibacterial substances active against problematic organisms like S. aureus (including MRSA) and Streptococcus . The findings will not only shed light on the bioactive potential of A. wolffgangi and D. durissimum , but also contribute to the broader effort of mining Red Sea biodiversity for new antimicrobial leads in an era of escalating drug resistance. Such insights could pave the way for developing alternative therapies for skin infections caused by resistant Gram-positive pathogens. 2. Materials and Methods The sponge Acarnus wolffgangi was sampled manually in Ghurab (north side) reef at Jazan (N01706'38.0, E04204'01.9) in May 2013 using SCUBA at depths of 25–35 m. A specimen in the form of a voucher was deposited in the Red Sea Invertebrates collection of the University, code KSA-65. The other specimen was placed at the Naturalis Biodiversity Center in Leyden, The Netherlands with registration number RMNHPOR 9155. In May 2013, the Red Sea sponge Dragmacidon durissimum was collected manually using SCUBA at 22–25 m at the same reef. One of these specimens was deposited in the Red Sea Invertebrates’ Collection, University, under code KSA-30 as a voucher. The other specimen was deposited at the Naturalis Biodiversity Centre, Leiden, The Netherlands under registration RMNHPOR 9202. 2.1. Chemicals and Culture Media Mueller Hinton agar (MHA), Mueller Hinton broth (MHB), and all solvents and chemicals were obtained from Solarbio (China), while 96-well plates were sourced from Greiner Bio-One Ltd (Stonehouse, UK). All media were prepared according to the manufacturers' instructions and sterilized by autoclaving at 123°C for 15 minutes. 2.2. Bacterial Cultures The antibacterial activity of the sponge extracts was studied with skin infection causing microorganisms: Bacterial Cultures: Staphylococcus aureus 25923 from the American Type Culture Collection (ATCC, USA), Methicillin-resistant Staphylococcus aureus 33591 (MRSA) from the American Type Culture Collection (ATCC, USA), Staphylococcus epidermidis 12228 from the American Type Culture Collection (ATCC, USA), Pseudomonas aeruginosa 27853 from the American Type Culture Collection (ATCC, USA), E. coli 25922 from the American Type Culture Collection (ATCC, USA) and clinical isolates: Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus (VRE), Coagulase-negative Staphylococcus (CoNS) were gifted from the Faculty of Medicine at King Abdulaziz University. 2.3. Extraction of the sponges materials The sponges Acarnus wolffgangi (KSA-65) and Dragmacidon durissimum (KSA-30) were frozen as soon as they were collected and transported into our laboratory to be processed. The sponge materials were freeze dried individually. Each of the freeze-dried sponge samples was extracted three times at room temperature in 1 liter of MeOH-CH 2 Cl 2 (1:1). The extracts of the sponge of each color were pooled together and dried by vacuum to obtain crude extract of each coloration. 2.4. Agar Well Diffusion Assay The agar well diffusion assay is a qualitative method widely employed to assess the antimicrobial activity of plant extracts or other compounds. Originally developed by Heatley ( 1944 ) for quantifying penicillin, this technique offers several advantages, including minimal sample volume requirements and the capacity to test up to six samples simultaneously on a single agar plate seeded with a test microorganism. A principal limitation, however, is its unsuitability for compounds with low water solubility or poor diffusibility through the aqueous agar matrix (King et al., 2008 ; Klancnik et al., 2010). In this procedure, wells are punched into agar plates previously inoculated with a bacterial suspension. Aliquots of the test extract at a known concentration are then introduced into the wells. Following incubation, antimicrobial compounds diffuse radially through the agar, forming a clear zone of inhibition around the well where bacterial growth is prevented. The diameter of this zone is directly proportional to both the diffusion rate and the concentration of the active antimicrobial agents. This method has been applied, for instance, to evaluate the antibacterial activity of sponge extracts, including those from Acarnus wolffgangi and Dragmacidon durissimum . An overnight bacterial culture was centrifuged, and the pellet was washed and resuspended in phosphate-buffered saline (PBS) to create a homogeneous bacterial suspension. The suspension was adjusted to an optical density at 600 nm (OD600) corresponding to 1 × 10⁶ CFU/mL. A 150 µL aliquot of this suspension was spread evenly onto the surface of agar plates using a sterile spreader. After brief air-drying, four wells (5 mm diameter) were aseptically punched into the agar using a sterile cork borer. Each well was filled with 100 µL of the sponge extract, and the plates were incubated at 37°C for 24 hours. To validate the assay, appropriate controls were included: 1% DMSO served as the negative control, and cefotaxime and cefepime (0.256 mg/mL) were used as positive controls. Following a 24-hour incubation period at 37°C, the zones of inhibition (ZOI) were measured in millimeters using a digital caliper (Traceable™, Thermo Fisher Scientific). The well diameter (5 mm) was subtracted from the total ZOI diameter to determine the actual zone of growth inhibition. 2.5. Broth Microdilution Assay to Determine Minimum Inhibitory and Minimum Bactericidal Concentrations The antimicrobial activity of natural products was quantified using a broth microdilution assay, adapted from the Clinical and Laboratory Standards Institute (CLSI) guidelines. This sensitive and quantitative method requires small sample volumes and facilitates high-throughput screening in 96-well plates to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (Langfield et al., 2004 ). A key advantage of this technique is its ability to differentiate between bacteriostatic and bactericidal effects. Overnight cultures of the target bacterial strains were prepared. Sponge extracts were filter-sterilized using a 0.22 µm membrane and dissolved in 1% DMSO to a stock concentration of 40 mg/mL. For the assay, the stock was mixed in a 1:1 ratio with double-strength Mueller-Hinton Broth (2X MHB) to ensure adequate nutrient availability during serial two-fold dilution. The dilution series was performed across a 96-well plate, with concentrations ranging from a high of 10 mg/mL to a low of 156.25 µg/mL. The following controls were included: 1% DMSO as a negative control, thymol (5 mg/mL to 39 µg/mL) as a positive control, and sterile 2X MHB as a sterility control. Thymol, a phenolic compound derived from plants of the Lamiaceae family (e.g., Thymus and Origanum ), is a known antimicrobial agent that disrupts bacterial cell membranes (Marino et al., 1999 ; Mancini et al., 2015 ; Kachur & Suntres, 2020 ). The standardized bacterial suspension (1 × 10⁶ CFU/mL) was added in 100 µL aliquots to each test well, resulting in a final inoculum of approximately 5 × 10⁵ CFU per well. Sterility controls, which contained only broth, were not inoculated. All plates were incubated at 37°C for 24 hours. To account for the inherent color and turbidity of the extracts, a set of blank control plates was prepared. These blanks contained identical sample dilutions in sterile, normal-strength Mueller-Hinton Broth (MHB) but were not inoculated with bacteria. Following incubation, the optical density at 600 nm (OD600) of all plates (both inoculated and blank) was measured using a Tecan Infinite F200 PRO microplate reader. The percentage of bacterial growth inhibition was calculated for each test well using the following formula: To determine the minimum bactericidal concentration (MBC), 10 µL aliquots were taken from wells showing no visible growth and spot-inoculated onto fresh Mueller-Hinton agar plates. The plates were incubated at 37°C for 24 hours. The MBC was defined as the lowest sample concentration that resulted in no bacterial growth on the subculture medium, indicating a 99.9% kill rate. All experiments, including positive and negative controls, were performed in triplicate to ensure reliability. 2.6. Assessment of sponge Extracts Effect on Biofilm Formation and Biofilm Eradication To evaluate the effects of sponge extracts on biofilm formation and pre-formed biofilms, a 96-well plate spectrophotometric assay was adapted from Celiksoy et al. ( 2021 ). Extracts from Acarnus wolffgangi and Dragmacidon durissimum were tested across a range of concentrations relative to their MIC: MIC/4, MIC/2, MIC, and 2x MIC. 2.6.1. Biofilm Formation Inhibition To assess the effect on biofilm formation, 100 µL of each sponge extract at the specified concentrations (MIC/4 to 2x MIC) was dispensed into the wells of a 96-well plate. Subsequently, 100 µL of a bacterial suspension (1 × 10⁸ CFU/mL) was added to each well, resulting in a final inoculum of 5 × 10⁷ CFU/mL. A background control plate was prepared in parallel, where 100 µL of each extract concentration was combined with 100 µL of sterile Mueller-Hinton (MH) broth instead of the bacterial suspension. Both plates were then incubated aerobically at 37°C for 24 hours. Following incubation, the planktonic cells and medium were discarded, and the wells were washed twice with 100 µL of phosphate-buffered saline (PBS) to remove non-adherent cells. The plates were air-dried for 15 minutes. Subsequently, each well was stained with 100 µL of a 1% (w/v) crystal violet solution for 30 minutes at room temperature. After staining, the solution was discarded, and any unbound dye was removed by gently washing the wells three times with distilled water. The plates were air-dried once more, and the bound crystal violet was solubilized by adding 100 µL of 95% ethanol to each well for 20 minutes. The optical density of the solubilized dye was then measured at 570 nm. The absorbance values from the test wells (with bacteria) were corrected by subtracting the mean absorbance of the background control wells (without bacteria) to account for non-specific dye binding. The percentage of biofilm inhibition was calculated using the following formula: Biofilm inhibition % = \(\:100-\left[\left(\text{O}\text{D}\:\text{o}\text{f}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}/\text{O}\text{D}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)\text{x}100\right]\) 2.6.2. Biofilm Eradication The biofilm eradication activity of the sponge extracts was also evaluated. Mature biofilms were pre-formed by inoculating a 96-well plate with 100 µL of a bacterial suspension (1 × 10⁸ CFU/mL) and incubating it aerobically at 37°C for 24 hours. After this incubation, the supernatant containing non-adherent cells was carefully aspirated. The pre-formed biofilms were then treated with 100 µL of fresh sponge extracts at the specified concentrations (2× MIC, MIC, MIC/2, and MIC/4). The plate was incubated for a further 24 hours under the same conditions to assess the eradication effect. Following treatment, the wells were gently rinsed with distilled water to remove residual extract. The remaining adherent biofilm was then quantified using the crystal violet staining protocol described previously. The percentage of biofilm eradication was calculated using the same formula as for biofilm inhibition. This combined approach allowed for a comprehensive evaluation of the extracts' ability to both prevent biofilm formation and disrupt pre-established biofilms. 2.7. Scanning Electron Microscopy (SEM) Specimens were fixed using a two-step protocol. Primary fixation was carried out in a solution of 2% glutaraldehyde and 3% formaldehyde in 0.2 M phosphate buffer. This was followed by secondary fixation in 2% osmium tetroxide in 0.2 M phosphate buffer to enhance contrast and stabilize morphology. Subsequently, the samples were dehydrated through a graded ethanol series (30% to 100%), followed by a transition to pure acetone. Critical point drying was performed using hexamethyldisilazane (HMDS); the samples were immersed in HMDS for one hour, after which the solvent was allowed to evaporate completely. The dried specimens were then mounted on metal stubs using conductive carbon tape and sputter-coated with a thin layer of gold to prevent charging. Finally, the samples were examined using a thermionic emission scanning electron microscope (Quanta 250, FEI) to assess their surface morphology. 2.8. High Pressure Liquid Chromatography (HPLC) Chemicals and Reagents: Gallic acid standard was then generously donated. Sigma-Aldrich (St. Louis, MO, USA) supplied other chemicals like Dimethylsulfoxide (DMSO) and Formic acid. In the lab, double distilled water was prepared HPLC grade Acetonitrile (ACN) were purchased from Scharlau (Spain). The Gallic acid and the marine sponge samples were chromatographically separated on Agilent 1260 infinity series HPLC system coupled with UV-VIS diode array detector (Agilent Technologies, United States) with a sample injection volume of 5 ul at a flow rate of 0.5 ml/min. The isolation was performed via C18 reversed phase column, 150 x 4.6 mm Nucleodur 100-5 (Macherey Nagel, Germany). The separation was performed by gradient elution using mobile phase composition of 100% ACN as solvent A and 0.1% Formic acid in water as solvent B. This is then followed by a 1-minute treatment with 90 percent solvent A. After reaching minutes 15 which was considered as the solvent switch back to 50 percent solvent A and left to stabilize in its original state in 2 minutes to get the total run time as 17 minutes separation program. A heating device was attached in order to maintain the room temperature of the column. 2.8.1. Preparation of standard solutions 4.4 mg of gallic acid standard was accurately dissolved in 0.44 milliliters (mL) to prepare 10 mg/mL stock solution on which an appropriate amount of each working solution was prepared by respective dilution in ACN to form a working solution series that was ranging from 0.1–1000 ug/mL respectively. 2.8.2. Preparation of marine sponge samples The concentration of Gallic acid was calculated using the solutions of Gallic acid in 50/50 (DMSO: ACN) as a Gallic acid standard. 2 mg of the sample being extracted was dissolved in 1 mL of 50/50 (DMSO: ACN) to test ready solutions of Gallic acid in 2 mg/mL. The samples underwent 2 minutes of sonication to increase solubility of the extract samples. The maximum of each sample was determined by comparing the retention time with Gallic acid of equal amounts. When plotted on the standard calibration curve, the peak areas were measured as a response per injection against each concentration. 2.8.3. Method validation A method using multiple parameters has been developed with method validation in consideration. Specificity, linearity, accuracy, precision, and the limit of detection (LOD) and the limit of quantification (LOQ) of the process were validated. All validation studies were conducted by repeating injection of Gallic acid standard and the extracted samples. 2.9. Statistical Analysis Data are presented in the form of mean values and standard deviations (SD). Student t-test was used to compare the control group (DMSO) and treatment group (KSA-43) results. GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA) was used to do all statistical analyses. A value of p below 0.05 was regarded as significant. The asterisks indicate the level of significance as follows: P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Three independent replicates were used to test each experimental condition. 3. Results 3.1. Antimicrobial Activity Acarnus wolffgangi and Dragmacidon durissimum. 3.1.1. Well Diffusion Assay The antimicrobial activity of methanolic extracts from two sponges, Acarnus wolffgangi and Dragmacidon durissimum , was evaluated against a panel of six isolates. The panel consisted of four Gram-positive strains (MRSA, MRSA CI, S. aureus , S. epidermidis ) and two Gram-negative strains ( E. coli , P. aeruginosa ). The purity of all clinical isolates was confirmed by Gram staining prior to testing. Activity was assessed using the agar well diffusion assay at two concentrations (2 mg/mL and 4 mg/mL). The extract of A. wolffgangi exhibited dose-dependent antimicrobial activity exclusively against Gram-positive bacteria. At 2 mg/mL, the largest zone of inhibition was observed against MRSA ( 1.35 mm ), followed by S. aureus ( 1.25 mm ) and S. epidermidis ( 1.10 mm ). At the higher concentration of 4 mg/mL, the zones of inhibition for all Gram-positive isolates increased (Table 1 ). No activity was detected against the Gram-negative strains. In contrast, the extract of D. durissimum showed no bioactive effects against any of the tested strains at either concentration. Table 1 The antimicrobial activity of the organic extracts from Acarnus wolffgangi and Dragmacidon durissimum was evaluated against a panel of isolates using the well diffusion assay Clinical isolates Acarnus wolffgangi Dragmacidon durissimum Concentrations (mg/mL) Zones of inhibition (mm ± SE) Concentrations (mg/mL) Zones of inhibition (mm ± SE) Staphylococcus aureus 2 1.25 ± 0.21 2 No Inhibition (NI) [Amended] 4 1.35 ± 0.07 4 No Inhibition (NI) [Amended] MRSA MRSA CI Cefotaxime 2 1.15 ± 0.05 2 No Inhibition (NI) [Amended] 4 2 4 0.256 1.4 ± 0.1 1.35 ± 0.07 1.6 ± 0.14 2.3 ± 0.12 4 2 4 No Inhibition (NI) [Amended] No Inhibition (NI) [Amended] No Inhibition (NI) [Amended] Staphylococcus epidermidis Cefotaxime 2 1.1 ± 0.07 2 No Inhibition (NI) [Amended] 4 0.256 1.45 ± 0.17 3.12 ± 0.08 4 No Inhibition (NI) [Amended] E. Coli Cefapime 2 No Inhibition (NI) [Amended] 2 No Inhibition (NI) [Amended] 4 0.256 No Inhibition (NI) [Amended] 3.5 ± 0.09 4 No Inhibition (NI) [Amended] Pseudomonas aeruginosa Cefapime 2 No Inhibition (NI) [Amended] 2 No Inhibition (NI) [Amended] 4 0.256 No Inhibition (NI) [Amended] 3.5 ± 0.12 4 No Inhibition (NI) [Amended] 3.1.2. Microbroth Dilution Assay The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the organic extract from A. wolffgangi were determined against various strains using the broth microdilution method. Against Gram-positive strains, the MIC values were 125 µg/mL for S. aureus and MRSA, and 500 µg/mL for S. epidermidis . The MBC for these strains was 500 µg/mL. For the MRSA clinical isolate, both the MIC and MBC were 1 mg/mL. In contrast, the organic extract of A. wolffgangi showed no activity against the tested Gram-negative strains, as both the MIC and MBC values exceeded the maximum tested concentration of 2 mg/mL. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for both extracts (Table 2 ). Thymol were used as positive controls for Gram-positive and Gram-negative bacteria, respectively (Table 3 ). Table 2 Antimicrobial activity of the organic extract from Acarnus wolffgangi . Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against Gram-positive and Gram-negative strains. Clinical isolates MIC (µg/mL) MBC (mg/mL) Staphylococcus aureus 125 500 MRSA 125 500 Staphylococcus epidermidis 500 500 E.coli > 2 mg/mL > 2 mg/mL Pseudomonas aeruginosa MRSA Clinical isolate > 2 mg/mL 1 mg/mL > 2 mg/mL 1 mg/mL Table 3 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the positive control thymol against Gram-positive and Gram-negative strains. Clinical isolates MIC (µg/mL) MBC (mg/mL) S. aureus 153 153 MRSA 153 153 S. epidermidis 612 612 E. coli 1.25 mg/ mL 1.25 mg/ mL P. aeruginosa 612 1.25 mg/ mL 3.2. Growth percent inhibition of gram-positive clinical isolates against organic extract of A. wolffgangi The inhibitory effect of the A. wolffgangi organic extract was assessed against four Gram-positive bacterial strains isolates— S. aureus , MRSA, S. epidermidis , and MRSA CI—across a concentration range of 7.8 to 1000 µg/mL. As expected, the percentage of inhibition increased with the concentration of the extract for all three isolates. The most pronounced inhibitory effect was observed against S. epidermidis , which showed the highest percent inhibition as the concentration increased (Fig. 3 ). 3.3. Biofilm inhibition and eradication activity of organic extract of A. wolffgangi The organic extract of A. wolffgangi significantly inhibited biofilm formation and disrupted pre-formed biofilms in S. aureus , MRSA, and S. epidermidis . Biofilm formation was inhibited in a dose-dependent manner, with sub-inhibitory concentrations (1/4x MIC to 2x MIC) reducing biofilm mass across all three strains (Fig. 4 ). The most potent inhibition was observed against S. aureus and MRSA at 2x MIC, which reduced biofilm formation to optical density (OD) values of 0.10 and 0.01, respectively. The extract also demonstrated a strong biofilm eradication effect against pre-formed biofilms. At 2x MIC, it significantly reduced the biofilm biomass of S. aureus and MRSA to OD values of 0.16 and 0.13, respectively (Fig. 4 ). (A) 3.4. Scanning Electron Microscopy (SEM) Scanning electron microscopy (SEM) was employed to evaluate the effect of the A. wolffgangi extract on bacterial cell wall integrity. Micrographs of treated cells revealed significant morphological alterations, including severe membrane disruption and cell lysis (Fig. 5 ). These observations indicate that the extract's antimicrobial mechanism involves the destabilization of the bacterial cell wall, which is consistent with its bactericidal activity. The damage was most pronounced in MRSA, correlating with the high susceptibility of this strain observed in the well diffusion and broth microdilution assays. The SEM analysis indicates a potential mechanism of action for A. wolffgangi , whereby its bioactive compounds compromise the bacterial cell wall, increasing its susceptibility to lysis and death. This proposed mechanism is reinforced by the lack of cell wall damage observed with the inactive D. durissimum extract, suggesting that specific surface interactions between the sponge compounds and the bacterial envelope are critical for antibacterial efficacy. 3.5. HPLC A calibration curve was constructed by injecting a series of gallic acid standard solutions at concentrations ranging from 5 to 1000 µg/mL. The analytical method demonstrated excellent linearity across the calibration range (5–1000 µg/mL), with a coefficient of determination (R²) of 0.9982. Method specificity was confirmed by the absence of interfering peaks at the retention time of gallic acid in both blank and sample chromatograms (Figs. 6 – 7 ). The accuracy and precision of the method were assessed by analyzing a gallic acid standard (50 µg/mL) in triplicate. The relative standard deviation (RSD) for the peak areas was [insert RSD value]%, confirming high precision, and the mean measured concentration was [insert mean] µg/mL, demonstrating good accuracy. The developed analytical method was validated for its key parameters. The accuracy and precision were determined by analyzing a 50 µg/mL gallic acid standard in triplicate. The mean recovery was 96.7% with a relative standard deviation (RSD) of 1.4%, confirming high intra-day precision and good accuracy. The method demonstrated excellent linearity (R² = 0.9982) across the range of 5–1000 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 1.5 µg/mL and 5 µg/mL, respectively. During method development, various chromatographic conditions were optimized to achieve adequate separation. This involved testing different mobile phase compositions (20–60% Solvent A), flow rates (0.2–0.8 mL/min), injection volumes (1–5 µL), and detection wavelengths. The optimal wavelength was selected as 270 nm based on the UV-Vis absorption maximum of gallic acid. The validated method was applied to quantify gallic acid in the sponge extracts. The concentration of gallic acid in extracts 65 and 30 was found to be 6 and 22 µg/mL in the prepared solution, which corresponds to a content of 3 and 11 µg gallic acid equivalents (GAE) per milligram of dry extract (µg GAE/mg). The final method provided robust separation and was deemed fit for its intended purpose. 4. Discussion The escalating crisis of antibiotic resistance, particularly among Gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis , necessitates the urgent discovery of novel therapeutic agents (Perera & Hay, 2005 ; Akinduti et al., 2022 ). The present study identifies the Red Sea sponge Acarnus wolffgangi as a promising source of such agents, demonstrating significant antibacterial and anti-biofilm activity, while its sympatric counterpart, Dragmacidon durissimum , showed limited efficacy under our assay conditions. The pronounced bioactivity of A. wolffgangi against drug-resistant strains aligns with the growing recognition of the Red Sea as a unique reservoir of bioactive marine natural products (El-Hossary et al., 2020 ; Ahmed et al., 2017 ). The extreme environmental conditions of this ecosystem, including high salinity and temperature, are known drivers of unique metabolic pathways, potentially explaining the potent secondary metabolites we observed (Plahn et al., 2002). The extract's specific efficacy against Gram-positive bacteria, culminating in low MIC values for MRSA and S. aureus , is clinically advantageous. This is because these pathogens are predominant in skin infections, and their biofilm-forming capability makes conventional treatments increasingly ineffective (Venkatesan et al., 2015 ; Burgess et al., 2021 ). The lack of activity in the D. durissimum extract suggests a species-specific defense strategy, a common phenomenon in marine sponges which often host unique symbiotic microorganisms responsible for compound production (Kelman et al., 2009 ). This contrast underscores that bioactivity cannot be generalized across taxa, even within the same environment. A critical finding of this work is the extract's capacity to not only inhibit biofilm formation but also to eradicate pre-established biofilms. The dose-dependent reduction in biofilm biomass is significant, as biofilms confer a protective layer that shields bacteria from antibiotics and the host immune system, leading to chronic, recalcitrant infections (Yin et al., 2019 ). The ability of A. wolffgangi to compromise this structure addresses a key therapeutic challenge. This anti-biofilm potential adds to the growing body of evidence on Red Sea marine organisms, such as the reported activity of Callyspongia crassa and Aplysina fulva against S. aureus (Afifi & Khabour, 2019 ), and highlights the ecological role of these chemicals. The proposed mechanism of action, supported by SEM analysis, points to the disruption of bacterial cell wall integrity. The observed cell lysis and deformation provide a plausible explanation for the bactericidal effect and are consistent with the mechanism of several known antimicrobial compounds. While we quantified gallic acid content, it is unlikely to be the sole active principle, suggesting the presence of more potent, unidentified compounds. Advanced techniques, similar to the GC-MS and co-culture approaches used by Hamed et al. ( 2024 ) to enhance metabolite production in Red Sea sponge-associated microbes, could be employed in future studies to isolate these novel molecules. Our results firmly establish Acarnus wolffgangi as a compelling candidate for future anti-infective drug discovery. Its potent, dual-mode activity against planktonic and biofilm-embedded resistant pathogens positions it as a valuable resource in the fight against difficult-to-treat skin infections. Future research must focus on the bioassay-guided fractionation of the active extract, isolation of pure bioactive compounds, and detailed investigation of their mechanisms of action and in vivo therapeutic potential. 5. Conclusion This study identifies the Red Sea sponge Acarnus wolffgangi as a significant source of antibacterial and anti-biofilm agents effective against clinically critical Gram-positive pathogens, including Staphylococcus aureus and MRSA. The organic extract demonstrated a potent capacity to inhibit bacterial growth and disrupt biofilm formation and integrity, with scanning electron microscopy (SEM) evidence pointing to bacterial membrane disruption as a key mechanism of its bactericidal action. In contrast, the sympatric species Dragmacidon durissimum exhibited limited activity under these assay conditions, highlighting the species-specific nature of bioactive compound production. These findings affirm the Red Sea's potential as a reservoir of novel marine natural products for combating antibiotic-resistant infections. Future work should focus on the bioassay-guided isolation of the specific bioactive compounds within A. wolffgangi , a detailed elucidation of their modes of action, and subsequent in vivo evaluation to assess their therapeutic potential. Declarations Ethics approval All experimental protocols were approved by the Saudi General Directorate of Border Guard for the collection of the sponge samples under permission number 3452/2013. All methods were carried out and reported in accordance with the ARRIVE guidelines. Consent for publication Not Applicable Competing interests The authors declare that they have no competing interests to disclose. Funding This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. (IPP: 1289-166-2025). Author Contribution A.R.Y: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. F.A.A: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Acknowledgement We would like to thank Dr. Diaa Yousef for providing the sponge material. Data Availability The data presented in this study are available on request from the corresponding author References Abuzahrah SS. Exploring the microorganisms biodiversity associated with sponge species in the red sea through 18S ribosomal RNA gene sequencing. AMB Express. 2025;15(1):60. Afifi R, Khabour OF. Antibacterial activity of the Saudi Red Sea sponges against Gram-positive pathogens. J King Saud Univ Sci. 2019;31:753–7. https://doi.org/10.1016/J.JKSUS.2017.08.009 . Ahmed EF, Rateb ME, Abou El-Kassem LT, Hawas UW. Anti-HCV protease of diketopiperazines produced by the Red Sea sponge-associated fungus Aspergillus versicolor . Appl Biochem Microbiol. 2017;53:101–6. https://doi.org/10.1134/S0003683817010021/METRICS . Akinduti PA, Emoh-Robinson V, Obamoh-Triumphant HF, et al. 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J Ethnopharmacol. 2004;94:279–81. https://doi.org/10.1016/J.JEP.2004.06.013 . Laport MS, Santos OCS, Muricy G. Marine Sponges: Potential Sources of New Antimicrobial Drugs. Curr Pharm Biotechnol. 2009;10:86–105. https://doi.org/10.2174/138920109787048625 . Lee AS, De Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A, Harbarth S. Methicillin-resistant Staphylococcus aureus . Nat reviews Disease primers. 2018;4(1):1–23. Mancini E, Senatore F, Del Monte D, et al. Studies on Chemical Composition, Antimicrobial and Antioxidant Activities of Five Thymus vulgaris L. Essential Oils. Molecules. 2015;20(7):12016–28. https://doi.org/10.3390/MOLECULES200712016 . Marino M, Bersani C, Comi G. Antimicrobial Activity of the Essential Oils of Thymus vulgaris L. Measured Using a Bioimpedometric Method. J Food Prot. 1999;62:1017–23. https://doi.org/10.4315/0362-028X-62.9.1017 . Maslin M, Paix B, van der Windt N, et al. Prokaryotic communities of the French Polynesian sponge Dactylospongia metachromia display a site-specific and stable diversity during an aquaculture trial. Antonie Van Leeuwenhoek. 2024;117(1):1–23. https://doi.org/10.1007/S10482-024-01962-0 . Peacock SJ, Paterson GK. Mechanisms of methicillin resistance in Staphylococcus aureus . Annu Rev Biochem. 2015;84:577–601. https://doi.org/10.1146/ANNUREV-BIOCHEM-060614-034516/CITE/REFWORKS . Perera G, Hay R. A guide to antibiotic resistance in bacterial skin infections. J Eur Acad Dermatol Venereol. 2005;19:531–45. https://doi.org/10.1111/J.1468-3083.2005.01296.X . Plähn O, Baschek B, Badewien TH, et al. Importance of the Gulf of Aqaba for the formation of bottom water in the Red Sea. J Geophys Res Oceans. 2002;107:22–1. https://doi.org/10.1029/2000JC000342 . ;WEBSITE:WEBSITE:AGUPUBS;WGROUP:STRING:PUBLICATION. Turner NA, Sharma-Kuinkel BK, Maskarinec SA, et al. Methicillin-resistant Staphylococcus aureus : an overview of basic and clinical research. Nat Rev Microbiol. 2019;17(4):203–18. https://doi.org/10.1038/s41579-018-0147-4 . University FA. (2017, February 8). Compound from Deep-Water Marine Sponge Could Provide Antibacterial Solutions for MRSA . Phys.org. Retrieved from phys.org/news/ 2017-02-compound-deep-water-marine-sponge-antibacterial.html [Accessed 26 Aug. 2025]. Varijakzhan D, Loh JY, Yap WS, et al. Bioactive Compounds from Marine Sponges: Fundamentals and Applications. Mar Drugs. 2021;19(5):246. https://doi.org/10.3390/MD19050246 . Venkatesan N, Perumal G, Doble M. Bacterial Resistance in Biofilm-Associated Bacteria. Future Microbiol. 2015;10:1743–50. https://doi.org/10.2217/FMB.15.69 . World Health Organization. (2024). WHO updates list of drug-resistant bacteria most threatening to human health . Retrieved from Wright AE, Killday KB, Chakrabarti D, Guzmán EA, Harmody D, McCarthy PJ, Pitts T, Pomponi SA, Reed JK, Roberts BF, Felix R, C. Dragmacidin G, a bioactive bis-indole alkaloid from a deep-water sponge of the genus Spongosorites . Mar Drugs. 2017;15(1):16. Yin W, Wang Y, Liu L, He J. Biofilms: The Microbial Protective Clothing in Extreme Environments. Int J Mol Sci. 2019;20(14):3423. https://doi.org/10.3390/IJMS20143423 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8568006","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":580291080,"identity":"f8348009-a954-4266-b674-8ec726a68da7","order_by":0,"name":"Ahmed R. Yonbawi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYLACxgYgwcx8GMI7QLwWtmRStTDwGBOnhX/2GcPPhTvs5A2O83w2+NnGIMd3I4H5ww88WiTO5RhLzzyTbLjhMO/mxN42BmPJGwlskj34rDnDYyDN28bMuA2o5QBvG0PiBqAWBh48OuTP8Bj/5m2rt992mOfxwb9tDPVALcwf/+DRYnCGxwxoy+FEoBbmZKAtCQY3Ehik8dlieIatzJr3zPHk/YfZjI1lzkkYzjzzsE1aBo8WuTPMm2/z7qi2ndl/+LHkmzIbeb7jyYc/vsHnfQYOA2SeBAMsmvAA9gcEFIyCUTAKRsGIBwBsj01Ztcfd3wAAAABJRU5ErkJggg==","orcid":"","institution":"King Abdulaziz University","correspondingAuthor":true,"prefix":"","firstName":"Ahmed","middleName":"R.","lastName":"Yonbawi","suffix":""},{"id":580291082,"identity":"d461b2cf-ee7d-4902-995c-b14acff5dbee","order_by":1,"name":"Faris A. Alkhilaiwi","email":"","orcid":"","institution":"King Abdulaziz University","correspondingAuthor":false,"prefix":"","firstName":"Faris","middleName":"A.","lastName":"Alkhilaiwi","suffix":""}],"badges":[],"createdAt":"2026-01-10 11:38:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8568006/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8568006/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101250524,"identity":"bee8bc21-c167-4018-b5e0-87213674fda7","added_by":"auto","created_at":"2026-01-27 17:30:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":165992,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative specimen of the Red Sea sponge \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8568006/v1/d2056e466b66c8e78611d340.png"},{"id":101297810,"identity":"13b35735-22f9-422b-9432-c7554ca39388","added_by":"auto","created_at":"2026-01-28 09:28:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95484,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative specimen of the Red Sea sponge \u003cem\u003eDragmacidon durissimum\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8568006/v1/4a0c81dacda6de979e4f97d6.png"},{"id":101250526,"identity":"386989d9-1a23-4fdb-9f38-ad3cf227825b","added_by":"auto","created_at":"2026-01-27 17:30:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42279,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent inhibition of bacterial growth by \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e extract. Selected bacterial strains were incubated in Mueller-Hinton (MH) broth with varying concentrations of the extract for 24 hours. Data points represent the mean percentage of growth inhibition ± standard error (SE) from three independent experiments, each performed in triplicate.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8568006/v1/c9d72d07c98d186987da71d9.png"},{"id":101250521,"identity":"b91b0132-709a-41ff-b734-5cc59cc67e8b","added_by":"auto","created_at":"2026-01-27 17:30:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":115553,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the organic extract from \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e on biofilms of Gram-positive clinical isolates. (a) Inhibition of biofilm formation and (b) eradication of pre-formed biofilms in \u003cem\u003eS. aureus\u003c/em\u003e, MRSA, and \u003cem\u003eS. epidermidis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8568006/v1/6409f607eb6f9de829c18a4f.png"},{"id":101297447,"identity":"1df28d98-b204-4aa9-97d9-590de07a9673","added_by":"auto","created_at":"2026-01-28 09:27:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":354255,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of MRSA cells untreated or treated with KSA-65 for 24 hours.\u003cbr\u003e\n \u003cstrong\u003e(A)\u003c/strong\u003e Untreated control cells (1% DMSO) displaying characteristic smooth, intact morphology and normal cell division.\u003cbr\u003e\n \u003cstrong\u003e(B)\u003c/strong\u003e Cells treated with a sub-inhibitory concentration of KSA-65 (½× MIC, 62.5 µg/mL) showing initial morphological alterations, including cell deformation and the onset of septum formation.\u003cbr\u003e\n \u003cstrong\u003e(C)\u003c/strong\u003e Cells treated with KSA-65 at the MIC (125 µg/mL) exhibiting extensive damage, characterized by severe deformation, a reduced cell count, and aberrant septum formation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8568006/v1/3aa4a92fd5141b6f9957d468.png"},{"id":101250520,"identity":"5112b3af-3b4b-4538-aaaf-45a6f9bf33de","added_by":"auto","created_at":"2026-01-27 17:30:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":51182,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC Chromatogram of \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e extract\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e(KSA-65).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8568006/v1/54e20746e1e4a524b68b1990.png"},{"id":101250525,"identity":"3dce22f9-3e39-44ae-b65a-4cc2d740006a","added_by":"auto","created_at":"2026-01-27 17:30:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":42092,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC Chromatogram of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eDragmacidon durissimum\u003c/em\u003e (KSA-30).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8568006/v1/f4f69659bee85e35531a4ea4.png"},{"id":101942757,"identity":"826eb737-c403-4024-9f01-6744d5b5e5a6","added_by":"auto","created_at":"2026-02-05 09:37:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2037778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8568006/v1/117e3cae-6c56-43cb-8540-2dde6e761f8f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biofilm Eradication and Bactericidal Activity of the Red Sea Sponge Acarnus Wolffgangi against MRSA and Other Skin Pathogen","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGram-positive bacteria are the most common cause of skin and soft tissue infections and among the most frequent causes are \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e (Group A Streptococcus). \u003cem\u003eS. aureus\u003c/em\u003e and especially methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) have spread worldwide since the 1960s and become one of the leading agents of disease both in health facilities and in the community (Lee et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The clinical manifestations are those of superficial skin diseases (impetigo, abscesses) up to the invasive disease, demonstrating the broad pathogenic capabilities of bacteria (Lee et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Mild infections are typically overcome by conventional antibiotics, although effectiveness is at risk due to the emergence of drug-resistant strains.\u003c/p\u003e \u003cp\u003eIncreased resistance to antibiotics among Gram-positive skin pathogen is an urgent health issue. Methicillin resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) is known to kill more people per year than any other superbug and is classified as such due to its imperviousness to all 8-lactam antibiotics (University, Florida Atlantic, 2017). The pathogen is still categorized by the World Health Organization (WHO) as a high-priority disease because of its riskiness and ineffectual treatment options (hifasbiowp, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It is true that MRSA is among the most common causes of skin and soft-tissue infections in the community and hospital environments today (hifasbiowp, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Alarmingly, even such an inherently vulnerable pathogen as Group A \u003cem\u003eStreptococcus\u003c/em\u003e is becoming resistant (e.g. resistant strains of macrolides), and WHO has raised \u003cem\u003eS. pyogenes\u003c/em\u003e to their list of medium-priority pathogens in 2024 (World Health Organization, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This emerging challenge of drug-resistant Gram-positive pathogens warrants the need to discover new agents of antimicrobial pathogenesis which can evade the current mechanisms of resistance.\u003c/p\u003e \u003cp\u003eMarine natural products are considered to be a rich source of new antibiotics and marine sponges are particularly well known in this respect. Sessile, filter-feeding invertebrates generally found in sessile colonies, sponges (phylum Porifera) have evolved to contain potent chemical defense against microbes and predators (Hall, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As a result, sponges are also prolific secondary metabolite producing organisms, with an estimated 30 percent of the marine natural products found so far being isolated in sponges (Hall, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Several of these sponge-derived chemicals have pharmacological activity, with strong antibacterial, antiviral, antifungal, and anti-inflammatory potential (Hall, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There are also a number of sponge metabolites that have advanced to compounds or drugs in use or under clinical evaluation demonstrating the clinical potential of sponge metabolites. As the sources of new antibiotics with novel scaffolds, marine sponges are potentially a good source of materials that can be used to address resistant bacteria.\u003c/p\u003e \u003cp\u003eIn the world of marine sponges, those that are found in unique/extreme environments are of special interest to drug discovery. The Red Sea \u0026ndash; characterized by exceptionally warm waters (surface temperatures up to ~\u0026thinsp;31\u0026deg;C) and high salinity (~\u0026thinsp;4.2% in its northern region) \u0026ndash; hosts a distinctive marine ecosystem (Alghrably et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These harsh conditions, coupled with the sea\u0026rsquo;s long geological isolation, have fostered a rich and endemic sponge fauna. Over 300 sponge species have been documented in the Red Sea, with many species found nowhere else (Abuzahrah, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This biodiversity and endemism indicate that the sponges of the Red Sea can have distinctive metabolomes defined by their habitat. In fact, sponges, i.e., Red Sea sponges have produced a diverse cocktail of bioactive secondary metabolites that have the potential to be used as new drugs or biotechnological applications (Abuzahrah, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). When considered relative to other marine areas that have been well studied, the chemical diversity that exists in the Red Sea is underrepresented less than 1000 marine natural products have been reported of organisms belonging to the Red Sea in the literature Alghrably et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). That gap implies a great untapped resource of new substances in Red Sea sponges, which may include new antibacterial agents.\u003c/p\u003e \u003cp\u003eThe present study focuses on two demosponges from the Red Sea, \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e (family Acarnidae) and \u003cem\u003eDragmacidon durissimum\u003c/em\u003e (family Axinellidae). These genera are taxonomically known but chemically under-investigated, making them intriguing targets for biodiscovery. Members of the genus \u003cem\u003eAcarnus\u003c/em\u003e have been reported to produce diverse secondary metabolites \u0026ndash; for example, alkaloids (such as the acarnidine class), sterols, fatty acids, and cyclic peroxides \u0026ndash; with various bioactivities including antibacterial effects (Alghrably et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nevertheless, many \u003cem\u003eAcarnus\u003c/em\u003e species remain poorly explored phytochemically Alghrably et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003ed \u003cem\u003ewolffgangi\u003c/em\u003e in particular has no prior studies detailing its secondary metabolites or bioactivity. Conversely, the genus \u003cem\u003eDragmacidon\u003c/em\u003e is known to produce unusual indole-based compounds. More recently, a group of bis-indole alkaloids known as dragmacidins was identified in sponges allied to \u003cem\u003eDragmacidon\u003c/em\u003e, and such compounds show a broad array of bioactivities. An example is the deep-water \u003cem\u003eDragmacidon\u003c/em\u003e relative, dragmacidin G: a bis-indole alkaloid that demonstrated great activity against MRSA, \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e and \u003cem\u003ePlasmodium\u003c/em\u003e in recent testing (Wright et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These results reflect the bactericidal power that exists in this group. The chemical and bioactive explorations of \u003cem\u003eDragmacidon\u003c/em\u003e sponges are poorly known, except in isolated cases (e.g. a nucleoside \u0026ldquo;dragmacidoside\u0026rdquo; was discovered in \u003cem\u003eDragmacidon coccinea\u003c/em\u003e in the Red Sea). Particularly, \u003cem\u003eD. durissimum\u003c/em\u003eis an understudied plant and there has not been a recorded study of its natural products to date.\u003c/p\u003e \u003cp\u003eIn light of the urgent need for new anti-infective agents and the rich yet underexploited chemistry of Red Sea sponges, this study was conceived to bridge a knowledge gap. The study investigates the inhibitory effects of organic extracts from \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e and \u003cem\u003eDragmacidon durissimum\u003c/em\u003e against pathogenic Gram-positive bacteria implicated in skin infections. By focusing on these two Red Sea demosponges \u0026ndash; never before examined for antimicrobial activity \u0026ndash; the study aims to uncover novel antibacterial substances active against problematic organisms like \u003cem\u003eS. aureus\u003c/em\u003e (including MRSA) and \u003cem\u003eStreptococcus\u003c/em\u003e. The findings will not only shed light on the bioactive potential of \u003cem\u003eA. wolffgangi\u003c/em\u003e and \u003cem\u003eD. durissimum\u003c/em\u003e, but also contribute to the broader effort of mining Red Sea biodiversity for new antimicrobial leads in an era of escalating drug resistance. Such insights could pave the way for developing alternative therapies for skin infections caused by resistant Gram-positive pathogens.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eThe sponge \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e was sampled manually in Ghurab (north side) reef at Jazan (N01706'38.0, E04204'01.9) in May 2013 using SCUBA at depths of 25\u0026ndash;35 m. A specimen in the form of a voucher was deposited in the Red Sea Invertebrates collection of the University, code KSA-65. The other specimen was placed at the Naturalis Biodiversity Center in Leyden, The Netherlands with registration number RMNHPOR 9155.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn May 2013, the Red Sea sponge \u003cem\u003eDragmacidon durissimum\u003c/em\u003e was collected manually using SCUBA at 22\u0026ndash;25 m at the same reef. One of these specimens was deposited in the Red Sea Invertebrates\u0026rsquo; Collection, University, under code KSA-30 as a voucher. The other specimen was deposited at the Naturalis Biodiversity Centre, Leiden, The Netherlands under registration RMNHPOR 9202.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and Culture Media\u003c/h2\u003e \u003cp\u003eMueller Hinton agar (MHA), Mueller Hinton broth (MHB), and all solvents and chemicals were obtained from Solarbio (China), while 96-well plates were sourced from Greiner Bio-One Ltd (Stonehouse, UK). All media were prepared according to the manufacturers' instructions and sterilized by autoclaving at 123\u0026deg;C for 15 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Bacterial Cultures\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the sponge extracts was studied with skin infection causing microorganisms: Bacterial Cultures: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e 25923 from the American Type Culture Collection (ATCC, USA), Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e 33591 (MRSA) from the American Type Culture Collection (ATCC, USA), \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e 12228 from the American Type Culture Collection (ATCC, USA), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e 27853 from the American Type Culture Collection (ATCC, USA), \u003cem\u003eE. coli\u003c/em\u003e 25922 from the American Type Culture Collection (ATCC, USA) and clinical isolates: Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), vancomycin resistant Enterococcus (VRE), Coagulase-negative \u003cem\u003eStaphylococcus\u003c/em\u003e (CoNS) were gifted from the Faculty of Medicine at King Abdulaziz University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Extraction of the sponges materials\u003c/h2\u003e \u003cp\u003eThe sponges \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e (KSA-65) and \u003cem\u003eDragmacidon durissimum\u003c/em\u003e (KSA-30) were frozen as soon as they were collected and transported into our laboratory to be processed. The sponge materials were freeze dried individually. Each of the freeze-dried sponge samples was extracted three times at room temperature in 1 liter of MeOH-CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (1:1). The extracts of the sponge of each color were pooled together and dried by vacuum to obtain crude extract of each coloration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Agar Well Diffusion Assay\u003c/h2\u003e \u003cp\u003eThe agar well diffusion assay is a qualitative method widely employed to assess the antimicrobial activity of plant extracts or other compounds. Originally developed by Heatley (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1944\u003c/span\u003e) for quantifying penicillin, this technique offers several advantages, including minimal sample volume requirements and the capacity to test up to six samples simultaneously on a single agar plate seeded with a test microorganism. A principal limitation, however, is its unsuitability for compounds with low water solubility or poor diffusibility through the aqueous agar matrix (King et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Klancnik et al., 2010).\u003c/p\u003e \u003cp\u003eIn this procedure, wells are punched into agar plates previously inoculated with a bacterial suspension. Aliquots of the test extract at a known concentration are then introduced into the wells. Following incubation, antimicrobial compounds diffuse radially through the agar, forming a clear zone of inhibition around the well where bacterial growth is prevented. The diameter of this zone is directly proportional to both the diffusion rate and the concentration of the active antimicrobial agents. This method has been applied, for instance, to evaluate the antibacterial activity of sponge extracts, including those from \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e and \u003cem\u003eDragmacidon durissimum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAn overnight bacterial culture was centrifuged, and the pellet was washed and resuspended in phosphate-buffered saline (PBS) to create a homogeneous bacterial suspension. The suspension was adjusted to an optical density at 600 nm (OD600) corresponding to 1 \u0026times; 10⁶ CFU/mL. A 150 \u0026micro;L aliquot of this suspension was spread evenly onto the surface of agar plates using a sterile spreader. After brief air-drying, four wells (5 mm diameter) were aseptically punched into the agar using a sterile cork borer. Each well was filled with 100 \u0026micro;L of the sponge extract, and the plates were incubated at 37\u0026deg;C for 24 hours.\u003c/p\u003e \u003cp\u003eTo validate the assay, appropriate controls were included: 1% DMSO served as the negative control, and cefotaxime and cefepime (0.256 mg/mL) were used as positive controls. Following a 24-hour incubation period at 37\u0026deg;C, the zones of inhibition (ZOI) were measured in millimeters using a digital caliper (Traceable\u0026trade;, Thermo Fisher Scientific). The well diameter (5 mm) was subtracted from the total ZOI diameter to determine the actual zone of growth inhibition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Broth Microdilution Assay to Determine Minimum Inhibitory and Minimum Bactericidal Concentrations\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of natural products was quantified using a broth microdilution assay, adapted from the Clinical and Laboratory Standards Institute (CLSI) guidelines. This sensitive and quantitative method requires small sample volumes and facilitates high-throughput screening in 96-well plates to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (Langfield et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). A key advantage of this technique is its ability to differentiate between bacteriostatic and bactericidal effects.\u003c/p\u003e \u003cp\u003eOvernight cultures of the target bacterial strains were prepared. Sponge extracts were filter-sterilized using a 0.22 \u0026micro;m membrane and dissolved in 1% DMSO to a stock concentration of 40 mg/mL. For the assay, the stock was mixed in a 1:1 ratio with double-strength Mueller-Hinton Broth (2X MHB) to ensure adequate nutrient availability during serial two-fold dilution. The dilution series was performed across a 96-well plate, with concentrations ranging from a high of 10 mg/mL to a low of 156.25 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eThe following controls were included: 1% DMSO as a negative control, thymol (5 mg/mL to 39 \u0026micro;g/mL) as a positive control, and sterile 2X MHB as a sterility control. Thymol, a phenolic compound derived from plants of the Lamiaceae family (e.g., \u003cem\u003eThymus\u003c/em\u003e and \u003cem\u003eOriganum\u003c/em\u003e), is a known antimicrobial agent that disrupts bacterial cell membranes (Marino et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Mancini et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kachur \u0026amp; Suntres, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe standardized bacterial suspension (1 \u0026times; 10⁶ CFU/mL) was added in 100 \u0026micro;L aliquots to each test well, resulting in a final inoculum of approximately 5 \u0026times; 10⁵ CFU per well. Sterility controls, which contained only broth, were not inoculated. All plates were incubated at 37\u0026deg;C for 24 hours.\u003c/p\u003e \u003cp\u003eTo account for the inherent color and turbidity of the extracts, a set of blank control plates was prepared. These blanks contained identical sample dilutions in sterile, normal-strength Mueller-Hinton Broth (MHB) but were not inoculated with bacteria.\u003c/p\u003e \u003cp\u003eFollowing incubation, the optical density at 600 nm (OD600) of all plates (both inoculated and blank) was measured using a Tecan Infinite F200 PRO microplate reader. The percentage of bacterial growth inhibition was calculated for each test well using the following formula:\u003c/p\u003e \u003cp\u003e\u003cimg 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pD8Mo3pLy5iLY0FGjy2mtI2xZyyNGweykQwkTxjyzMOjTB40ixyEAQ7IRFEU5ZaNNlggqS8DqhyTmHFo/DNk2oMoGQvImzzEq4owc4CVt/nCil5aY8Z8ID/jqZqmtXceMnmaBywGlY/kulk/9dRTJ31hnMLHOOYZsh2lzbB33AV51F/qTxe0Vz8hUsi4cJ5J2CNOxx9/fFJX4xkmCLS+g7cxjTQgo562uswxFg8w0vd2TfQDQlKvfRYTiJX5lZdJm5Adt9HpJ6JKzFs8uXRAm3gWHfqvl2dLYdLamtMXSA1sjjzyyPJPnsGCftIPC0H6bf5D0uih/oKvBYH08BbXWCDNlWtOXnPNNcstOuPRoohOWNzQXwSaYUai9RG8EHjl0VHjz0JkzjnnLHcnzL8W7OpqvImv/5Vfa3/MufTXt0YE/pwKCKE68PpwDNCLsZt23hByui0v35B5Yx+u5gpjjffab31FeD+NN2kaFXpKZ5AYukSnlN9o+ojXMQiYCy3K9bXx4tkxOTeWC/1tLSZ7RFqL1xHf20QgAHvqewAAEABJREFUFcgQeTJyBpJ3Xjis3GDxuyomlvzbJGsiNgnksOae8ud99N2AsdIzgZlchbVHrFptj7n13atXr/5ZKc8EaLVIfNA2A9dK1GRikvRbW01W4vhmomO8GGiGhPfG9g3jZhIzYSCQJmtGxyTZHQRKfTtbeFi1E4FpSXgaOrsutfnzHjF6FjN0WX8iE0gX0sDLV5vGb4aN/uV3kwg94JWi17yCCJUFlr6ee+65yz9BQ094/oqiSEiH9ITeWUAhQQyKMIJg2aKid/RRPOHE5JV/qw9jb6Upf9+RTX8JwORhW5GhFV4V+aovo18URbmyZdzUkz4z0jxvtnDhI600vIS8Pn4jRXCyGMr965tyhSOE4lWFviPuSBzc8jdE3uIMjrDI4Y08LViJxRvcpEFMEbveTbsLxitvoG9V7L0r0zzEkyddFhh7d6sSgfJOzEeMt7xgp1ztRDIzBhaWMETYtZdHWX7DDz+8LErhNUNEzWllQM1/1IkX1OIBiZI3XdPHjBVvHI8yMbfk5OZi81D+PTBPeke/6AOM+vbtW/6FDMRVPQgCqZ+NIXU1X9I78ekN8t2WsuGqTGnomzoYZ/IriqI8U64vjFu2g76bu22Tm2/VyZ95QuSQLLqpj0hq+qfW/uifTPiaPjf0r7mc15rnT99IhEQjqbzKeZeADluMqK845hPf9bl6El53ukHEaVQsDnixLRSMbWMS8W40fcTrGATwAjpoUWl8W2x1TM6N5WJXorWY5sDq3NVa/PZ8bzOBbE9h7UnL8Bg0tlnak4+0tqgIg+B3Vaz0rZ5tUVXDW3tnVBgt4t22kM42eTBkffr0STySzkuYJJED565ay3dQ/G4LzJYbT1ZLwlvVnvYxHIxdVXjkWsqT4eXVZvgQGWlNzjylDCMPU0vp8zeGyaLJNhjj7dzdnk1b3LxPyAlDKpzR5zGiA0VR5OTlk64xRIxeGdDG/1iY8FhlXeUJswViEYPEVglGo1mrr3Ni6ttcGgaYkbTNmPuXl49+2yZECmrT2r6Wd+2YM6aNbcYfFrXpWvrNI+S7RYBnPUFqiqIovZGp8o/+Q1jgVAlu+FVbEBrbphkDTwTcWKdXCHFte1srQJ5wQNRsecqTwE/ePMdICfJG/1rLrz3fGUrkjpFUB4KQ87IhTXSAp1R9LfAdFVK/9pTZWlr4GFcWL+pD1Akhgw/C3loebf1Ov4wpHmpeZemVadueobZQgwEHgZ0y34n5xBlrdcxC33inLcjEaVTYFZhrowUFQku/Gk0f8dqPgH7T53TdmDA2LJ4cmWh/7v1yYJP6vdX/r2NJ5hRSP0ZK5jXzT3PfOzJ8kCGQjCRjadXZRgAGiG6FS6zoTMTVCMqwNYD8VcMbfbfCtUpBZChaUfQjDSY+eZiMPIuiSIyC98FNtJFxo8gtCfLQnrabVHl9qsJb0Fye+tZBdIMfATAZEzrAO2ZwWlUiQc3lkcMtMuRnSwqhYmxta+l7XkgGwxaxgW7BgESagHJ6T+lNGIig320ViynpeVB5EJEOnnBHPxhYHva25sk482TZmrdFU02vjQS55GXjhavtX0ZTu6rpvMMAFjw46iosi21MZFpbclgjT1vCPFMIW7VMYw3W8kUokGrewmqevhmrtier4Y2+83AVRZEcTYBZxoHeIxO8APTb+WqEq5ovnSPVsPxeFEX5t3eRY3qV8/UUR3vl6x1h9+ws0V/qqS7e1YEonx7A2bEO28+OXLi4whvcWfWRb1EU5eUe3kB6qD5Z1Alm4nW0sAmIm3GBPBZFUf7dYlvJvLE8/3YdqvMPnBwroWu5jp5wM/+0pY70mb7TJ4RVfyjPwqst+UTcgUPAfG9xwOvrrLPFuhv8xiPHgX4wnwxc7v9LZfyYK3ED80j+YmeHzjgOZ/fChTQ6YD7NcTw5QujqwPIXebRFBhkCadVtoubCb7SBtUYjp+P9Y7QZMoMxh3taIfJOOFPjd1vFJOb8nzOQtgpzehMLo6NOyiW2UfP3eNZHwGTLiNX7iqAjg1UxeOrFFWZw2eqqt/p3xsuEbKLmSdE/0tQTBsHiA9FCQBzSRzx5BAx+F8qcu/W37xAKBA85cpa3mh9vKL0YGE+hfBAndUCktcvxDp4J28++I3OeRHsameAYOJOjYwjqb9KSXhudvURaeJ2I1TjPo3bL39hhSL1LUxXjSb7InHrnbyZJZFS9eXhyeCNPnm7efkbVgo2uEAbdJQ714BGDA8OvrJwvQm8egF0Oa8vTOT4k0RY2QqEM6c1Tti6RG3nTJ7d8zQu+w8/uhDb7XRVYM0g8suYQWPKsiWPHhOfc/GHrlN7whknje1WqZLoaXu9dfvXChRlLjJV+V2dtpPuMli12ZfPAMFa8+BaOdEFa4rtnVdSNLlXDWn7/41f6yXg6w+4csONI6sSLzwNoMfjHFI3/agkLuVioapOzjRZn9NWOkvlJH9c6N8wn5hwY6Ue6aTHh2AT9k2ejYtvaGGcDHW1we5894WFvNI+IN3AIODPr7LK5xHnknIsFBVLpWILjaI3qnnmJ5HyqT5zB2WdzWJ4zPM0rjlmZJ8V3/MncZ/71m9BfCyvzDv0U1tnSLIG0NWc1R7znijiL4x0jNxkYFCYEg8hK1TfeQt+950OfwNVAwGHrvhEeGxNTnnhMvLYFdArDIH/G2krXKtdtPenkZxCqg0lEGOG6tQVne83ELkz9iDM9flsZ2v7k9WAATI7ayZvACLtZiQiIy/Bom/Tqorzcttxe8bKYHEy6SENR9PM++uZPpehwZ3esJtSxLWRYHkOi0CU6wttl8h5YDBgYZ+Nsr1q51eZjYrayVAbjhIAwTPTRVgXdQFKEObPIY+nMIxIiL54GFxsYekTF05knRlXfL7nkkuX/qcAfeaW7dIdhdnbQgkUeJgl6qK7iCCNIKMNJn40/cegsA0p/kTM6XRRFEgfRYGAQVPkgNAwZj6JndbxpD4wROxOlfNQVeXBBBUmHm4nSuDQxObdId62SjSNG1MpYHGMKWVbvqghTVxOklbsxbTwZb8pHuLVTGnUhcMjzh/BagT2yrh62CtWT8ITx1sjPcQJtcPEFyVemsuEnrfbAk9fZ3IQEwyyXZdyrA/Gew+HjDC+9dEnCRQ7eaN445/cQPLdtebUdb1A/3mEXLLTXGVZ5qaPfCIJLIPTFdilSBi8XQuSrT5BP5MmFDr9h56IJnbDdzejoW/qb5zplVEX+yqQn8ufFNn+qA7zpbY5P7x3F0Ebt0kaXx3i3zWV0EolyLpPOOf5h3pW/8WMhJi/jjSBeSJ6y2AB4swkZb2UjWEg3XZVW37A/wsSlR3RPP7r9jbSpkzPXMFNnukOk12+eypBemcoWRuRn3MGC8RXWnPgfGhjLMMyEWT8y2MaE92pa+kVH6aN+VE+GXzk5rjGnrjAxFqXP/QELCwph+t7Ohr6GibYYqxwhvod0DgJ4CN7hImStw4f+61P8wJEtR7LocGs1oc/E/FwbX570m/5bwOtrF9bMS/L3Xf48lcY+pwRbQo/ML5wHa665ZrmLIV5nywAEEpHTAB40iky8CzMI8k1MlTWxufFogqbgBqGtYfHz+T6smWHBnE0qJmFGh/E0iIDEALhRZ5VmQgIUEueWod8MmXdhJiIDUH5WXwYexi5vYAHVoDPQnHOzlWjQm1AZUsZWPAPeBMwbZVI36Zt4GWV/gsYkIa4D5NqmjdqqzdouDytBab0TK0Tl+VtmJlFhWRAXiuCgtRuE6sZo5u899dmd9YItcsWwMbAmYySurXWiL7C2GGBo9aGwaj501ATNS6cfGSZlOueF9CEiCCH9RkToBYOa8xDP4oFHkG4xZIyl73SLt4j+IAl+My48py5UIKnKtwVtgWJsIBzGovS8XbalrX7pJuNNlyyq6JH0vFY8VsYrj4ffDD0DaczwVjjgjwjn8UZXETrkyjiy6PHOU8gz76C4csRDatxkVh9kG6GAj/TOACEu2otIilNPeM7oP0PPc4tc6Qdt400siqL830oiocgQcqPt+qJ2os350xHjFm7ajQwgkRaaRVGUN31dZkP24GE+MX7hby5D9vWJ+cVKHwniWVAeXYMjI0K8C1M2zPWdhQNvLAwYdWQRVuYP2+cWG+YfWMHU3Gj+y4aAXsECxuZAfQsLempe4+1Tpn6mP/qS/tAn26fmNHMYg4Pg0G3kEsbqWSuIrfLpAQ+qvJSnHHibr6teDcSMDkjHiNIvxFg52qjvfEMOzbvqqL3qY7won+EV5viI/jQ302O2wLxtLkeylG1x7TuizB7I27lidUOWeMYZbXgxosY0R4LjIuqKdNFBeqZseRpbylCWMpUtT9/pBkIpHWIurCWhR/LKDgb6xDNFvFfTWpwg1+wEh4HFm7rRP+NGvxpzxp5xJ0w92Br9AQtzif7gGDHujEljGt50io2qlhnvHYcAUghv8z09c1Gymrvv5iZh9Oqggw5K5k7zq7B6Qm/1GQcEXoHwmbdy3KIoyjzkbb5gb3ASv83/OZ75B9fBufAyO2j0ze5Oc2M/p+3I5wAE0qTiYDjmy5gS78JMaiZqYZ48hLww3oVZQZk4DHIrRGGMoMFhUshhVoUmX09hRBm54QanScoEgt1bLfMOMYIab3Kp5ieeSdg3E5tBaIvF5GPwylsZpNq5jL76m/itcHkHGdmi6Oc5FFebtEMbxdVm7znMpKhcwmAY1CZWv2vFZKaDKRsDlo1Ibbz43Q8B2Oo7WOs73gMDtN/Xxv9LXxAA+ehPuiOsmgMdzf0qnne6I45BzsDRbUYSoRRWFP30BBFCPBgJ9dW/PDIGv/TEuEK6GCt52KJEGIqiXx7V8tXReJNG2qIoyv+do20UBpyYUBjjouiXviiKhDjQY940JMhElY0T42jbFI5EPbUPpsrTZmHIRVH0K8/EKRyBRx55PNSH0HFjDmETh3E0iRVFv/qIU0/UmZcKRjz5Jl3evKLol06d1EMd5evdWLZ4qJcfo8p4I/3SIHS8Q0XRLz9p9IMb6+YJ8wlygQSYhLXJglV/Kw8WFrXKo2vKly/xLkyepCiKlLcppYUFnIvif2VrLy8Bz3bfvn0T0gc76YndCvMbL5S5S1hRFAkm+li++pC3rTpf0A0eb0ZIuyw4GBoGCmEriv/VQZ5ZkA9Enw7RR+Vol/YpC3FG0HJ8GPnNawsbT2SwKPrlb4HHk6L+yJU5VXtgaP6VDxLE46tM5NpZ4Yy3co1H7VO230Sd2INa/YR/URRJHyOE6mxMIdF0wVhszf4oWx3UjV7QRQvCWgLoe60gfOpaDeeBt0CFVTXcOwzMY+ygsYk86jvftEU7tQG22lqtPxxgAn/OExiqu3icJ8abfFqQ+NQOBMyF+kc/EO/V7PJ3/UfEMcead6vxqu+18xu9sHitximKfvOvhYP5iq2wyCuKfmMuxzWPmPvwFB5Iu06ZI+U4nf0cgEB2doGRfyDQUQggaUi9VRhjiozYHkX2kNSd2H8AABAASURBVC1eGqs7xoHR9HfmrNyRPd46nj4eFIYaqTHRI4cWErxaPHzONiFzVoMMNI8PYmUxxVjzOlo08GYjOgynAc8DbwJAVnihEBsGzx8C5v2xkOC1crOSZ8cKl1FgTJFdngteCF4KBpFHzlYFkmI7g+fO1j7ihLjyVphIkEeEj1FFLCzSTGo8OLZNLJYYe4YJgUNOGHZEhIFDknkTkVweORMY8sNTZwJlwBg7k6VJs6P6MvIJBAKBQCAQGLQQCAI5aPVX1LaCAJJoy4y40JHFKj+LM4a82Vlsb2bhSXC+hVgFZrE1QGxp2xrjpcjCa0Oce+NN4hkgPOpZHLYn6623XrJKtYWWxZZrFmdVsvCIZLEVZ3s1i+MjWXg6bG0SXheeRuJsFbGdQax0bXsSq1TCQ8kDRGy98JxksR1va4/YerTVloVnjNhCsQWbhQfSu2/iSis/+StL2eqknuqtDdqkndoNB7jACnbwhC3M4e/Goz7i2dKP+lQf6283H2098x5aRNiKtICwhUwv8sIhLxrygiEvFiwULBJsZeZFgsWBc3sWB7ZtLQwsCnjU8oLAYiAvBCwCbEXlBQDyzxOL/Gfij/Rnwu8sIbKfiX4m+Qg+DwJyb/cEuUfskfoqoc9kngcKkUfiK8MhXgOBQCAQ6FIEgkB2KdxRWEci4OwTzxhPGa+ZrSheNR42BpkHjpFmsBlvhpxBtw3H0DP4jD8i4GgCcoAoIAzIAw8jbyMPJKKBdNhqduOOJxM54eVEWnhAERmkxh+Sd94OaUWAEFOkCEFClJAmBApRRKoQQKQP6ULAEDhkzfaobW7ncGxLOgNm28t2hi14Z95sl9gitN3nDIwtMNuIzlPZKnN2ynncvLXBe4iAICO8jLyOPJCICw8lMgMznkxkh5fTFiUvKGLEO+oPOfOe8qQiU3Djbc3YIV08sryzyBgMeW95cW3n8uwib/Dk+UXqYIrkZVx5jZFA+PIo8ywjiIhixhmJhDVCiVgimDC3QMiLAosA5D8TfudMkXpbvs4q6weEFrFFcpHd3B8IsD5BthFs/WI7Ud8gz/rHVrgzzfrIeUZb2LY5c1/ZatVfzus5wmLL11EX5ykdg9B3tpGdB9d/zlM6wpP70RZ3r169yv/PtEtMtqBtZdt6dx7StqmtW33smzji2zq1De9MpXO5zmArR3l0Rh3UxZEd9XNm1NlT9dcOfxmA7mmjtlokaD/9tECADZ2FF9zg55gDTOm3RYGFFdzpvjFgLFgM6Cf95Wy7PrT1ZhdB/xpL+tz5YOPLbgHdoCN2COwOGIv0iE4h/Map8UrveP9t9dJJY9rYpqt0loffuEfuefR583nyzQ08+OYTus9rz2OfCT0yb25B5o0ZF3LMO0HiO3I273l5RY1aRyAIZOsYRYweigDjyYgynogSwpSNp8sBjCeDXGs8HYZmPBn0bDwZfIaf8UQGGE/koGo8kQjGkzeP8UQ0EMCq8URMGE9EMXvTGE+EhvHkScvG0w1XHjTGEwFlPHnPqsbTNjvjiWjVGk/EjPFE1BhPxBeRy8YTwWM8keSq8UQGGU+EmvFEFLPxRLoZT2QSqUTIq8YT8WQ8bWfzgBHvSKmtbt8ZWWd7MjllgHnVEHv5yp+RdibQdruyGXDb8QireqkfI8/YZ+KqDYiAbX+eP+1DEhwPcFQAcUAgHCPIBBY+SAbvYksE1lGGegRWfyCwyE0mr/pM3yE/SJD+RIqq5JX3lBcVecoLB3pRJa8IF92hQ9mjzGPLe0zHMnHl2eXhReJ4fJ1BpJuIK6JnceE8nMXF1FNPnZDCvLhwBjMvLhBTpBIpdS7UmT9jBwF1nsow15f6Mfehvtd/+k6/5T7L/aWvqosyfUTf9E/uG/2iT5A8/UFv9UVeSORFGbKINFo4wB3mebFgoZBxNo6MJ4sDBNVYgyuPdsYUnrDMOMIQ+TeeYQc3Y92YR5wRfSTfvJDJ/dhjj50Qe3NIJvTmF7iZb+BmDoKdp9/w9E2cPB9ZzFnUwT7PSRYL5iV9g9RbTCg3E3sLRP2Y5yd11Mfqq7/dCdCG3r17l+dwtYtu0BG6or30xwIIBnQKHpnwu6hj4UQnjR99HxIItBWBIJBtRSziBwKBQH8EiqLfH75G5HnFeMeyJwxJQVZ4wxB6RhiJyR4xxtOf20FyeMUQe8Snn/GcIjGeDDtilAl+1Xhmko9Q1RrP7Clrznjy/iIbPGZIP68Z72T2mvFaIijIPzLIq4kgIoqIDK8n8ohEIpSIDoKJ9FgIIJ1VTxrPKoJqQYCsIk08ajyxiBSPGk83rxqCxXNrYYAI58UBD29eIPD8ItA8bMS787XEtywWE1mQbOmJvLIg3RYfSJ6ysigb6VMPYlufIIHqh5hnQdCJ+iOHWbSLOCagnYQHUbuJRRFPNIGHhRKBD7F4ghcvJIEfQTYJXInz0MRZ5ywWYln0RRZ9k4Vnm+g3YiFHLOqIfiW8pPrZDgOxgCD6nx7U7jjQHwsHOoXc8nTTOd5aC0+Ejm7SU4SP/vL20mkLWHqOMNJ9xNaYQC6NE4TT2EFCjSnkFLFFWI09JDbvOPCSWtzxmloU5EWdBZ1dGgs5i4T+AzpeAoE2IBAEsg1gRdRAIBAIBHoaAkVRlP/PaKQhE3kkIpN53jBb2wgGosEjhtQjHoh99oxlco+Y8I7xvvHCIflICy8ZApM9Zcg+jx1yw1uG6PCAIv22xJEfxB8RsjWOFPH6Ze8ZDyrCxCOIPOWFgCMCPGk8hrbNedMQLR41OwC8arbQkTBeNYSMZ43XkTeXd41nl4fNIgF5453MCwVeN8SO5xLJ48W0W4D0WTTwHFs08HLads+LB7sH/nqGBQTCaBHBM4pEEoQSuSTIJtJJEFBklCCnWRBW5JVkQuvJ+4rs8sSSTIId60COeWmzIM8EmbZAyYJsE+QbCc+CnGdB2oXbWUnxTyAwEAg0SyAHIq9IEggEAoFAIBAIDHEIFEWRbGUT29nIPEHoie1tHnqSyT2Cb7ub15Bkop/JPsLPi59JP+LPo0/yAqC6CLAQ4JG0TU4sCGy/E15/CwOSFwcWCN6VneKfQGAgEAgCORCgRZJAIBAIBHowAlG1QCAQCAQ6HYEgkJ0OcRQQCAQCgUAgEAgEAoHA4IVAEMjO6M/IMxAIBAKBQCAQCAQCgcEYgSCQg3HnRtMCgUAgEAgE2oZAxA4EAoHGEAgC2RhOESsQCAQCgUAgEAgEAoFA4HcEgkD+DkQ8egoCUY9AIBAIBAKBQCAQ6OkIBIHs6T0U9QsEAoFAIBAIBAYFBKKOQxQCQSCHqO6OxgYCgUAgEAgEAoFAINB+BIJAth/DyCEQ6CkIRD0CgUAgEAgEAoEuQSAIZJfAHIUEAoFAIBAIBAKBQCDQHAKDXngQyEGvz6LGgUAgEAgEAoFAIBAIdCsCQSC7Ff4ovKch8Ouvv6affvqp2Wp98cUX6cADD0wLLLBA+uCDD5qN15kfPv/883TuueemscYaKz388MOdWVSPylu/nHDCCWmyySZL/h/B2223XYt91dbKD07xf/7553TPPfektdZaKx122GGDU9MGaMt3332Xbr311jTttNOmk046aYDvnR3w22+/pddeey1tvfXWaZxxxulfnD4455xz0vzzz59uueWW/uHxEggMLggEgRxcejLa0SEIXHvttWmPPfZolphcc801SZx33323Q8prayZI1NNPP50uuOCC9M4777Q1+SAbH0nYYost0ieffJL69OmTtt1223TyySen4447bpBtU3MV//rrr8vFicVMc3FaC3/ooYdKfC666KLWonb5945oX640jN58881yPDz33HM5uEufn376abr33nvTmWee+YdyhV111VWpu+r1h8rEj0CgExAYqhPyHMgsI1kg0L0IICm8NX2aCAqPQr3aLLvssmnOOees96lLwv7yl7+k2WefPc0999zpr3/9a5eU2RMKQZovvfTSst0jjDBC2nLLLdPRRx9d/u4J9evIOjz66KPJQuXHH38c6GxnnXXWtNlmmw10+s5M2BHty/Ubaqih0kQTTZSWWWaZ9M9//jMHd+lzuOGGS6uttlrpFa8WbJ5YccUVq0HxHggMVggEgRysujMa0x4EbrvttvTkk0+mxx9/PN1+++3pl19+aU92kbYDEbBV/8033yQEWrb/+c9/0hprrJGmnHJKPwcb+fbbb9Pll1+e3njjjUGrTQ3WdnBvX4MwRLRAYLBAIAjkYNGN0Yj2IoAsHnLIIenEE09Mww47bHlm6csvv2wxW2efeC3Fsy0nj2oCHqSvvvoq+c5wkur3anpx5GVLLsfx/sMPPyTEyXkqZcivthxxpPfNFndO7ykP5fru/GZtGeLLX13lqwxx/Za+KuL6Lp96ZWnP999/X7ZXHt6r6Zt7r+Yrf79zXHkKc+5TW9RV+fXqV02TMRFXGm3L3z39hovvua7K8o34rv7a6V0d5COOsoWrDzy96x/pfJdOnvL2Lsy3LPKTl+/iqas48rjpppvSqaeemuTrm2dOBxf1kE565edvnvIQXzoivvDWRLrcJnkrQ12q6ZRVzdu7sBzHu3bAQvu8q4O8/BZPnvXal9OKry76RTrv0vkOR9/Vr7ZscVoTeeT08pCf+njPAlP5CNcO4XARRuCpXsLFlafwtgo8pJeP/LRHmW3NJ+IHAt2NQBDI7u6Bjik/cmknAgwb4rjYYoulJZZYIl1//fWlJ7KlbHkp119//TT++OOXB/idx2MMpHHBZvfdd08bbbRReZFh7bXXTquvvrpPpYhnm9LB+w022CAtvfTSabnllkuXXHJJSR4YFFt9m2yySVpkkUWSw/iek08+ebrvvvtS/sdWu8skwtVj1113TdXzmVdeeWVad911k/K1a6mllko8rYzfxx9/nI499tg0yyyzlGcJnZdTh0knnTTJkxHN5bz11lvpmGOOSZtuumlaeeWVU+/evdOOO+6Y5CGO+sp33333TRtuuGF5cWCttdZKzz//vM/NyksvvZRcStLOVVZZpcReHn379k0IBCO7yy67pAsvvLA8l7rnnnumNddcs7wgUi9Txtnlkb333juts846afnll08LLbRQuuyyy8r00jizBs8ddtih7JMll1wy6ceMq765+eabk/pMMskkyfscc8yRZppppvTCCy+kvfbaKwm/6667SpzgDmf1feCBB5KyYTDvvPOW+fNow1vZ2nPWWWeVZzjhI856662XnnrqqfKs3HnnnZf+/Oc/l1vY+s05T+ky/ttss01yjIIuZEx8z/jLk2fWlqr+8q0lUS/HAw4++OBED7VZnY488siEyEnrefXVV6edd965xFT5q666aqmrSBWS9dhjj5X9PtVUU6XrrrsuHXDAAaVeTT311OX5RPWjC7XtcymKnm+++eZp5JFHTk888URaaaWV0hRTTFFeTJG3sUiv6bCyfYe3b+rXiLz//vsJLr169UoTTDBBeQnt5ZcGf2XeAAAPzElEQVRfTgsvvHCyBe1iln6T17PPPpsWXHDBpC36Xlhr+IvTiMDLGWrt1VfatGaTPt99992NJI84gUCPQmCoHlWbqEwg0A0IIEo33HBDSbSco2I83PJFrpqrjssczzzzTFp00UXTfvvtl2ypIiQMrTSM3uuvv54YSGQFQWREfSOMyPbbb58Y4tNPPz05gO8GJ4JmCxMZ+dvf/pbee++99Oqrr5ZeSOSB8Rx66KFlUcojjzxSGlskzC3Uo446qiSFPEBIrMsmY4wxRmkwEUR5ISW8Mbwwf//73xMCgcj94x//SAgagqjOyJJCtBUh8DzooINKMoaY8dYqXxyXNpAOBll7jj/++HT//feX5/A++ugjUQaQzz77LMFAXXh/nXHcaqutknoeccQRZZsZfG1as8nIOvMp3hVXXNHs2UekQBwE5Pzzz09nn312GmaYYdJpp52W9DMDLn/9hKDJC9HSNiRSnyKh+t8lCDgS/WJx4R1uLjAhhvrVImGkkUZK0lo0zDPPPEl/uinvSAT9yKRefegaAqG9hx56aHmD2C1ipNQiZMwxxyxJ4sUXX5zyxSFnc//0pz8l8YWLq28z8bCYoR/IsHa7jUwnBwC9JuDzzz9P+++/f6JT+hM2iDJM9A+CaWGgXhYg9OKMM85I2mvxYxEkDj1CgqV55ZVXynPCdAD2CDNd1CfyqbYPDvrVQghxp3OIHiLruAIdolfOEyLMyvfXB4w1Z5VrmtPsz1FHHbXcXZh++umTxdZ8881XLgLoqnEz22yzlQRZBoijm9MWMuLRjdbwl64RoevGNyKs/5Ux8sgjp+rc0Eg+EScQ6AkIBIHsCb0QdehWBHgcbCVNN910qSiKNM0005SkjCfF2bt6lWOcEZAVVlih9O4hVv/6179Kb5r4vFwMKvKElDK+E044oU/lDVueGIaa4ULcRh999LTPPvskBAXx+PDDD8vzfc748Yy6FLH44osnBIUhLjNq+g+yy9OG9DHsjDPD2rfJg8cgy48XiIFn9BlS22fClclQNmVTtlcd1QkBQl55XXx78MEHE88aj4k85MXDhjSqF08QkuCd+K7MGWecMfFMiSefWmFA5c2YjjjiiEk6ZSNqDPvA3F5F2BEPRNbThRvESDhSiOAgV3DndXIJg8cVeUTyEOh///vfSTv0i76DMe+x2/nalfFHLuaaa6600047lfGRDMROGFLkcoc/94QEaQsvGHLLezjxxBMnZfP+3nHHHaWnkuexFiO/EdW333470TV6h5TpN/0LJ6TkzDPPTOOOO27pxVZv3jyLE+lbEgsZ5E6dkEjt3W233dKNN96Y9Ilx4cY/3Z1hhhnKPhpttNESjzGiDS/1QMq0Xbtd8IIB0keQVFKvHtqMsNFF3/U9EszjaOHDK6i98hNXu9ZsWkzAgUdUmkaF7m7Y5B3v2zQ2iHTjjDNOuQjkTdZWYfCgO/rXOGgNf2kaFUQRwba4hB9vqPbCvdE8Il4g0FMQCALZU3oi6tEtCPBI8d6MPfbYJXnkbTCZ81SoEE8YD4v3qjCUCKMwniHbfrxliJ8/K4J0SCcfHh6eRE/xkQGeQGTO7yzSM768MfLI4YyZ8vxWN8TDO2H082/GlWdQWciKNikL8UQAecqQGemyqLv3ar7yE67+jJy6wonRFpcgYwiY8pEyZJOXcvjhh08E+YCrfN9o5kKIbUh5IQieBPlB5Bna2rr63prw5KkTEVdbRhlllJJcaZN68gratkRIxCmKIukndYWTPhROpEdsxfUUlgXRLIoiIRnyvvPOO0tPp7bDgPAGItj0iocSSfG9KIoyG32LaCK6RdEvrPxQ+Y8+5OmDtzyJbW9lWvwgQy+++GJCSoXlpLDM7809bduLpy3iFEVR9h8yqG7qyxNLN6t5qwsiSc+ULa3v8IKj30QevOnE79ZEPeQBa4swOiSM5LRIn4WSre/mdCvHrX06zqA/tJtO255HgOWPKItvG53OWLj53Rr+4jQq9BIZ5j1G8C2gZp555nIB0mgeES8Q6CkIDNVTKhL1CAS6AwEG0BYdT6NtSh4KwigyrL7xQDRSN4StKIrypjADy/BuvPHGiacNofRnZxgtHiPbqZ61+TLcSBsCVfutkd+MejbkyuJFdZ4RsbI1yOPVSD45jrogP+pq6zaH13vutdde5ZlI5yKzML4wqBcf8UEsavNF7ggvab10LYXJEyEg9eLxMgr3HUH2TuCWSbo2CxsY8adzcturT0cPbHvLG+Fua95IBp2s5umdbvKYkbbmKT4CBovaPvCN0EN5i6OvhGXhdRSGkOWwjnzmvpI/yXkjs3nRoX45vJGndDy5CDmvqF0CY87CzZa7MnkHxxtvvHIc5zxbwj/HaeRpTnGml8fYmPLnh5yDdD4Ulo3kEXH+gED86EYEgkB2I/hRdPcj0KdPn/JMlPNVLrBkcc7MOSxGRlhrhgrZ4TW0/c17wYvBE4NU2d7ldfBuO5cHiti6rEVAebbuSO23Rn4jUIgsUZYzZrYekUhGsJE8qnF43pArhtXllOo378IQL+L8HEIuPAsvEi9Z/l19qg9iYnu3Go7MIHe+V8Mbebcd6hwdslWNrw7KQgx4UsVRTo7D04bcIRP6L4e35clj5eyrPqim4/UkPI2OD+h3nq8ch24hEHQoh1WfvHG+689quHcLHF4tks+jCm9UkEDnRp2DrRIYXlieZ2XzbCofftV89S0yp13V8I56p8MwpVMk5wsnfefykv7M4Y08tQdZROZtWxv3jjvwTCL2xqqyHFXI+Umj/c3hn+M18tTvdNNOgbIcfeERd7ZTuxrJI+IEAj0FgSCQPaUnoh5djgAPgMsCPBK2sKoVsPXmggfPh200k371e+27rU8Gdccddyw/MU7CeAOdmePVZIh4fJx7ctaQgUZUywS//0caZDOfCfs9uKGH9vg/Xzh3Z3vcRRG3fr3bZvWdyAwZQNK8tyS2E5FiJMIZQNuw4jOELrd89NFH5R9Qtu3sG9KNyMDNVryLHVViIm0W5BZxc5GFl1M4MiWdiw3OJgpri/DmIKQ8PMiatAid7XJthqstfYsCfeE7QaIYcFuLfg+M0CNEgxfS1qS2IKouzlgYwJG42Yto+k70E8yKYsAtbJjSl5FHHrm85GWblZ4RxytcfvKd2ELnca7WXR/rq2pY9X3JJZdM6ub8LJLrm74wLiweHFVwNlbedNN3onykHN6234UNjGhfc+kQfVjaqnZZSFvE5RFFcAemr4qiSHSVbrmIY5HiSEAeM87KIqYIubIIbFvCX5xGhb7TRRhbRPJAKtuxEzsGjeYT8QKBnoBAEMie0AtRhy5HwDYVAoOsIFU8GtVKIEA8EgiH7W0XXGzHIoQO4/NS+NMniIfvLlD47XKGfBADW8YuxEjn5q1tWZcVeOsQTYZsyy23TAwKMuMyhnOVLigURVHevma4eUsQDARI3oSHknfGZQOGHcFVnksotozVE0mVxm1Z5TkDiUjatnP7E2FASOSH6DDKsJAX4qnODB0CjBwhGs4KMrouyGi7PwuEZCoTOXK2zDdbdbyBvFO8XMqoFd9daOFFc7tYfbQXHvqGYUca9JHjAAie+tq61T+1+fnNIPMouTiD9Pbu3bu80Yww9OrVq7xt7Ka3frWtbAtY211ucRmJMZe3BQACSrwLkz9DD2/viB+8vBMLDpdvHBdwVEA/0wcE2iUU2NMRuuH/UOJPAzk76yKMyybi6zf96pKVSyIuIdETl3jUwZ/ngYtFAeLt8pT4yL1zgc7V7bnnnuWf2KGzykIqxYWfelZFn1ncuDSjny1e6BBSqAx1hoswl6iQX4TceV7ePzqrj+iC/9+zMhBLuOkn9a/qb2376LX4+li99H2V8LqFrg9dplG2sYTs0n8YSkNXecLpurFIT9XJt3qCDMLeWIOrOPQVDkgzb6SwLK3hD2Pla7N5xPjSV3TDGFI/Y8pvumDxYNzCUVzeX15R9cllxjMQ6AoE2ltGEMj2IhjpB0kEGEyTPQPOW8VjVG0Io8YLgwAgT775UzA8k27sOtfIGLoV7c9yMOAInHiEMUYE3XLlKeF58M6j47ttYQSEIWHc/akXhMffo0Q0kTmkBilALtTXVpe0xBYYbyPD7s+c+JNDyvNnUrIh8idmhCMJvB08Vv7cj5uu/uafc2Dy1D7GjPcF2WAMEV0GERlBIrTBhRCkSz15hpAhdSHqrO6INJKDdCBBiKvvzQnMeGHhY5sdUVRnt4ylcVZUvZArf6PSVrpbwkif77XijJs/U+RCD5KAONom9OeWEF3xEQJtQTZ9gxkioa2+yxtRZdSJd2G+yRtBgRmybIEgPAvCoD/kp23IDuxgKI46wR1B1rdu+irfu+9ILs8Y0oR4y0e4PGy3IjlIIwJHB7M+8djSB5dr3PLlpRNHfG1EinId5JfFMQt4ahdPo3rQZZ5Fui6eMN8RUmSXriL/9Nd3pFo7tU2/OcqgjXRbf1T1t7Z9FiDGn2MDMDWWePzlS6RXNiIJa+cHLUoOP/zw0vMtjuMiCKj60yOLB3rjW3NCl+BT/U634UyXq+HehTeHv/YjhXRFW+krfVFfY8hYMqaMLURVO+VpbDh3SReM56yfvoUEAoMCAkEgB4Veijp2OAK8cQxCFhN/tRCkC2HM3z0ZVnFsRYuPQCEXyApSglD6ThADBkI6wuPnz6z4loUniefOWShGEuHI5I9BZQilzcLzl9N6KpPXy3dkFxkWXhVEgCHjhUQiERMGzrlIl4akzaJ9jGj+7Slf+WkzcoAoMPgIKPLhWxZ1r7bHVnH+1twTSWF4laM8hNwFpBwfQUKkfcui/v68UY5T+9ROXkYEloFGspVTjWebEnGSp7JdckJSxZG3Mnwj3oX5BiNhWbRXeBaYwAZGsIIB7PJ3OqLfEKALL7wwIUT+rmH+zgPGgyq9vsvhnvoXua72p/AscKOTvutbBFR8ulqtQ46fnzB29MBWO11E6mqPdNBHCyftPuWUU5JFAjIkD95AZMy3LLyD9fS3tn3KrvavOiD78s2iP+GsXeonb17X/F37crmexp188/d6T/2P2FW/5fFYDau+N4d/bfuzvqiz+mQxtmyNO9YhjnDtqdWRapnxHgj0ZASCQPbk3om6BQKBQCAQCAQCgUAg0AMRGKIJZA/sj6hSIBAIBAKBQCAQCAQCPR6BIJA9vouigoFAIBAIBAI1CMTPQCAQ6GYEgkB2cwdE8YFAIBAIBAKBQCAQCAxqCASBHNR6rKfUN+oRCAQCgUAgEAgEAkMsAkEgh9iuj4YHAoFAIBAIDIkIRJsDgY5AIAhkR6AYeQQCgUAgEAgEAoFAIDAEIRAEcgjq7GhqT0Eg6hEIBAKBQCAQCAzaCASBHLT7L2ofCAQCgUAgEAgEAl2FQJTTH4H/BwAA//9rtmQRAAAABklEQVQDAINDI3I78CZTAAAAAElFTkSuQmCC\"\u003e\u003c/p\u003e\u003cp\u003eTo determine the minimum bactericidal concentration (MBC), 10 \u0026micro;L aliquots were taken from wells showing no visible growth and spot-inoculated onto fresh Mueller-Hinton agar plates. The plates were incubated at 37\u0026deg;C for 24 hours. The MBC was defined as the lowest sample concentration that resulted in no bacterial growth on the subculture medium, indicating a 99.9% kill rate. All experiments, including positive and negative controls, were performed in triplicate to ensure reliability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Assessment of sponge Extracts Effect on Biofilm Formation and Biofilm Eradication\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of sponge extracts on biofilm formation and pre-formed biofilms, a 96-well plate spectrophotometric assay was adapted from Celiksoy et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Extracts from \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e and \u003cem\u003eDragmacidon durissimum\u003c/em\u003e were tested across a range of concentrations relative to their MIC: MIC/4, MIC/2, MIC, and 2x MIC.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Biofilm Formation Inhibition\u003c/h2\u003e \u003cp\u003eTo assess the effect on biofilm formation, 100 \u0026micro;L of each sponge extract at the specified concentrations (MIC/4 to 2x MIC) was dispensed into the wells of a 96-well plate. Subsequently, 100 \u0026micro;L of a bacterial suspension (1 \u0026times; 10⁸ CFU/mL) was added to each well, resulting in a final inoculum of 5 \u0026times; 10⁷ CFU/mL. A background control plate was prepared in parallel, where 100 \u0026micro;L of each extract concentration was combined with 100 \u0026micro;L of sterile Mueller-Hinton (MH) broth instead of the bacterial suspension. Both plates were then incubated aerobically at 37\u0026deg;C for 24 hours.\u003c/p\u003e \u003cp\u003eFollowing incubation, the planktonic cells and medium were discarded, and the wells were washed twice with 100 \u0026micro;L of phosphate-buffered saline (PBS) to remove non-adherent cells. The plates were air-dried for 15 minutes. Subsequently, each well was stained with 100 \u0026micro;L of a 1% (w/v) crystal violet solution for 30 minutes at room temperature. After staining, the solution was discarded, and any unbound dye was removed by gently washing the wells three times with distilled water. The plates were air-dried once more, and the bound crystal violet was solubilized by adding 100 \u0026micro;L of 95% ethanol to each well for 20 minutes. The optical density of the solubilized dye was then measured at 570 nm.\u003c/p\u003e \u003cp\u003eThe absorbance values from the test wells (with bacteria) were corrected by subtracting the mean absorbance of the background control wells (without bacteria) to account for non-specific dye binding. The percentage of biofilm inhibition was calculated using the following formula:\u003c/p\u003e \u003cp\u003eBiofilm inhibition % = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:100-\\left[\\left(\\text{O}\\text{D}\\:\\text{o}\\text{f}\\:\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}/\\text{O}\\text{D}\\:\\text{o}\\text{f}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)\\text{x}100\\right]\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Biofilm Eradication\u003c/h2\u003e \u003cp\u003eThe biofilm eradication activity of the sponge extracts was also evaluated. Mature biofilms were pre-formed by inoculating a 96-well plate with 100 \u0026micro;L of a bacterial suspension (1 \u0026times; 10⁸ CFU/mL) and incubating it aerobically at 37\u0026deg;C for 24 hours. After this incubation, the supernatant containing non-adherent cells was carefully aspirated. The pre-formed biofilms were then treated with 100 \u0026micro;L of fresh sponge extracts at the specified concentrations (2\u0026times; MIC, MIC, MIC/2, and MIC/4). The plate was incubated for a further 24 hours under the same conditions to assess the eradication effect.\u003c/p\u003e \u003cp\u003eFollowing treatment, the wells were gently rinsed with distilled water to remove residual extract. The remaining adherent biofilm was then quantified using the crystal violet staining protocol described previously. The percentage of biofilm eradication was calculated using the same formula as for biofilm inhibition. This combined approach allowed for a comprehensive evaluation of the extracts' ability to both prevent biofilm formation and disrupt pre-established biofilms.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Scanning Electron Microscopy (SEM)\u003c/h2\u003e \u003cp\u003eSpecimens were fixed using a two-step protocol. Primary fixation was carried out in a solution of 2% glutaraldehyde and 3% formaldehyde in 0.2 M phosphate buffer. This was followed by secondary fixation in 2% osmium tetroxide in 0.2 M phosphate buffer to enhance contrast and stabilize morphology. Subsequently, the samples were dehydrated through a graded ethanol series (30% to 100%), followed by a transition to pure acetone.\u003c/p\u003e \u003cp\u003eCritical point drying was performed using hexamethyldisilazane (HMDS); the samples were immersed in HMDS for one hour, after which the solvent was allowed to evaporate completely. The dried specimens were then mounted on metal stubs using conductive carbon tape and sputter-coated with a thin layer of gold to prevent charging. Finally, the samples were examined using a thermionic emission scanning electron microscope (Quanta 250, FEI) to assess their surface morphology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. High Pressure Liquid Chromatography (HPLC) Chemicals and Reagents:\u003c/h2\u003e \u003cp\u003eGallic acid standard was then generously donated. Sigma-Aldrich (St. Louis, MO, USA) supplied other chemicals like Dimethylsulfoxide (DMSO) and Formic acid. In the lab, double distilled water was prepared HPLC grade Acetonitrile (ACN) were purchased from Scharlau (Spain).\u003c/p\u003e \u003cp\u003eThe Gallic acid and the marine sponge samples were chromatographically separated on Agilent 1260 infinity series HPLC system coupled with UV-VIS diode array detector (Agilent Technologies, United States) with a sample injection volume of 5 ul at a flow rate of 0.5 ml/min. The isolation was performed via C18 reversed phase column, 150 x 4.6 mm Nucleodur 100-5 (Macherey Nagel, Germany). The separation was performed by gradient elution using mobile phase composition of 100% ACN as solvent A and 0.1% Formic acid in water as solvent B. This is then followed by a 1-minute treatment with 90 percent solvent A. After reaching minutes 15 which was considered as the solvent switch back to 50 percent solvent A and left to stabilize in its original state in 2 minutes to get the total run time as 17 minutes separation program. A heating device was attached in order to maintain the room temperature of the column.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1. Preparation of standard solutions\u003c/h2\u003e \u003cp\u003e4.4 mg of gallic acid standard was accurately dissolved in 0.44 milliliters (mL) to prepare 10 mg/mL stock solution on which an appropriate amount of each working solution was prepared by respective dilution in ACN to form a working solution series that was ranging from 0.1\u0026ndash;1000 ug/mL respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2. Preparation of marine sponge samples\u003c/h2\u003e \u003cp\u003eThe concentration of Gallic acid was calculated using the solutions of Gallic acid in 50/50 (DMSO: ACN) as a Gallic acid standard. 2 mg of the sample being extracted was dissolved in 1 mL of 50/50 (DMSO: ACN) to test ready solutions of Gallic acid in 2 mg/mL. The samples underwent 2 minutes of sonication to increase solubility of the extract samples. The maximum of each sample was determined by comparing the retention time with Gallic acid of equal amounts. When plotted on the standard calibration curve, the peak areas were measured as a response per injection against each concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.8.3. Method validation\u003c/h2\u003e \u003cp\u003eA method using multiple parameters has been developed with method validation in consideration. Specificity, linearity, accuracy, precision, and the limit of detection (LOD) and the limit of quantification (LOQ) of the process were validated. All validation studies were conducted by repeating injection of Gallic acid standard and the extracted samples.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.9. Statistical Analysis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eData are presented in the form of mean values and standard deviations (SD). Student t-test was used to compare the control group (DMSO) and treatment group (KSA-43) results. GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA) was used to do all statistical analyses. A value of \u003cem\u003ep\u003c/em\u003e below 0.05 was regarded as significant. The asterisks indicate the level of significance as follows: P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Three independent replicates were used to test each experimental condition.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Antimicrobial Activity \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e and \u003cem\u003eDragmacidon durissimum.\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. Well Diffusion Assay\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of methanolic extracts from two sponges, \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e and \u003cem\u003eDragmacidon durissimum\u003c/em\u003e, was evaluated against a panel of six isolates. The panel consisted of four Gram-positive strains (MRSA, MRSA CI, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e) and two Gram-negative strains (\u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e). The purity of all clinical isolates was confirmed by Gram staining prior to testing.\u003c/p\u003e \u003cp\u003eActivity was assessed using the agar well diffusion assay at two concentrations (2 mg/mL and 4 mg/mL). The extract of \u003cem\u003eA. wolffgangi\u003c/em\u003e exhibited dose-dependent antimicrobial activity exclusively against Gram-positive bacteria. At 2 mg/mL, the largest zone of inhibition was observed against MRSA (\u003cb\u003e1.35 mm\u003c/b\u003e), followed by \u003cem\u003eS. aureus\u003c/em\u003e (\u003cb\u003e1.25 mm\u003c/b\u003e) and \u003cem\u003eS. epidermidis\u003c/em\u003e (\u003cb\u003e1.10 mm\u003c/b\u003e). At the higher concentration of 4 mg/mL, the zones of inhibition for all Gram-positive isolates increased (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No activity was detected against the Gram-negative strains. In contrast, the extract of \u003cem\u003eD. durissimum\u003c/em\u003e showed no bioactive effects against any of the tested strains at either concentration.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe antimicrobial activity of the organic extracts from \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e and \u003cem\u003eDragmacidon durissimum\u003c/em\u003e was evaluated against a panel of isolates using the well diffusion assay\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClinical isolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAcarnus wolffgangi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDragmacidon durissimum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentrations (mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZones of inhibition (mm\u0026thinsp;\u0026plusmn;\u0026thinsp;SE)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConcentrations (mg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eZones of inhibition (mm\u0026thinsp;\u0026plusmn;\u0026thinsp;SE)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMRSA\u003c/p\u003e \u003cp\u003eMRSA CI\u003c/p\u003e \u003cp\u003eCefotaxime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e0.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e\u003c/p\u003e \u003cp\u003eCefotaxime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e0.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003cp\u003e3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eE. \u003cem\u003eColi\u003c/em\u003e\u003c/p\u003e \u003cp\u003eCefapime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e0.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003eCefapime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e0.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo Inhibition (NI) [Amended]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. Microbroth Dilution Assay\u003c/h2\u003e \u003cp\u003eThe minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the organic extract from \u003cem\u003eA. wolffgangi\u003c/em\u003e were determined against various strains using the broth microdilution method.\u003c/p\u003e \u003cp\u003eAgainst Gram-positive strains, the MIC values were 125 \u0026micro;g/mL for \u003cem\u003eS. aureus\u003c/em\u003e and MRSA, and 500 \u0026micro;g/mL for \u003cem\u003eS. epidermidis\u003c/em\u003e. The MBC for these strains was 500 \u0026micro;g/mL. For the MRSA clinical isolate, both the MIC and MBC were 1 mg/mL. In contrast, the organic extract of \u003cem\u003eA. wolffgangi\u003c/em\u003e showed no activity against the tested Gram-negative strains, as both the MIC and MBC values exceeded the maximum tested concentration of 2 mg/mL. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for both extracts (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thymol were used as positive controls for Gram-positive and Gram-negative bacteria, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial activity of the organic extract from \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against Gram-positive and Gram-negative strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical isolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMBC (mg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRSA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE.coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;2 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;2 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003eMRSA Clinical isolate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;2 mg/mL\u003c/p\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;2 mg/mL\u003c/p\u003e \u003cp\u003e1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMinimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the positive control thymol against Gram-positive and Gram-negative strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical isolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMBC (mg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRSA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. epidermidis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e612\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e612\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25 mg/ mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.25 mg/ mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e612\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.25 mg/ mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Growth percent inhibition of gram-positive clinical isolates against organic extract of \u003cem\u003eA. wolffgangi\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe inhibitory effect of the \u003cem\u003eA. wolffgangi\u003c/em\u003e organic extract was assessed against four Gram-positive bacterial strains isolates\u0026mdash;\u003cem\u003eS. aureus\u003c/em\u003e, MRSA, \u003cem\u003eS. epidermidis\u003c/em\u003e, and MRSA CI\u0026mdash;across a concentration range of 7.8 to 1000 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eAs expected, the percentage of inhibition increased with the concentration of the extract for all three isolates. The most pronounced inhibitory effect was observed against \u003cem\u003eS. epidermidis\u003c/em\u003e, which showed the highest percent inhibition as the concentration increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Biofilm inhibition and eradication activity of organic extract of \u003cem\u003eA. wolffgangi\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe organic extract of \u003cem\u003eA. wolffgangi\u003c/em\u003e significantly inhibited biofilm formation and disrupted pre-formed biofilms in \u003cem\u003eS. aureus\u003c/em\u003e, MRSA, and \u003cem\u003eS. epidermidis\u003c/em\u003e. Biofilm formation was inhibited in a dose-dependent manner, with sub-inhibitory concentrations (1/4x MIC to 2x MIC) reducing biofilm mass across all three strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The most potent inhibition was observed against \u003cem\u003eS. aureus\u003c/em\u003e and MRSA at 2x MIC, which reduced biofilm formation to optical density (OD) values of 0.10 and 0.01, respectively. The extract also demonstrated a strong biofilm eradication effect against pre-formed biofilms. At 2x MIC, it significantly reduced the biofilm biomass of \u003cem\u003eS. aureus\u003c/em\u003e and MRSA to OD values of 0.16 and 0.13, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e(A)\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Scanning Electron Microscopy (SEM)\u003c/h2\u003e \u003cp\u003eScanning electron microscopy (SEM) was employed to evaluate the effect of the \u003cem\u003eA. wolffgangi\u003c/em\u003e extract on bacterial cell wall integrity. Micrographs of treated cells revealed significant morphological alterations, including severe membrane disruption and cell lysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These observations indicate that the extract's antimicrobial mechanism involves the destabilization of the bacterial cell wall, which is consistent with its bactericidal activity. The damage was most pronounced in MRSA, correlating with the high susceptibility of this strain observed in the well diffusion and broth microdilution assays.\u003c/p\u003e \u003cp\u003eThe SEM analysis indicates a potential mechanism of action for \u003cem\u003eA. wolffgangi\u003c/em\u003e, whereby its bioactive compounds compromise the bacterial cell wall, increasing its susceptibility to lysis and death. This proposed mechanism is reinforced by the lack of cell wall damage observed with the inactive \u003cem\u003eD. durissimum\u003c/em\u003e extract, suggesting that specific surface interactions between the sponge compounds and the bacterial envelope are critical for antibacterial efficacy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5. HPLC\u003c/h2\u003e \u003cp\u003eA calibration curve was constructed by injecting a series of gallic acid standard solutions at concentrations ranging from 5 to 1000 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eThe analytical method demonstrated excellent linearity across the calibration range (5\u0026ndash;1000 \u0026micro;g/mL), with a coefficient of determination (R\u0026sup2;) of 0.9982. Method specificity was confirmed by the absence of interfering peaks at the retention time of gallic acid in both blank and sample chromatograms (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The accuracy and precision of the method were assessed by analyzing a gallic acid standard (50 \u0026micro;g/mL) in triplicate. The relative standard deviation (RSD) for the peak areas was [insert RSD value]%, confirming high precision, and the mean measured concentration was [insert mean] \u0026micro;g/mL, demonstrating good accuracy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe developed analytical method was validated for its key parameters. The accuracy and precision were determined by analyzing a 50 \u0026micro;g/mL gallic acid standard in triplicate. The mean recovery was 96.7% with a relative standard deviation (RSD) of 1.4%, confirming high intra-day precision and good accuracy. The method demonstrated excellent linearity (R\u0026sup2; = 0.9982) across the range of 5\u0026ndash;1000 \u0026micro;g/mL. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 1.5 \u0026micro;g/mL and 5 \u0026micro;g/mL, respectively.\u003c/p\u003e \u003cp\u003eDuring method development, various chromatographic conditions were optimized to achieve adequate separation. This involved testing different mobile phase compositions (20\u0026ndash;60% Solvent A), flow rates (0.2\u0026ndash;0.8 mL/min), injection volumes (1\u0026ndash;5 \u0026micro;L), and detection wavelengths. The optimal wavelength was selected as 270 nm based on the UV-Vis absorption maximum of gallic acid.\u003c/p\u003e \u003cp\u003eThe validated method was applied to quantify gallic acid in the sponge extracts. The concentration of gallic acid in extracts 65 and 30 was found to be 6 and 22 \u0026micro;g/mL in the prepared solution, which corresponds to a content of 3 and 11 \u0026micro;g gallic acid equivalents (GAE) per milligram of dry extract (\u0026micro;g GAE/mg). The final method provided robust separation and was deemed fit for its intended purpose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe escalating crisis of antibiotic resistance, particularly among Gram-positive pathogens like methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, necessitates the urgent discovery of novel therapeutic agents (Perera \u0026amp; Hay, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Akinduti et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The present study identifies the Red Sea sponge \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e as a promising source of such agents, demonstrating significant antibacterial and anti-biofilm activity, while its sympatric counterpart, \u003cem\u003eDragmacidon durissimum\u003c/em\u003e, showed limited efficacy under our assay conditions.\u003c/p\u003e \u003cp\u003eThe pronounced bioactivity of \u003cem\u003eA. wolffgangi\u003c/em\u003e against drug-resistant strains aligns with the growing recognition of the Red Sea as a unique reservoir of bioactive marine natural products (El-Hossary et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ahmed et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The extreme environmental conditions of this ecosystem, including high salinity and temperature, are known drivers of unique metabolic pathways, potentially explaining the potent secondary metabolites we observed (Plahn et al., 2002). The extract's specific efficacy against Gram-positive bacteria, culminating in low MIC values for MRSA and \u003cem\u003eS. aureus\u003c/em\u003e, is clinically advantageous. This is because these pathogens are predominant in skin infections, and their biofilm-forming capability makes conventional treatments increasingly ineffective (Venkatesan et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Burgess et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The lack of activity in the \u003cem\u003eD. durissimum\u003c/em\u003e extract suggests a species-specific defense strategy, a common phenomenon in marine sponges which often host unique symbiotic microorganisms responsible for compound production (Kelman et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This contrast underscores that bioactivity cannot be generalized across taxa, even within the same environment.\u003c/p\u003e \u003cp\u003eA critical finding of this work is the extract's capacity to not only inhibit biofilm formation but also to eradicate pre-established biofilms. The dose-dependent reduction in biofilm biomass is significant, as biofilms confer a protective layer that shields bacteria from antibiotics and the host immune system, leading to chronic, recalcitrant infections (Yin et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The ability of \u003cem\u003eA. wolffgangi\u003c/em\u003e to compromise this structure addresses a key therapeutic challenge. This anti-biofilm potential adds to the growing body of evidence on Red Sea marine organisms, such as the reported activity of \u003cem\u003eCallyspongia crassa\u003c/em\u003e and \u003cem\u003eAplysina fulva\u003c/em\u003e against \u003cem\u003eS. aureus\u003c/em\u003e (Afifi \u0026amp; Khabour, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and highlights the ecological role of these chemicals.\u003c/p\u003e \u003cp\u003eThe proposed mechanism of action, supported by SEM analysis, points to the disruption of bacterial cell wall integrity. The observed cell lysis and deformation provide a plausible explanation for the bactericidal effect and are consistent with the mechanism of several known antimicrobial compounds. While we quantified gallic acid content, it is unlikely to be the sole active principle, suggesting the presence of more potent, unidentified compounds. Advanced techniques, similar to the GC-MS and co-culture approaches used by Hamed et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) to enhance metabolite production in Red Sea sponge-associated microbes, could be employed in future studies to isolate these novel molecules.\u003c/p\u003e \u003cp\u003eOur results firmly establish \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e as a compelling candidate for future anti-infective drug discovery. Its potent, dual-mode activity against planktonic and biofilm-embedded resistant pathogens positions it as a valuable resource in the fight against difficult-to-treat skin infections. Future research must focus on the bioassay-guided fractionation of the active extract, isolation of pure bioactive compounds, and detailed investigation of their mechanisms of action and \u003cem\u003ein vivo\u003c/em\u003e therapeutic potential.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study identifies the Red Sea sponge \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e as a significant source of antibacterial and anti-biofilm agents effective against clinically critical Gram-positive pathogens, including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and MRSA. The organic extract demonstrated a potent capacity to inhibit bacterial growth and disrupt biofilm formation and integrity, with scanning electron microscopy (SEM) evidence pointing to bacterial membrane disruption as a key mechanism of its bactericidal action. In contrast, the sympatric species \u003cem\u003eDragmacidon durissimum\u003c/em\u003e exhibited limited activity under these assay conditions, highlighting the species-specific nature of bioactive compound production. These findings affirm the Red Sea's potential as a reservoir of novel marine natural products for combating antibiotic-resistant infections. Future work should focus on the bioassay-guided isolation of the specific bioactive compounds within \u003cem\u003eA. wolffgangi\u003c/em\u003e, a detailed elucidation of their modes of action, and subsequent \u003cem\u003ein vivo\u003c/em\u003e evaluation to assess their therapeutic potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eAll experimental protocols were approved by the Saudi General Directorate of Border Guard for the collection of the sponge samples under permission number 3452/2013. All methods were carried out and reported in accordance with the ARRIVE guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot Applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. \u003cb\u003e(IPP: 1289-166-2025).\u003c/b\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.R.Y:\u0026nbsp;Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.\u0026nbsp;\u0026nbsp;F.A.A:\u0026nbsp;Writing \u0026ndash; review \u0026amp; editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Dr. Diaa Yousef for providing the sponge material.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data presented in this study are available on request from the corresponding author\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbuzahrah SS. 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Int J Mol Sci. 2019;20(14):3423. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/IJMS20143423\u003c/span\u003e\u003cspan address=\"10.3390/IJMS20143423\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Marine sponges, Red Sea, Acarnus wolffgangi, Dragmacidon durissimum, Methicillin-resistant Staphylococcus aureus (MRSA), skin infections","lastPublishedDoi":"10.21203/rs.3.rs-8568006/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8568006/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe escalating threat of antibiotic-resistant Gram-positive bacteria, such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (including MRSA) and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, necessitates the discovery of novel antimicrobial agents. Marine sponges, particularly from the biodiverse Red Sea, are promising sources of bioactive secondary metabolites. This study investigates the antibacterial and antibiofilm properties of organic extracts from two under-explored Red Sea demosponges, \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e and \u003cem\u003eDragmacidon durissimum\u003c/em\u003e, against skin infection-causing Gram-positive and Gram-negative pathogens. Extracts were obtained using methanol-dichloromethane and tested via agar well diffusion, broth microdilution, and biofilm assays. \u003cem\u003eAcarnus wolffgangi\u003c/em\u003e exhibited significant antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e, MRSA, and \u003cem\u003eS. epidermidis\u003c/em\u003e, with MIC values as low as 125 \u0026micro;g/mL and MBC values of 500 \u0026micro;g/mL, alongside notable biofilm inhibition and eradication at sub-MIC concentrations. In contrast, \u003cem\u003eDragmacidon durissimum\u003c/em\u003e showed no significant activity. Scanning electron microscopy revealed bacterial cell wall disruption by \u003cem\u003eA. wolffgangi\u003c/em\u003e extracts, suggesting a bactericidal mechanism. High-performance liquid chromatography confirmed the presence of gallic acid (10 \u0026micro;g/mL) in both extracts. These findings highlight \u003cem\u003eA. wolffgangi\u003c/em\u003e as a potential source of novel antimicrobial agents to combat resistant Gram-positive pathogens, underscoring the therapeutic potential of Red Sea sponges. Further research is needed to isolate and characterize active compounds for clinical development.\u003c/p\u003e","manuscriptTitle":"Biofilm Eradication and Bactericidal Activity of the Red Sea Sponge Acarnus Wolffgangi against MRSA and Other Skin Pathogen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 17:30:30","doi":"10.21203/rs.3.rs-8568006/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f672eb7-f0e1-492a-8d10-a41ee78c3a11","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-31T09:40:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 17:30:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8568006","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8568006","identity":"rs-8568006","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}