Unraveling the anti-biofilm properties of Laurinterol on pioneer biofouling bacteria from the red seaweed Laurencia johnstonii 

Unraveling the anti-biofilm properties of Laurinterol on pioneer biofouling bacteria from the red seaweed Laurencia johnstonii | 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 Unraveling the anti-biofilm properties of Laurinterol on pioneer biofouling bacteria from the red seaweed Laurencia johnstonii Martha Patricia Agúndez-Salas, Ruth Noemí Aguila-Ramírez, Ana Laura González-Castro, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5227932/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract A biofilm is a complex microbial community, representing the initial stage in biofouling formation. Consequently, it is responsible for significant economic losses in several industrial sectors worldwide. Therefore, there is a constant need for safer and environmentally friendly coatings, particularly those derived from new marine sources such as seaweeds with antifouling properties. Red algae produce metabolites that prevent bacterial attachment and biofilm formation by disrupting microbial membranes, inhibiting quorum sensing, or interfering with extracellular matrix production, among them Laurencia johnstonii. This species has particular ecological or geographical advantages that make it more accessible or abundant for research in the Bay of La Paz, BCS. This study aimed to assess the anti-biofilm potential of the red seaweed Laurencia johnstonii. To identify bioactive compounds, the anti-biofilm activity of the ethanolic extract was evaluated against marine biofilm-forming strains: Bacillus altitudinis, Bacillus pumilus, Bacillus subtilis, and Bacillus cereus. The ethanolic extract of L. jonhstonii exhibited the highest percentage of inhibition. Subsequent chromatographic fractionation led to the isolation and identification of laurinterol, the primary compound responsible for the anti-biofilm activity (>97 %) and antibacterial activity (MIC <3.9 µg/mL). To our knowledge, this is the first report of the activity of laurinterol against biofilm-forming strains. algae biofouling marine natural products metabolites sesquiterpene Figures Figure 1 Figure 2 Introduction Biofilms are complex microbial communities that attach to a substrate surface or an interphase. Furthermore, microbial biofilms can adhere to a living surface or tissues growing in a self-secreted extracellular matrix (Donlan 2002 ). In healthcare, biofilm formation complicates the treatment of infectious diseases and contributes to bacterial resistance to common antibiotics, the main cause of persistent infections (Zhao et al. 2023 ). In marine environments, microbial biofilms are present on a wide range of artificial objects, from bottles to oil platforms, resulting in significant economic losses in several sectors worldwide (Qian et al. 2022 ). Moreover, the formation of this matrix facilitates microbial aggregation, a crucial step in the colonization of surfaces by microorganisms such as barnacles, mussels, and macroalgae among others. Biofilm represents the initial stage in the development of biofouling (Wang et al. 2022 ). Marine fouling organisms cause a variety of damages beyond increased drag and reduced energy efficiency in ships and vessels (Schultz et al. 2010 ). Their attachment to the surfaces such as piers, docks, and underwater pipelines accelerates corrosion and wear. Additionally, the weight and abrasive nature of these organisms can compromise the structural integrity of boats, offshore oil rigs, and subsea infrastructure (Hopkins et al. 2021 ). In aquaculture systems, fouling organisms compete for space and nutrients with farmed species, leading to reduced growth rates and increased stress. Furthermore, biofouling also facilitate the dissemination of pathogens and diseases within aquaculture systems. Organisms that settle on nets, cages, and tanks may harbor harmful microorganisms, which can infect the farmed species, resulting in economic losses and potential environmental harm (Bannister et al. 2019 ). Antifouling coatings are a common solution to this problem; however, they often contain toxic compounds such as copper, other metals, or the broad-spectrum biocide TBT, which causes significant damage to the marine environment (Abioye et al. 2019 ). Therefore, novel environmentally friendly coatings derived from marine sources are being developed as safer alternatives. Furthermore, marine biofilms contribute to ecological challenges due to their ability to colonize diverse biological surfaces, including plankton, algae, and animals. Seaweeds have evolved in the marine environment over millions of years, where biofouling is a constant threat. To counteract this, they have developed adaptive traits that inhibit microbial adhesion, reducing vulnerability to fouling and ensuring optimal access to sunlight and nutrients. This mechanism probably involves complex evolutionary pressures, including the need to remain free of microbial colonies that could obscure the algal surface or interfere with the photosynthetic process, growth, and distribution (Qian et al. 2022 ). As a result, some seaweeds have evolved effective chemical defenses against microbial epibionts, producing secondary metabolites with antibacterial and antifouling properties (Hellio & Yebra 2009 ; Zammuto et al. 2022 ). Some of the compounds produced by algae are particularly effective against pioneer bacterial species— the first to colonize and form biofilms. By inhibiting the attachment of these initial settlers, algae reduce the risk of more complex biofilm formation, which could involve a broader range of microorganisms and potentially harmful organisms such as barnacles or other larger marine invertebrates. These metabolites interfere with bacterial attachment and biofilm formation by disrupting microbial cell membranes (Feng et al. 2022 ), inhibit quorum sensing (the signaling mechanism that bacteria use to coordinate biofilm formation) (Goecke et al 2010 ; Behzadnia et al. 2024 ), or affecting the production of extracellular matrix components that bind bacteria together (Rima et al. 2022 ; Behzadnia et al. 2024 ). These antimicrobial compounds act as a chemical defense, preventing microbial colonization and biofilm formation on the algal surface. These antifouling properties may offer an alternative to current methods and could help to reduce the toxic effects observed in the oceans. Previous reports on antifouling activity highlight the potential of red seaweeds (Da Gama et al. 2008 ; Aguila-Ramírez et al. 2012 ; Pinteus et al. 2021 ), particularly the genus Laurencia , which is considered one of the main sources of active metabolites with around 800 identified compounds, mainly terpenoids (Yamagishi et al. 2024). These compounds often demonstrate potent properties, making Laurencia an ideal candidate for biofilm inhibition and antimicrobial activity research. Our previous research demonstrated the anti-biofilm potential of an ethanolic extract of Laurencia johnstonii (Aguila-Ramírez et al. 2012 ). These findings suggest Laurencia as a promising candidate for further investigation. Therefore, this study aims to evaluate the anti-biofilm activity of the red seaweed Laurencia johnstonii and identify the active compounds. Materials and methods Algal material and ethanolic extract preparation Laurencia johnstonii was collected at Coyote Beach (24° 21′ 09.2′′ N–110° 16′ 23.5′′ W) in Bahia de La Paz, Mexico. Taxonomic identification was assessed by morphological characters (Abbott & Hollenberg 1976) and confirmed by Dr. Juan Manuel López Vivas at Marine Botany Laboratory (Universidad Autónoma de Baja California Sur, Mexico). For the ethanolic extract, 100 g of air-dried seaweed were ground to 40-mesh size and macerated with 250 mL of distilled ethanol for 24 h. The ethanolic extract was concentrated using an RII rotavapor evaporator (Buchi, Switzerland) at 40°C. The extract was stored at -20°C until further analysis. Fractionation of Laurencia johnstonii For isolation, 3 g of L. johnstonii extract were separated by solid-liquid extraction using an elution gradient of n -hexane, dichloromethane, ethyl acetate, and methanol. This process yielded four fractions: F1, F2, F3, F4. Fraction F2 (1 g) was further fractionated on a silica gel column (70–230 mesh) with an elution gradient of n -hexane, dichloromethane, and methanol, resulting in five fractions: F2C1, F2C2, F2C3, F2C4, F2C5. To isolate the anti-biofilm compounds, the active fraction F2C2 was further fractionated on a silica gel column (70–230 mesh) with an elution gradient of n -hexane, dichloromethane, and methanol, yielding two final fractions: F2C2A, F2C2B. The sesquiterpene laurinterol (8 mg) was isolated from the F2C2A fraction by high-performance liquid chromatography (HPLC) (Fig. 1 ). Preparative chromatography was conducted on an HPLC system consisting of a 2335 quaternary gradient module and a 2998 photodiode array detector (Waters Corporation). Sample was injected manually with a 500 µL loop. Separation was conducted on a C18 prep OBD column (5 µm, 10 x 250 mm, Waters), using a mobile phase consisted of 10 mM ammonium acetate buffer (solvent A) and acetonitrile (solvent B) with the following elution gradient: 0–5 min, 60% B; 40 min, 100% B; 50 min, 100% B, 55 min, 60% B. The column was operated at ambient temperature with a flow rate of 3.5 mL/min, and the run time was 55 min. UV detection was set at 280 nm. Figure 1 . Bio-guided fractionation of Laurencia johnstonii extract to obtain laurinterol. Chemical characterization of laurinterol NMR spectrum was recorded on a Bruker AVANCE 500 MHz by dissolving the sample in CDCl 3 (99.9%), with chemical shifts reported relative to solvent (7.26 ppm) and TMS as an internal pattern. The NMR data were compared with those previously reported in the literature to confirm the structure (García-Davis et al. 2019 ; González-Castro et al. 2024 ). Laurinterol: C 15 H 19 BrO, 1 H NMR (CDCl 3 ) 0.54 (1H, t, J = 3.9, H-12), 0.57 (1H, m H-12), 1.14 (1H, dt, J = 8.1, 4.2 Hz, H-3), 1.28 (1H, d, J = 4.5 Hz, H-5), 1.40 (3H, s, H-13), 1.57 (3H, s, H-14), 1.66 (1H, dd, J = 12.4, 8.0 Hz, H-4), 1.94 (1H, tdd, J = 12.3, 8.2, 4.4, H-4), 2.08 (1H, dd, J = 13.2, 8.1 Hz, H-5), 2.29 (3H, s, H-15), 5.13 (1H, Br, s, 7-OH), 6,61 (1H, s, H-8), 7.60 (1H, s, H-11). Figure 2 . Laurinterol sesquiterpene isolated from Laurencia johnstonii. Bacteria and culture media Pioneer bacteria were isolated from acrylic and fiberglass plates submerged in the sea for 6 h. To efficiently select strains that could adhere and form biofilms, a rapid method based on the crystal violet staining of biofilms formed in 96-well microtiter plates was employed to identify bacterial strains with high biofilm-forming abilities. The bacterial strains were identified as Bacillus altitudinis , B. pumilus , B. subtilis , and B. cereus Bacterial cultures were grown on tripticasein soy agar with 2.7% NaCl (TSA-NaCl). Before the assay, the bacterial strains were suspended in saline solution and adjusted to 1 × 10 8 cells mL − 1 (Merck SQ118). Anti-biofilm activity assay The in vitro anti-biofilm activity was assessed using the crystal violet assay in a 96-well flat-bottom plate. Each well was inoculated with 100 µL of bacterial suspension and 100 µL of extract (1 mg mL − 1 ). The plates were incubated at 35 ºC for 48 h (Heratherm Thermo Scientific). All assays were performed in triplicate, with a blank control (broth without bacteria) and a negative control (broth with bacterial suspension) included. After incubation, the wells were washed with distilled water to remove non-adherent cells, and the cells were fixed with 250 µL of methanol for 15 min. After removing the methanol, the plates were air-dried for 45 minutes. Next, 200 µL of 1% crystal violet solution was added, and the plates were left at room temperature for 20 minutes. The crystal violet was removed, and the plates were rinsed with distilled water until any excess dye was cleared and the plates were allowed to dry. Finally, 250 µL of ethanol (96%) was added to solubilize the crystal violet adhered to the plate. The biofilm mass was quantified by measuring the absorbance of the destaining solution at 595 nm using a microtiter plate reader (Infinite M1000 PRO, Tecan) (Shukla & Rao 2017 ). The biofilm inhibition (%) was calculated using the following equation: $$\:\left[1-\left(\frac{{A}_{extract}-{A}_{blank}}{{A}_{control}}\right)x\:100\right]$$ Where, A control : negative control absorbance, A blank : blank absorbance, A extract : extract absorbance Minimal inhibitory concentration (MIC) determination The extract was serially diluted to create a concentration gradient ranging from 1000 µg mL − 1 to 0.97 µg mL − 1 . All samples were subjected to triplicate analysis, along with the blank (broth without bacteria) and the negative control (broth with bacterial suspension), following the same methodology mentioned above. Results Anti-biofilm activity The ethanolic extract exhibited strong activity against biofilm formation in most tested strains, and fraction F2C2 showed the highest inhibition of biofilm formation across all bacterial strains (Table 1 ). Consequently, this fraction was further fractionated, and the two resulting fractions, F2C2A, and F2C2B, were also evaluated. Both fractions showed strong inhibition of biofilm formation at a concentration of 0.06 mg · mL − 1 . Table 1 Percentage of inhibition of biofilm formation (%) of the ethanolic extract of Laurencia johnstonii and its fractions [1 mg mL − 1 ]. *Calculated at a concentration of [0.06 mg mL − 1 ], (n = 3). B. altitudinis B. pumilus B. subtilis B. cereus Ethanolic extract 79.40 ± 3.9 100 ± 3.3 100 ± 0.5 12.11 ± 0.8 F2C1 93.33 ± 3.7 61.47 ± 0.4 90.78 ± 2.8 87.15 ± 2.1 F2C2 94.24 ± 3.5 100 ± 0.5 100 ± 1.0 98.42 ± 0.5 F2C3 80.46 ± 1.5 0 65.86 ± 2.3 72.06 ± 0.7 F2C4 76.01 ± 1.8 46.47 ± 1.2 100 ± 0.5 32.04 ± 1 F2C5 52.99 ± 2.9 0 73.28 ± 3.5 82.49 ± 3.2 F2C2A* 100 ± 0.3 99.92 ± 1.3 97.97 ± 1.2 100 ± 0.8 F2C2B* 96.68 ± 1.0 99.98 ± 1.3 96.84 ± 1.0 99.18 ± 1.2 Minimum Inhibitory Concentration (MIC) The sesquiterpene laurinterol, isolated from the active fraction F2C2A, was evaluated using the microdilution assay to establish its MIC (Table 2 ). Table 2 Minimum inhibitory concentration (µg mL − 1 ) of L . johnstonii fractions against biofilm formation. B. altitudinis B. pumilus B. subtilis B. cereus Ethanolic extract 1.9 1.9 1.9 <0.9 F2C2A 3.9 3.9 3.9 <3.9 Laurinterol <0.97 <0.97 <0.97 <0.97 Discussion A biofilm is a complex, self-sustaining ecosystem where organisms exhibit cooperation and competition relationships (Nadell et al. 2016 ). Moreover, several studies have indicated that bacterial cells within biofilms resist various stresses that are 1,000 times greater than those observed in the planktonic form (Ashrafudoulla et al. 2019). In marine environments, biofilms can modify or mask surface topographies and properties, leading to macrofouling colonization (Qian et al. 2022 ). Consequently, the prevention of biofilm formation represents a safer and more optimal strategy for the inhibition of bacterial proliferation and the avoidance of all the problems associated with biofilms. In recent decades, there has been a growing interest in marine natural products with anti-biofilm attributes. In particular, algae synthesize a range of diverse biogenic compounds, which has been identified as an effective strategy for disrupting biofilm structures and eliminating biofilms, without causing harm to other organisms within the ecosystem (Behzadnia et al. 2024 ). Several publications have examined the anti-biofilm activity of organic extracts. However, the majority of these studies have focused on biofilms of human pathogenic bacteria, including Escherichia coli , Salmonella , Pseudomonas aeruginosa , and Staphylococcus aureus , and have not identified the active molecules (Caudal et al. 2024 ). Conversely, recent research has placed a greater emphasis on the anti-fouling properties of diverse algal groups (Nag et al. 2022 ). Most of the antifouling metabolites isolated to date belong to terpenoids, alkaloids, and steroids (Al-Lihaibi et al. 2019 ). Compounds containing halogenated furanones serve as potent inhibitors of quorum sensing across a wide range of bacterial species. This inhibition prevents the formation of sessile microcolonies on both living and non-living surfaces. The red genus Laurencia is found in tropical zones and represents a rich source of brominated compounds with antifouling activity against mussel and barnacle larvae, such as omaezallene (Umezawa et al. 2014 ), aplysin-20 aldehyde, and 13-dehydroxyisoaplysin-20 (Fukada et al. 2023 ). From Laurencia viridis the compound 15,16-epoxythyrsiferol B; 15,16-epoxythyrsiferol A; 28-hydroxysaiyacenol B saiyacenol C show antibiofilm properties (Al-Lihaibi et al. 2015 ). A previous evaluation of Laurencia johnstonii extract exhibited antimicrobial activity against strains of marine bacteria, with MIC values ranging from 0.1 to 1 µg mL − 1 (Aguila-Ramírez et al. 2012 ), as well as against pathogenic strains (García-Davis et al. 2018 ). In the present study, the MIC values of the ethanolic extract of L. johnstonii from anti-biofilm activity were found to range between 0.9 and 1.9 µg mL − 1 . Therefore, we aimed to isolate the active compound, identified as the sesquiterpene laurinterol, with MIC values below 0.97 µg mL –1 against all the bacterial strains tested. The species of L. johnstonii , native to the Baja California Pacific coastline, was found to contain higher concentrations of laurinterol, compared to related species from the same Pacific coastal region (Arberas-Jimenéz et al. 2020). Previously, laurinterol also showed activity against some marine bacteria isolated from algal habitats (Vairappan et al. 2001 ). This is important because the bacterial communities within biofilm are critical to the process of larval settlement in marine environments. Therefore, inhibiting bacterial growth on surfaces is essential to preventing larval settlement of macrofouling organisms (Rajitha et al. 2020 ). In this context, the antifouling activity of laurinterol, along with other 13 Laurencia -derived compounds, was previously evaluated against larvae of the barnacle Amphibalanus amphitrite (EC 50 = 0.65 µg ml –1 , LC 50 = 5.8 µg ml –1 ). Although laurinterol showed a higher EC 50 compared to the control (CuSO 4 ), its selectivity index (LC 50 /EC 50 ), calculated according to its ecotoxicity (EC 50 ) against the marine crustacean Tigriopus japonicus , was higher (Oguri et al. 2017 ). These studies highlight the relevance of laurinterol as a structural model for anti-biofilm studies. Moreover, it is bioavailability and a broad spectrum of bioactive properties have proven useful for structure-activity relationship analysis (Arberas‑Jiménez et al. 2020). From this perspective, our study suggests that the sesquiterpene laurinterol could be a potential candidate for inhibiting marine biofilms and may provide a valuable starting point for rational design of coatings with anti-biofilm properties. Conclusions This study confirms the anti-biofilm activity of Laurencia johnstonii extract and its main compound, laurinterol, against all biofilm-forming marine bacterial strains tested. These findings not only support the potential of L. johnstonii as a valuable natural source of bioactive compounds with potent anti-biofilm properties but also underscore its significance in marine biotechnology and antimicrobial research. The extract and laurinterol demonstrated consistent efficacy in inhibiting biofilm formation, suggesting their potential use in preventing bacterial colonization in aquatic environments, where biofilm-related issues are common. Furthermore, the promising results of this study highlight the importance of exploring marine resources, particularly algae, as an untapped reservoir for bioactive compounds. As biofilm formation is a major contributor to chronic infections, fouling, and other industrial challenges, identifying natural substances like laurinterol offers a sustainable and environmentally friendly alternative to traditional chemical treatments. The potential application of L. johnstonii extract in various industries, such as healthcare, marine engineering, and environmental conservation, warrants further investigation. Declarations Acknowledgments The authors would like to acknowledge the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) for their support of this project, including the post-doctoral fellowship (3813579), as well as the COFAA program of the Instituto Politécnico Nacional (IPN). Funding Not applicable. Competing interests The authors declare that there is no conflict of interest. Availability of data and materials All relevant data are within the manuscript and supplementary material file. Any additional data is available from the corresponding author upon reasonable request. Authors' contribution Writing original draft preparation: MPAS. Methodology: RNAR, SGD, MMO. Data curation and formal analysis: MPAS, ALGC, SGD. Writing-review and editing: ALGC, RNAR, SGD, MMO. Project administration and supervision: RNAR, MMO. Ethical Approval Not applicable. References Abbott I, G J Hollenberg (eds) (1976) Marine algae of California. Stanford University Press, Redwood City. p 844 Abioye OP, Loto CA, Fayomi OSI (2019) Evaluation of anti-biofouling progresses in marine application. J Bio Tribocorros 5:1-8. 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Front Cell Infect Microbiol, 13, 1137947. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 11 Dec, 2024 Reviewers agreed at journal 26 Nov, 2024 Reviewers agreed at journal 21 Nov, 2024 Reviewers invited by journal 21 Nov, 2024 Submission checks completed at journal 21 Nov, 2024 First submitted to journal 20 Nov, 2024 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. 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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-5227932","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":380986785,"identity":"24c85043-491c-4989-a598-55952f995bcb","order_by":0,"name":"Martha Patricia Agúndez-Salas","email":"","orcid":"","institution":"Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas (IPN-CICIMAR)","correspondingAuthor":false,"prefix":"","firstName":"Martha","middleName":"Patricia","lastName":"Agúndez-Salas","suffix":""},{"id":380986786,"identity":"9adbbb25-5325-4d89-aa38-a974df868855","order_by":1,"name":"Ruth Noemí Aguila-Ramírez","email":"","orcid":"","institution":"Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas (IPN-CICIMAR)","correspondingAuthor":false,"prefix":"","firstName":"Ruth","middleName":"Noemí","lastName":"Aguila-Ramírez","suffix":""},{"id":380986787,"identity":"fca940af-55c9-442c-b1e0-419cc7ad873d","order_by":2,"name":"Ana Laura González-Castro","email":"","orcid":"","institution":"Universidad Autónoma de Baja California Sur","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Laura","lastName":"González-Castro","suffix":""},{"id":380986788,"identity":"f0d427f9-4d97-4a24-b7a8-846828d63984","order_by":3,"name":"Sara García-Davis","email":"","orcid":"","institution":"Instituto Universitario de Bio-Orgánica Antonio González (IUBO AG), Universidad de La Laguna (ULL)","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"García-Davis","suffix":""},{"id":380986789,"identity":"7639a36d-9a4f-499c-bf74-5ae75c717c69","order_by":4,"name":"Mauricio Muñoz-Ochoa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAlElEQVRIiWNgGAWjYBADOTaStRiTriWxgWilutPOmD348McmvU+69wDTzTYitJjdzjE3nMGTltsmcy6BOecMcVrMpHkkDue2SeQYMOdUEK3F4HA6G1iLAdFaEg4nsJFgS1qZ5IwDaYZtMmcMDhPpl+RtEsAQk5ef3WP4OJeYEEMACQaGAyRpAGsZBaNgFIyCUYAVAADuiS9TTgnIbwAAAABJRU5ErkJggg==","orcid":"","institution":"Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas (IPN-CICIMAR)","correspondingAuthor":true,"prefix":"","firstName":"Mauricio","middleName":"","lastName":"Muñoz-Ochoa","suffix":""}],"badges":[],"createdAt":"2024-10-08 21:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5227932/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5227932/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69862895,"identity":"fdca3887-647e-46d4-a6f9-9bd179813d24","added_by":"auto","created_at":"2024-11-26 06:09:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":177696,"visible":true,"origin":"","legend":"\u003cp\u003eBio-guided\u003cstrong\u003e \u003c/strong\u003efractionation of \u003cem\u003eLaurencia johnstonii\u003c/em\u003e extract to obtain laurinterol.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5227932/v1/e24ed025f7307dbf8fccdc97.png"},{"id":69862894,"identity":"83660a7c-75f0-4cd9-94dc-009a29b0bde7","added_by":"auto","created_at":"2024-11-26 06:09:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50235,"visible":true,"origin":"","legend":"\u003cp\u003eLaurinterol sesquiterpene isolated from \u003cem\u003eLaurencia johnstonii.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5227932/v1/2f2a8a6b3da8fbaeee47942a.png"},{"id":69863004,"identity":"ff47347a-ef1c-47cf-9151-78ca74485b52","added_by":"auto","created_at":"2024-11-26 06:17:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":712097,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5227932/v1/4cd1b3e2-f64c-49f7-bd9b-68a6e589c05f.pdf"},{"id":69862897,"identity":"56ab7c84-8ed1-4b3b-b288-789ca79e1312","added_by":"auto","created_at":"2024-11-26 06:09:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":381016,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5227932/v1/f56fd6a23c080b1b438ae7c2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unraveling the anti-biofilm properties of Laurinterol on pioneer biofouling bacteria from the red seaweed Laurencia johnstonii ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiofilms are complex microbial communities that attach to a substrate surface or an interphase. Furthermore, microbial biofilms can adhere to a living surface or tissues growing in a self-secreted extracellular matrix (Donlan \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In healthcare, biofilm formation complicates the treatment of infectious diseases and contributes to bacterial resistance to common antibiotics, the main cause of persistent infections (Zhao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In marine environments, microbial biofilms are present on a wide range of artificial objects, from bottles to oil platforms, resulting in significant economic losses in several sectors worldwide (Qian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, the formation of this matrix facilitates microbial aggregation, a crucial step in the colonization of surfaces by microorganisms such as barnacles, mussels, and macroalgae among others. Biofilm represents the initial stage in the development of biofouling (Wang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMarine fouling organisms cause a variety of damages beyond increased drag and reduced energy efficiency in ships and vessels (Schultz et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Their attachment to the surfaces such as piers, docks, and underwater pipelines accelerates corrosion and wear. Additionally, the weight and abrasive nature of these organisms can compromise the structural integrity of boats, offshore oil rigs, and subsea infrastructure (Hopkins et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn aquaculture systems, fouling organisms compete for space and nutrients with farmed species, leading to reduced growth rates and increased stress. Furthermore, biofouling also facilitate the dissemination of pathogens and diseases within aquaculture systems. Organisms that settle on nets, cages, and tanks may harbor harmful microorganisms, which can infect the farmed species, resulting in economic losses and potential environmental harm (Bannister et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Antifouling coatings are a common solution to this problem; however, they often contain toxic compounds such as copper, other metals, or the broad-spectrum biocide TBT, which causes significant damage to the marine environment (Abioye et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, novel environmentally friendly coatings derived from marine sources are being developed as safer alternatives.\u003c/p\u003e \u003cp\u003eFurthermore, marine biofilms contribute to ecological challenges due to their ability to colonize diverse biological surfaces, including plankton, algae, and animals. Seaweeds have evolved in the marine environment over millions of years, where biofouling is a constant threat. To counteract this, they have developed adaptive traits that inhibit microbial adhesion, reducing vulnerability to fouling and ensuring optimal access to sunlight and nutrients. This mechanism probably involves complex evolutionary pressures, including the need to remain free of microbial colonies that could obscure the algal surface or interfere with the photosynthetic process, growth, and distribution (Qian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As a result, some seaweeds have evolved effective chemical defenses against microbial epibionts, producing secondary metabolites with antibacterial and antifouling properties (Hellio \u0026amp; Yebra \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zammuto et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome of the compounds produced by algae are particularly effective against pioneer bacterial species\u0026mdash; the first to colonize and form biofilms. By inhibiting the attachment of these initial settlers, algae reduce the risk of more complex biofilm formation, which could involve a broader range of microorganisms and potentially harmful organisms such as barnacles or other larger marine invertebrates. These metabolites interfere with bacterial attachment and biofilm formation by disrupting microbial cell membranes (Feng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), inhibit quorum sensing (the signaling mechanism that bacteria use to coordinate biofilm formation) (Goecke et al \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Behzadnia et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), or affecting the production of extracellular matrix components that bind bacteria together (Rima et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Behzadnia et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These antimicrobial compounds act as a chemical defense, preventing microbial colonization and biofilm formation on the algal surface. These antifouling properties may offer an alternative to current methods and could help to reduce the toxic effects observed in the oceans. Previous reports on antifouling activity highlight the potential of red seaweeds (Da Gama et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Aguila-Ram\u0026iacute;rez et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pinteus et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), particularly the genus \u003cem\u003eLaurencia\u003c/em\u003e, which is considered one of the main sources of active metabolites with around 800 identified compounds, mainly terpenoids (Yamagishi et al. 2024). These compounds often demonstrate potent properties, making \u003cem\u003eLaurencia\u003c/em\u003e an ideal candidate for biofilm inhibition and antimicrobial activity research.\u003c/p\u003e \u003cp\u003eOur previous research demonstrated the anti-biofilm potential of an ethanolic extract of \u003cem\u003eLaurencia johnstonii\u003c/em\u003e (Aguila-Ram\u0026iacute;rez et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These findings suggest \u003cem\u003eLaurencia\u003c/em\u003e as a promising candidate for further investigation. Therefore, this study aims to evaluate the anti-biofilm activity of the red seaweed \u003cem\u003eLaurencia johnstonii\u003c/em\u003e and identify the active compounds.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAlgal material and ethanolic extract preparation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLaurencia johnstonii\u003c/em\u003e was collected at Coyote Beach (24\u0026deg; 21\u0026prime; 09.2\u0026prime;\u0026prime; N\u0026ndash;110\u0026deg; 16\u0026prime; 23.5\u0026prime;\u0026prime; W) in Bahia de La Paz, Mexico. Taxonomic identification was assessed by morphological characters (Abbott \u0026amp; Hollenberg 1976) and confirmed by Dr. Juan Manuel L\u0026oacute;pez Vivas at Marine Botany Laboratory (Universidad Aut\u0026oacute;noma de Baja California Sur, Mexico).\u003c/p\u003e \u003cp\u003eFor the ethanolic extract, 100 g of air-dried seaweed were ground to 40-mesh size and macerated with 250 mL of distilled ethanol for 24 h. The ethanolic extract was concentrated using an RII rotavapor evaporator (Buchi, Switzerland) at 40\u0026deg;C. The extract was stored at -20\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFractionation of Laurencia johnstonii\u003c/h3\u003e\n\u003cp\u003eFor isolation, 3 g of \u003cem\u003eL. johnstonii\u003c/em\u003e extract were separated by solid-liquid extraction using an elution gradient of \u003cem\u003en\u003c/em\u003e-hexane, dichloromethane, ethyl acetate, and methanol. This process yielded four fractions: F1, F2, F3, F4.\u003c/p\u003e \u003cp\u003eFraction F2 (1 g) was further fractionated on a silica gel column (70\u0026ndash;230 mesh) with an elution gradient of \u003cem\u003en\u003c/em\u003e-hexane, dichloromethane, and methanol, resulting in five fractions: F2C1, F2C2, F2C3, F2C4, F2C5. To isolate the anti-biofilm compounds, the active fraction F2C2 was further fractionated on a silica gel column (70\u0026ndash;230 mesh) with an elution gradient of \u003cem\u003en\u003c/em\u003e-hexane, dichloromethane, and methanol, yielding two final fractions: F2C2A, F2C2B. The sesquiterpene laurinterol (8 mg) was isolated from the F2C2A fraction by high-performance liquid chromatography (HPLC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Preparative chromatography was conducted on an HPLC system consisting of a 2335 quaternary gradient module and a 2998 photodiode array detector (Waters Corporation). Sample was injected manually with a 500 \u0026micro;L loop. Separation was conducted on a C18 prep OBD column (5 \u0026micro;m, 10 x 250 mm, Waters), using a mobile phase consisted of 10 mM ammonium acetate buffer (solvent A) and acetonitrile (solvent B) with the following elution gradient: 0\u0026ndash;5 min, 60% B; 40 min, 100% B; 50 min, 100% B, 55 min, 60% B. The column was operated at ambient temperature with a flow rate of 3.5 mL/min, and the run time was 55 min. UV detection was set at 280 nm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Bio-guided fractionation of \u003cem\u003eLaurencia johnstonii\u003c/em\u003e extract to obtain laurinterol.\u003c/p\u003e\n\u003ch3\u003eChemical characterization of laurinterol\u003c/h3\u003e\n\u003cp\u003eNMR spectrum was recorded on a Bruker AVANCE 500 MHz by dissolving the sample in CDCl\u003csub\u003e3\u003c/sub\u003e (99.9%), with chemical shifts reported relative to solvent (7.26 ppm) and TMS as an internal pattern. The NMR data were compared with those previously reported in the literature to confirm the structure (Garc\u0026iacute;a-Davis et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gonz\u0026aacute;lez-Castro et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLaurinterol: C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eBrO, \u003csup\u003e1\u003c/sup\u003eH NMR (CDCl\u003csub\u003e3\u003c/sub\u003e) 0.54 (1H, t, J\u0026thinsp;=\u0026thinsp;3.9, H-12), 0.57 (1H, m H-12), 1.14 (1H, dt, J\u0026thinsp;=\u0026thinsp;8.1, 4.2 Hz, H-3), 1.28 (1H, d, J\u0026thinsp;=\u0026thinsp;4.5 Hz, H-5), 1.40 (3H, s, H-13), 1.57 (3H, s, H-14), 1.66 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.4, 8.0 Hz, H-4), 1.94 (1H, tdd, J\u0026thinsp;=\u0026thinsp;12.3, 8.2, 4.4, H-4), 2.08 (1H, dd, J\u0026thinsp;=\u0026thinsp;13.2, 8.1 Hz, H-5), 2.29 (3H, s, H-15), 5.13 (1H, Br, s, 7-OH), 6,61 (1H, s, H-8), 7.60 (1H, s, H-11).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Laurinterol sesquiterpene isolated from \u003cem\u003eLaurencia johnstonii.\u003c/em\u003e\u003c/p\u003e\n\u003ch3\u003eBacteria and culture media\u003c/h3\u003e\n\u003cp\u003ePioneer bacteria were isolated from acrylic and fiberglass plates submerged in the sea for 6 h. To efficiently select strains that could adhere and form biofilms, a rapid method based on the crystal violet staining of biofilms formed in 96-well microtiter plates was employed to identify bacterial strains with high biofilm-forming abilities. The bacterial strains were identified as \u003cem\u003eBacillus altitudinis\u003c/em\u003e, \u003cem\u003eB. pumilus\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, and \u003cem\u003eB. cereus\u003c/em\u003e Bacterial cultures were grown on tripticasein soy agar with 2.7% NaCl (TSA-NaCl). Before the assay, the bacterial strains were suspended in saline solution and adjusted to 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Merck SQ118).\u003c/p\u003e\n\u003ch3\u003eAnti-biofilm activity assay\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e anti-biofilm activity was assessed using the crystal violet assay in a 96-well flat-bottom plate. Each well was inoculated with 100 \u0026micro;L of bacterial suspension and 100 \u0026micro;L of extract (1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The plates were incubated at 35 \u0026ordm;C for 48 h (Heratherm Thermo Scientific). All assays were performed in triplicate, with a blank control (broth without bacteria) and a negative control (broth with bacterial suspension) included. After incubation, the wells were washed with distilled water to remove non-adherent cells, and the cells were fixed with 250 \u0026micro;L of methanol for 15 min. After removing the methanol, the plates were air-dried for 45 minutes. Next, 200 \u0026micro;L of 1% crystal violet solution was added, and the plates were left at room temperature for 20 minutes. The crystal violet was removed, and the plates were rinsed with distilled water until any excess dye was cleared and the plates were allowed to dry. Finally, 250 \u0026micro;L of ethanol (96%) was added to solubilize the crystal violet adhered to the plate. The biofilm mass was quantified by measuring the absorbance of the destaining solution at 595 nm using a microtiter plate reader (Infinite M1000 PRO, Tecan) (Shukla \u0026amp; Rao \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe biofilm inhibition (%) was calculated using the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\left[1-\\left(\\frac{{A}_{extract}-{A}_{blank}}{{A}_{control}}\\right)x\\:100\\right]$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere, A\u003csub\u003econtrol\u003c/sub\u003e: negative control absorbance, A\u003csub\u003eblank\u003c/sub\u003e: blank absorbance, A\u003csub\u003eextract\u003c/sub\u003e: extract absorbance\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMinimal inhibitory concentration (MIC) determination\u003c/h2\u003e \u003cp\u003eThe extract was serially diluted to create a concentration gradient ranging from 1000 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 0.97 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. All samples were subjected to triplicate analysis, along with the blank (broth without bacteria) and the negative control (broth with bacterial suspension), following the same methodology mentioned above.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnti-biofilm activity\u003c/h2\u003e \u003cp\u003eThe ethanolic extract exhibited strong activity against biofilm formation in most tested strains, and fraction F2C2 showed the highest inhibition of biofilm formation across all bacterial strains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Consequently, this fraction was further fractionated, and the two resulting fractions, F2C2A, and F2C2B, were also evaluated. Both fractions showed strong inhibition of biofilm formation at a concentration of 0.06 mg\u003cb\u003e\u0026middot;\u003c/b\u003emL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\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\u003ePercentage of inhibition of biofilm formation (%) of the ethanolic extract of \u003cem\u003eLaurencia johnstonii\u003c/em\u003e and its fractions [1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]. *Calculated at a concentration of [0.06 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e], (n\u0026thinsp;=\u0026thinsp;3).\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=\"char\" char=\"\u0026plusmn;\" 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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eB. altitudinis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. pumilus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthanolic extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e79.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e12.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2C1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e93.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e90.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e87.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2C2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e94.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e98.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2C3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e80.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e65.86\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e72.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2C4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e76.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e32.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2C5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e52.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e73.28\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e82.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2C2A*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e97.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2C2B*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e96.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e96.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e99.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\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=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMinimum Inhibitory Concentration (MIC)\u003c/h2\u003e \u003cp\u003eThe sesquiterpene laurinterol, isolated from the active fraction F2C2A, was evaluated using the microdilution assay to establish its MIC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\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\u003eMinimum inhibitory concentration (\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ejohnstonii\u003c/em\u003e fractions against biofilm formation.\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eB. altitudinis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. pumilus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthanolic extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2C2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;3.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaurinterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;0.97\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"},{"header":"Discussion","content":"\u003cp\u003eA biofilm is a complex, self-sustaining ecosystem where organisms exhibit cooperation and competition relationships (Nadell et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, several studies have indicated that bacterial cells within biofilms resist various stresses that are 1,000 times greater than those observed in the planktonic form (Ashrafudoulla et al. 2019). In marine environments, biofilms can modify or mask surface topographies and properties, leading to macrofouling colonization (Qian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, the prevention of biofilm formation represents a safer and more optimal strategy for the inhibition of bacterial proliferation and the avoidance of all the problems associated with biofilms.\u003c/p\u003e \u003cp\u003eIn recent decades, there has been a growing interest in marine natural products with anti-biofilm attributes. In particular, algae synthesize a range of diverse biogenic compounds, which has been identified as an effective strategy for disrupting biofilm structures and eliminating biofilms, without causing harm to other organisms within the ecosystem (Behzadnia et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Several publications have examined the anti-biofilm activity of organic extracts. However, the majority of these studies have focused on biofilms of human pathogenic bacteria, including \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and have not identified the active molecules (Caudal et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConversely, recent research has placed a greater emphasis on the anti-fouling properties of diverse algal groups (Nag et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Most of the antifouling metabolites isolated to date belong to terpenoids, alkaloids, and steroids (Al-Lihaibi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Compounds containing halogenated furanones serve as potent inhibitors of quorum sensing across a wide range of bacterial species. This inhibition prevents the formation of sessile microcolonies on both living and non-living surfaces. The red genus \u003cem\u003eLaurencia\u003c/em\u003e is found in tropical zones and represents a rich source of brominated compounds with antifouling activity against mussel and barnacle larvae, such as omaezallene (Umezawa et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), aplysin-20 aldehyde, and 13-dehydroxyisoaplysin-20 (Fukada et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). From \u003cem\u003eLaurencia viridis\u003c/em\u003e the compound 15,16-epoxythyrsiferol B; 15,16-epoxythyrsiferol A; 28-hydroxysaiyacenol B saiyacenol C show antibiofilm properties (Al-Lihaibi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA previous evaluation of \u003cem\u003eLaurencia johnstonii\u003c/em\u003e extract exhibited antimicrobial activity against strains of marine bacteria, with MIC values ranging from 0.1 to 1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Aguila-Ram\u0026iacute;rez et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), as well as against pathogenic strains (Garc\u0026iacute;a-Davis et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, the MIC values of the ethanolic extract of \u003cem\u003eL. johnstonii\u003c/em\u003e from anti-biofilm activity were found to range between 0.9 and 1.9 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Therefore, we aimed to isolate the active compound, identified as the sesquiterpene laurinterol, with MIC values below 0.97 \u0026micro;g mL\u003csup\u003e\u0026ndash;1\u003c/sup\u003e against all the bacterial strains tested. The species of \u003cem\u003eL. johnstonii\u003c/em\u003e, native to the Baja California Pacific coastline, was found to contain higher concentrations of laurinterol, compared to related species from the same Pacific coastal region (Arberas-Jimen\u0026eacute;z et al. 2020). Previously, laurinterol also showed activity against some marine bacteria isolated from algal habitats (Vairappan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). This is important because the bacterial communities within biofilm are critical to the process of larval settlement in marine environments. Therefore, inhibiting bacterial growth on surfaces is essential to preventing larval settlement of macrofouling organisms (Rajitha et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this context, the antifouling activity of laurinterol, along with other 13 \u003cem\u003eLaurencia\u003c/em\u003e-derived compounds, was previously evaluated against larvae of the barnacle \u003cem\u003eAmphibalanus amphitrite\u003c/em\u003e (EC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.65 \u0026micro;g ml\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, LC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.8 \u0026micro;g ml\u003csup\u003e\u0026ndash;1\u003c/sup\u003e). Although laurinterol showed a higher EC\u003csub\u003e50\u003c/sub\u003e compared to the control (CuSO\u003csub\u003e4\u003c/sub\u003e), its selectivity index (LC\u003csub\u003e50\u003c/sub\u003e/EC\u003csub\u003e50\u003c/sub\u003e), calculated according to its ecotoxicity (EC\u003csub\u003e50\u003c/sub\u003e) against the marine crustacean \u003cem\u003eTigriopus japonicus\u003c/em\u003e, was higher (Oguri et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These studies highlight the relevance of laurinterol as a structural model for anti-biofilm studies. Moreover, it is bioavailability and a broad spectrum of bioactive properties have proven useful for structure-activity relationship analysis (Arberas‑Jim\u0026eacute;nez et al. 2020). From this perspective, our study suggests that the sesquiterpene laurinterol could be a potential candidate for inhibiting marine biofilms and may provide a valuable starting point for rational design of coatings with anti-biofilm properties.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study confirms the anti-biofilm activity of \u003cem\u003eLaurencia johnstonii\u003c/em\u003e extract and its main compound, laurinterol, against all biofilm-forming marine bacterial strains tested. These findings not only support the potential of \u003cem\u003eL. johnstonii\u003c/em\u003e as a valuable natural source of bioactive compounds with potent anti-biofilm properties but also underscore its significance in marine biotechnology and antimicrobial research. The extract and laurinterol demonstrated consistent efficacy in inhibiting biofilm formation, suggesting their potential use in preventing bacterial colonization in aquatic environments, where biofilm-related issues are common.\u003c/p\u003e \u003cp\u003eFurthermore, the promising results of this study highlight the importance of exploring marine resources, particularly algae, as an untapped reservoir for bioactive compounds. As biofilm formation is a major contributor to chronic infections, fouling, and other industrial challenges, identifying natural substances like laurinterol offers a sustainable and environmentally friendly alternative to traditional chemical treatments. The potential application of \u003cem\u003eL. johnstonii\u003c/em\u003e extract in various industries, such as healthcare, marine engineering, and environmental conservation, warrants further investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge the Consejo Nacional de Humanidades, Ciencias y Tecnolog\u0026iacute;as (CONAHCYT) for their support of this project, including the post-doctoral fellowship (3813579), as well as the COFAA program of the Instituto Polit\u0026eacute;cnico Nacional (IPN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are within the manuscript and supplementary material file. Any additional data is available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWriting original draft preparation: MPAS. Methodology: RNAR, SGD, MMO. Data curation and formal analysis: MPAS, ALGC, SGD. Writing-review and editing: ALGC, RNAR, SGD, MMO. Project administration and supervision: RNAR, MMO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbott I, G J Hollenberg (eds) (1976) Marine algae of California. Stanford University Press, Redwood City. p 844\u003c/li\u003e\n\u003cli\u003eAbioye OP, Loto CA, Fayomi OSI (2019) Evaluation of anti-biofouling progresses in marine application. 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Chem Biodiver e202400833\u003c/li\u003e\n\u003cli\u003eZammuto V, Rizzo MG, Span\u0026ograve; A, Genovese G, Morabito M, Spagnuolo D, Capparucci F, Gervasi C, Smeriglio A, Trombetta D, Guglielmino S, Nicol\u0026ograve; MS, Gugliandolo C (2022) In vitro evaluation of antibiofilm activity of crude extracts from macroalgae against pathogens relevant in aquaculture. Aquaculture 549:737729.\u003c/li\u003e\n\u003cli\u003eZhao A, Sun J, Liu Y (2023) Understanding bacterial biofilms: From definition to treatment strategies. Front Cell Infect Microbiol, 13, 1137947.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"algae, biofouling, marine natural products, metabolites, sesquiterpene","lastPublishedDoi":"10.21203/rs.3.rs-5227932/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5227932/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"A biofilm is a complex microbial community, representing the initial stage in biofouling formation. Consequently, it is responsible for significant economic losses in several industrial sectors worldwide. Therefore, there is a constant need for safer and environmentally friendly coatings, particularly those derived from new marine sources such as seaweeds with antifouling properties. Red algae produce metabolites that prevent bacterial attachment and biofilm formation by disrupting microbial membranes, inhibiting quorum sensing, or interfering with extracellular matrix production, among them Laurencia johnstonii. This species has particular ecological or geographical advantages that make it more accessible or abundant for research in the Bay of La Paz, BCS. This study aimed to assess the anti-biofilm potential of the red seaweed Laurencia johnstonii. To identify bioactive compounds, the anti-biofilm activity of the ethanolic extract was evaluated against marine biofilm-forming strains: Bacillus altitudinis, Bacillus pumilus, Bacillus subtilis, and Bacillus cereus. The ethanolic extract of L. jonhstonii exhibited the highest percentage of inhibition. Subsequent chromatographic fractionation led to the isolation and identification of laurinterol, the primary compound responsible for the anti-biofilm activity (\u003e97 %) and antibacterial activity (MIC \u003c3.9 µg/mL). To our knowledge, this is the first report of the activity of laurinterol against biofilm-forming strains.","manuscriptTitle":"Unraveling the anti-biofilm properties of Laurinterol on pioneer biofouling bacteria from the red seaweed Laurencia johnstonii ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 06:09:54","doi":"10.21203/rs.3.rs-5227932/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-12-12T04:14:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21732776240608018802139945264370033465","date":"2024-11-26T15:04:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42860018834943121896565202898979727251","date":"2024-11-21T14:17:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-21T05:54:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-21T05:39:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2024-11-20T19:38:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"974692a1-c342-4110-a7dc-e56be85a8f03","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-12-31T05:53:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-26 06:09:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5227932","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5227932","identity":"rs-5227932","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}