Bioactivity of amphidinol-like extracts of Amphidinium carterae grown under varying cultivation conditions

Bioactivity of amphidinol-like extracts of Amphidinium carterae grown under varying cultivation conditions | 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 Bioactivity of amphidinol-like extracts of Amphidinium carterae grown under varying cultivation conditions Maria Elena Barone, Maria Elena Barone, Elliot Murphy, David Fierli, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3048270/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 Microalgae have attracted interests from the bioenergy, bioremediation, cosmetics and pharmaceutical sectors due to produce biofuels, pigments, omega-3 fatty acids, antioxidants or antimicrobial compounds. Among others, amphidinol-like compounds biosynthesised by dinoflagellate species of the genus Amphidinium have shown promising antimicrobial properties. Here, a two-stage cultivation regime was used to grow A.carterae (subclade III), which was exposed to different conditions, including an illumination regime switch to LED blue light, a salinity enhancement treatment, sodium carbonate or hydrogen peroxide supplementation. A solid phase extraction of the dried biomass was carried out and returned 6 fractions for each treatment, which were analysed by LC-HRMS. Amphidinol-like compounds AM-B, AM-C, AM-22 and AM-A were detected through their fragmentation patterns, mostly in Fraction-4 samples. AM-B was the major analogue in Fraction-4 ( m/z 1463.69, 42.3 + / - 16.5 % of amphidinol composition), followed by AM-C ( m/z 1343.84, 21.4 + / - 6.6 %). In Fraction-5, along with an unidentified analog with m/z AM-1320, AM-C was the most prevalent amphidinol (15.5 + / - 3.0 %) which was on average as abundant as in Fraction-4. This returned the highest antimicrobial activity against the pathogens S.aureus , E.faecalis and C.albicans , with MBC ranging from 1 to 512 µg×mL -1 . Further analysis using the Bocillin competition assay showed that the antibacterial activity was not associated with the presence of compounds targeting penicillin binding proteins (PBPs) in the cell wall of bacteria. Results indicate that the modulation of both amphidinol profile and fraction bioactivity can be induced by adjusting the cultivation parameters used to grow two-stage batch cultures of A. carterae . microalgae MNPs antimicrobials amphidinols LC/HRMS Amphidinium carterae Staphylo-coccus aureus Enterococcus faecalis Candida albicans. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key points Amphidinols were most prevalent in SPE-derived/Fraction-4 samples and mainly included AM-B, AM-C, AM-22 and AM-A, as determined by LC-HRMS/MS. Antimicrobial activities were observed for some fractions against Staphylococcus aureus , Enterococcus faecalis and Candida albicans and were the highest in Fraction-4 samples (eluted in H 2 O/MeOH 25:75). The content in amphidinols was enhanced on average 7-fold for the carbonate supplementation treatment compared to the control. Introduction Microalgae can biosynthesise a wide diversity of metabolites, which include for example antioxidants, pigments, fatty acids, vitamins, proteins or carbohydrates. Those have applications in industrial sectors associated with the nutraceutical, bioenergy, pharmacological or cosmetics fields [Pisoschi et al. 2018 ; Montes D’Oca et al. 2011 ; Saide et al. 2021 , Yarkent et al. 2015; Khan et al. 2018 ]. The cultivation of marine microalgae offers some advantages over terrestrial plant crops in that their physico-chemical environment can be carefully controlled in photobioreactors, that they do not compete for arable lands or that they can contribute to safeguarding freshwater resources [Saide et al. 2021 ; Khan et al. 2018 ]. The metabolic plasticity of microalgae can also be exploited to trigger the accumulation by cells of high-value compounds. For example, some species can overproduce lipids or carotenoids under conditions of high salinity, high illumination exposure or nutrient limitation [Sun et al. 2018 ; Illman et al. 2000 ; Khotimchencko et al. 2005; Renaud et al. 2002 ; Wensel et al. 2014 ; Liu et al. 2008 ]. In addition, two-stage cultivation modes have increasingly been considered to produce biomass under favorable conditions then to trigger, later in the growth of the culture using a stressor, the production of sought-after compounds [Sun et al 2018 ]. Microalgae are therefore viewed as promising sources of metabolites. Several natural products of microalgal origins such as macrolides (e.g. amphidinolide Q), alkaloids (e.g., eucapsitrione), aromatic compounds (e.g., cyanobacterin), pigments (e.g., fucoxanthin), fatty acids (e.g., EPA), phenolic derivatives (e.g., cyanobacterial 4-40-hydroxybiphenyl), polyketides (e.g., amphidinin B) or terpenoids have shown promising bioactivity against a wide range of pathogens [Liu et al. 2015 ; Swain et al. 2017 ; Karpiński et al. 2019; Desbois et al. 2008 ; Kubota et al. 2006; Venugopal et al. 2020 ]. Among microalgae, some marine dinoflagellates have been shown to produce bioactive polyhydroxylated polyketides known as amphidinols [Satake et al. 1991 ]. These complex polyketides have been extracted from several species within the genus Amphidinium (e.g., A. carterae , A. gibbosum , A. massartii , A. operculatum ) [Wellkamp et al. 2020 ]. In excess of 60 derivatives have been isolated from Amphidium sp. and classified in different groups such as amphidolactones, lasonolide, iriomoteolides, amphirions, colopsinols, amphezonol, luteophanol, karatungiols, amphidinolides, amphidinols, amphidirins or lingshuiol [Takahashi et al. 2007 , McConnell et al. 1994, Song et al. 2003 , Blunt et al. 2015 , Kumagai et al. 2017 , Kumagai et al. 2014, Akakabe et al. 2014, Kobayashi et al. 2003, Kobayashi et al. 2006, Kubota et al. 2005 , Washida et al. 2006 , Kobayashi et al. 2008, Huang et al. 2004 ]. Several purified compounds such as karatungiol A and lutheophanol D have shown potent bioactivity against bacterial and fungal pathogens, including for instance Staphylococcus sp., Micrococcus sp., Aspergillus niger or Candida albicans , as well as several human tumoral cell lines [Barone-Murphy et al. 2021; Martinez et al. 2019 ; Wellkamp et al. 2020 ; Kubota et al. 2005 ; Kobayashi et al. 2006]. Previous work carried out with extracts of CCAP 1102/8 Amphidinium carterae isolated from the west of Ireland showed the presence of AM-A, AM-B, AM-22, and a new derivative later identified as AM-C [Murphy et al. 2022 ]. Fractions obtained by solid phase extraction enriched in AM-A and AM-C exhibited bactericidal effects on the Gram-positive pathogens Staphylococcus aureus and E. faecalis. Here, the objective of this study was to cultivate CCAP 1102/8 A. carterae was under varying conditions using a two-stage batch regime to assess potential variation in its amphidinol profile determined by LC-HRMS, and changes in the bioactivity of the corresponding fraction against Gram-positive bacteria and the fungus Candida albicans . Materials and Methods Microalgal cultivation Five photobioreactor (GroTech) were prepared using Guillard f/2 medium (final volume of 6.3 L) with an initial seeding of 0.5 mg/mL (wet weight) of the marine dinoflagellate CCAP 1102/8 Amphidinium carterae . The reactors were incubated for 20 days at 20 ± 1 o C under ca. 60–80 µmol m 2 s- 1 illumination provided by white light LED panels using a 14:10 light:dark photoperiod. The culture was aerated (210 mL/min) through a sterile 0.22 µm-pore size syringe filter. On day 20, after the culture had reached the late exponential growth phase, the cultivation conditions were modulated in terms of exposure to blue light, hydrogen peroxide, sodium carbonate and sodium chloride (Table 1 ). To do so, the reactors were supplemented with 630 mL of filter-sterilised seawater supplemented where appropriate with the relevant chemicals. On day 30, the microalgal biomass was collected from each reactor after centrifugation (2000 rpm , 5 min) and desalted with ammonium formate (0.5 M). The samples were then freeze-dried overnight (Scanvac. MillRock) and stored at -20 o C until further processing. Table 1 Second stage cultivation conditions applied on day-20 for CCAP 1102/8 Amphidinium carterae . Abbreviation Condition Description References BL Blue Light LED 100 µmol m- 2 s- 1 Humphrey et al. 1983 HS High Salinity 0.8 g/L of NaCl (from 30 up to 55 mS/cm) Boukhris et al. 2017 NaHCO 3 Sodium Carbonate 2.5 mmol l -1 Kitaya et al. 2008 H 2 O 2 Hydrogen Peroxide 0.5 mM Barone et al. 2021 C+ Control seawater n/a Solid Phase Extraction (SPE) Extracts were prepared for each A. carterae reactor, from which 500 mg of dried biomass was extracted with 60 mL (3 x 20 mL) of methanol aided by sonication (Elma P Sonic) (3 x 15 min freq. 37% power 90%) followed by centrifugation (3 x 10 min at 4500 rpm). Each crude extract was filtered (VWR Qualitative Retention 40 µm), transferred to pre-weighed vials and dried at 40°C under N 2 stream. The extracts were resuspended in 10 mL of MeOH (purity?) and 500 mg of C 18 powder (POLYGOPREP 50–60 C 18 ) was added to each. The extracts were then partially dried under reduced pressure. A C 18 SPE cartridge was conditioned with MeOH (2 x 6 mL) followed by H 2 O (2 x 6 mL). Once conditioned, the dried samples bound to C 18 was loaded onto the column. Six fractions (12 mL) were then recovered by eluting solvent mixtures of decreasing polarity: (A) 100% H 2 O, (B) H 2 O:MeOH (75:25 v/v ), (C) H 2 O:MeOH (50:50 v/v ), (D) H 2 O:MeOH (25:75 v/v ), (E) MeOH 100% and (F) MeOH:CH 2 Cl 2 (50:50 v/v ). The fractions were dried under reduced pressure and weighed prior to storage for further chemical and antimicrobial activity analyses. Liquid Chromatography Mass Spectrometry (LC-HRMS) Approximately 0.2 mg of dried fractions was resuspended in the corresponding fraction solvent at a concentration of 1 mg⋅mL − 1 prior to chemical profiling. Samples were run in positive MS (200–3000 m/z ) and MS/MS modes. High resolution mass spectra data were obtained with an Agilent 6540 qTof mass spectrometer UHPLC-DAD-HRMS. MS/MS data used a cone voltage of 40 V, and a collision energy of 75 V. The cone and desolvation gas flows were set at 300 and 12 L/min, respectively, and the source temperature was 300 C. A binary gradient elution was used, with phase A consisting of water and phase B of acetonitrile in water (both containing 6.7 mM ammonium formate). The column used was a 50 mm × 2.1 mm i.d., 1.7 µm, Acquity UPLC BEH C 18 (Waters). The gradient was from 30–90% B over 11 min at 0.4 mL/min, held for 1 min, and returned to the initial conditions over 1 min and held for 2 min to equilibrate the system. The injection volume was 5 µL and the column and sample temperatures were 40°C and 10°C, respectively. HRMS spectra were obtained using the LCMS conditions described by Welkamp et al. [2020]. All fractions were analysed by UHPLC-HRMS/MS. Microbial strains Microbiological assays were carried out using the Gram-positive strains S. aureus ATCC 25923, E. faecalis ATCC 29212 and Candida albicans ATCC 90028. The strains were grown in selective agar media (Bile Aescuilin Azide (BEA) for E. faecalis , Mannitol Salt Agar (MSA) for S. aureus ) then incubated at 37°C overnight prior to carrying out the bioassays. Sabouraud Dextrose Agar (SDA) and overnight incubation at 35 o C were used for C. albicans . All media were manufactured by Oxoid (Basingstoke, UK). Determination of Minimum Inhibitory Concentrations (MIC) The susceptibility tests were carried out using the broth microdilution assay with kanamycin as positive bacterial break point control according to the CLSI guidelines [Clinical & Laboratory Standard Institute, 2020]. C. albicans ATCC 90028 was analysed according to the CLSI M27-A2 guidelines using Amphotericin B as control. A single colony of the ATCC strains was inoculated in 5 mL of BHI (Brain Heart Infusion broth) for bacterial strains, and RPMI-1640 for C. albicans , for 4 to 6 hours, to achieve the exponential phase. Then, 10 5 CFU/mL was inoculated to in 96-well plate in 0.1 mL of sterile Muller-Hinton broth (MHB). All of the extracts were re-suspended in acetone:deionised water (10:90 v/v). Prior to bacterial inoculation, scalar dilutions (1:2) of the extracts were performed from the 1st to the 11th wells of a row of a 96-well plate with a final volume of 100 µL of MHB. The 12th well was used as a growth control. Control wells of MHB and solvents were also included. Triplicate wells were used for all the samples and the plates were incubated at 37 o C for 14–18 h. Wells inoculated with C. albicans were incubated at 35 o C. The MIC values were determined after 24 h and confirmed after 48 h. Nitro blue-tetrazolium (1 mg/mL) was then added to each well and incubated for 30 minutes to detect the MIC points. Determination of Minimum Bactericidal and Fungicidal Concentration A volume of 50 µL from all the wells above the MIC values were inoculated in Muller-Hinton agar media. The plates were further incubated for 24 h and 48 h for bacteria and yeast, respectively. The minimum bactericidal/fungicidal concentrations were then identified as the lowest concentration of extract returning no growth. All analyses were performed in triplicates. Penicillin-binding Protein (PBP) competition assay The potential binding of the compound at different MBC concentrations (0.5X, 1X, 2X) to Penicillin Binding Proteins (PBPs) was evaluated in a competition assay with BOCILLIN™ FL (Invitrogen™), a fluorescent reporter molecule analog to Penicillin V. The membrane fractions for the detection of PBPs were prepared based on Djoric’ et al. (2020) and Arbeloa et al. ( 2004 ) methodologies. Protein samples were incubated at room temperature for 10 min with 5 µg/mL (7.5 µM) of BocillinTM FL (Invitrogen™), followed by SDS-PAGE on a Criterion™ TGX™ (Tris-Glycine eXtended) 10% polyacrylamide gel. Electrophoresis was carried out for 2 h at a constant voltage of 100V. The fluorescent Bocillin covalently bound to the PBPs was detected with excitation at 488 nm and emission at 520 nm (Typhoon FLA 9500; filter Alexa 488; PMT 1000; pixel 50 µm). Data treatment Data collected was analysed using IBM SPSS Statistics 26 package 2. Principal Component Analysis (PCA) was carried out to assess the relationship between peak heights and bioactivity using a correlation matrix in a Varimax rotation mode. Results Liquid chromatography and mass spectrometry analysis The fractions were first analysed by UPLC-DAD-HRMS/MS for the presence of amphidinol-like compounds for each of the five growth conditions. Amphidinol type compounds were detected through their characteristic fragmentation patterns in Fractions-4 (H 2 O/MeOH 25:75) and − 5 (MeOH) (Fig. 1 ). These compounds were below the level of detection for fractions 1, 2, 3 and 6. Retention times for the compounds ranged from 3.4–5.6 min with m/z of 1361, 1463, 1667, 1343, 1320 and 1454 during a 11-min UHPLC run. Compounds with m/z 1361 and 1463 shared identical fragmentation patterns with AM-A and AM-B, respectively, and were concluded to be both compounds from analysis of data previously published by Welkamp et al. [2020]. Both compounds shared key fragments at m/z 1085 (C-29/C-30) and 687 (C-29/C-30 and C-1/C-1’) as well as a characteristic loss of water molecules. AM-22 was also identified in Fraction 4 through the m/z 1667 [M + Na] + . Further, the recently characterised AM-C previously reported as N16 as mentioned in Murphy et al. [ 2022 ] and Welkamp et al. [2020] was detected with the m/z 1343 [M + Na] + . The fragmentation pattern of AM-C and its m/z 1343 revealed key fragments at m/z 1085 and 945. A comparative study was performed on Fractions 4 and 5 for each growth condition and the control sample. Ionisation potential was assumed to be shared between analogues so that the relative integration of the amphidinol derivatives can be assessed for each fraction. AM-B appeared as the major analogue in Fraction-4 while a derivative of AM-A with m/z 1320 and a very close fragmentation pattern was found to be the major amphidinol in Fraction-5. In Fraction 4, the control treatment exhibited the lowest relative abundance in targeted amphidinols, while the highest abundance was for the NaHCO 3 and H 2 O 2 treatments. Overall, the highest abundance in AMs was observed in Fraction-5 extracts, in particular for the H 2 O 2 and NaCl treatments, where the unknown amphidinol derivative at m/z 1320 was very prominent (Fig. 1 ). The unknown derivative AM-1320 (with a sodium adduct at m/z 1342) exhibited the highest concentration of all analogues present. The analogue presented a key fragment at m/z 1084 instead of 1085 for AM-A and -B, indicating the possible substitution of a hydroxyl group for an NH 2 between C-44 and C-65 as the 945 fragment is still present (Fig. 2 ). An unreported m/z 1454.84 was also identified with a similar elution time to AM-A. They both shared similar fragments. This may be a new derivative analogue coeluting with AM-A but showing a distinct fragmentation pattern as AM-B (Fig. 2 ). Bioactivity: determination of Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal-Fungicidal Concentrations (MB-FCs) The bioactivity of the six fractions generated via solid phase extraction from the five reactors was assessed against three pathogens (Fig. 3 ). The bioactivities varied for MBCs between 8 and 512 µg⋅L − 1 for S. aureus and between 128 and 4096 µg⋅mL − 1 for E. faecalis , while the MFCs varied between 16 and 512 µg⋅mL − 1 for C. albicans (5 to 6 scalar dilutions overall). Fractions-1 and − 2 did not return appreciable antimicrobial activity against the pathogens tested. The Fraction-6 samples gave very weak MICs against E. faecalis . For Fraction-3, only the samples from the NaHCO 3 and BL treatments returned bactericidal and fungicidal activities against S. aureus and C. albicans , respectively. The Fraction-5 extracts inhibited E. faecalis , with a MIC value of 16 µg⋅mL − 1 . However, MBCs could not be determined for any of the extracts analysed, indicating a bacteriostatic effect. The main antimicrobial activity was observed in Fraction-4, which returned significant MICs and MB-FCs against each microbial pathogen. S. aureus reacted the most to the Fraction-4 sample of the NaHCO 3 treatment (MBC of 8 µg⋅mL − 1 ). C. albicans appeared more susceptible to the Fraction-4 from the blue light treatment (MFC of 1 µg⋅mL − 1 ). E. faecalis was less sensitive to Fraction-4 than the other two pathogens (MBC > 128 µg⋅mL − 1 ). Penicillin-binding Protein (PBP) competition assay Fraction-4 extracts gave the highest bioactivities against the bacterial pathogens tested and were selected for analysis by the PBP competition assay. The extracts used for PBP labeling with Bocillin FL showed no demonstrable binding affinity for PBPs with the extracts tested against ATCC 29212 E. faecalis and ATCC 25923 Staphylococcus aureus strains, suggesting that no compound in these fractions showed affinity for PBPs at the tested concentrations ranging from 0.5X to 4X MBC values (Fig. 4 ). Principal Component Analysis (PCA) This analysis focused on the most bioactive fractions (Fractions3, 4 and 5) containing amphidinol-like compounds to delineate patterns of bioactivity in the extracts tested against the three pathogens used (Fig. 5 ). Fraction-5 were predominantly grouped together along the positive domain of Component 1 (PC1) while Fraction-3 extracts clustered along the negative domain. Fraction-4 extracts were distributed between both the negative and positive domains of component 2 (PC2). Some amphidinols identified in the fraction by LC-HRMS were clearly associated with Fraction-3 (AM-1348, AM-1371), Fraction-4 (AM-B, AM-C, AM-22, AM-1454, AM-1225, AM-A) and Fraction-5 (AM-C, AM-1320, AM-1325). Noticeably, the bioactivity vectors were mostly oriented toward the positive domain of PC2 and Fraction-4 extracts (except that of MIC for E. faecalis along the positive domain of PC1). Discussion Microalgae have attracted interests due to their capacity to produce high-value compounds with potential applications in the nutraceutical and cosmetics sectors [Fabris et al. 2020 ]. They have also been increasingly considered as potential sources of new antimicrobial compounds, the development of which is urgent to alleviate the emerging antibiotic resistance of various pathogens in health settings worldwide [WHO 2021]. Multiple bioprospecting studies for new microalgal species and strains have been conducted and various extracts and compounds tested against a range of pathogens, including bacteria and fungi [McGee et al., 2020; Desbois et al.2008; Falaise et al. 2019]. Of interest here, polyketide compounds of the amphidinol type, which are biosynthesised by Amphidinium spp., have shown promising bioactivity against such pathogens Amphidinol-containing fractions of a strain of A. carterae (sub-clade III) previously isolated from Ireland were shown to exhibit antimicrobial activity against Gram-positive bacteria [Kubota et al. 2006, Martinez et al. 2019 , Barone et al. 2021 ]. Here, this dinoflagellate was grown under varying conditions to assess whether or not they could influence the amphidinol-like compound profile and the antimicrobial properties of extracts. Variations in amphidinol profile Amphidinium carterae strain CCAP 1102/8 was grown via a two-stage process. LC-MS analysis was carried out on SPE-derived fractions in which variations in the concentration of several amphidinol-like compounds were observed. The extracts which indicated the highest concentrations of amphidinols were obtained from the biomass retrieved from cultures exposed to stressors. However, when harvested, these cultures returned lesser amounts of biomass compared to the control treatment. This is in agreement with previous studies carried out on a variety of microalgal species, which have indicated that the modulation of cultivation conditions can often increase the intra-cellular yields of particular metabolites but hamper growth [Liyanaarachchi et al. 2021 , Pan et al. 2019 , Humphrey et al. 1983; Das et al. 2011 , Boukhirs et al. 2017, Qiao et al. 2020]. For instance, relevant to Amphidinium sp., Tsirigoti et al. [ 2020 ] demonstrated the benefits of applying low temperature to increase the production of DHA, an ω-3 fatty acid which has previously shown antimicrobial activity against S. aureus and MRSA strains [Desbois et al. 2006]. Several amphidinols with similar and characteristic fragmentation patterns were detected by LC-HRMS/MS analysis, with variations in their concentration in Fractions-4 and − 5. In Fraction-4, the control treatment exhibited the lowest relative concentration of the amphidinols, while the highest amounts were obtained for the NaHCO 3 and H 2 O 2 samples. AM-B, AM-C, AM-22 and AM-A were prominent in Fraction-4. Their abundance in the extracts of the NaHCO 3 , Salinity, Blue Light and H 2 O 2 treatments were on average 2- to 4-fold higher than the control, respectively. AM-1320 followed by AM-C were the major identified amphidinol in Fraction-5. The concentration in AM-C was the greatest for the H 2 O 2 treatment, with a 2.1-fold enhancement compared to the control treatment. Future work should consider the extent by which further modulation in supplement concentrations, light intensity or photoperiod patterns could affect the profile and abundance of amphidinols. Likewise, different time points could be analysed during the second stage of cultivation. Antimicrobial activity of amphidinol-containing fractions Several amphidinol-like compounds have been isolated from Amphidium species [Satake et al. 1991 , Kobuta et al. 2004, Kobayashi et al. 2005, Welkemp et al. 2020, Martinez et al. 2019 , Barone et al. 2021 ]. Some purified compounds such as Amphidinol-22, Amphidinol-A or Amphidinol-20 have shown different bioactivities against various pathogens and cancer cell lines [Martinez et al. 2019 , Welkamp et al. 2020]. Crude methanolic extracts of A. carterae biomass and several purified amphidinols have previously shown antifungal activities, with MICs ranging from 560 µg⋅mL − 1 to 9 µg⋅mL − 1 against Aspergillus spp. or Candida albicans [Echigoya et al. 2005 ; Martinez et al. 2019 ; Nuzzo et al.; Cutignano et al. 2017 ]. Some antibacterial properties have also previously been attributed to amphidinol-like molecules such as Lutheophanol D, Amphidinin C, Amphidinin E, Amphidinin F and Amphidinin G extracted from A. carterae against Micrococcus lentus , S. aureus , B. subtilis and E. coli [Kubota et al. 2005 , Kubota et al. 2014 ]. Amphidinols have not been tested as extensively against bacteria. Recent work showed however that methanolic extracts from A. carterae (strain CCMP1314 from the USA, sub-clade III) containing AM-22 elicited antimicrobial activity against E. coli , Methicillin-Resistant S. aureus (MRSA), Methicillin-Susceptible S. aureus (MSSA) and M. tuberculosis [Martinez et al. 2019 ]. This antibacterial potential was also supported with our CCAP 1102/8 Amphidinium carterae strain from the West of Ireland [Barone et al. 2021 ]. The present study corroborates these findings against Gram-positive bacteria and the fungus C. albicans , further detailing how the bioactivity and amphidinol signature of extracts could be impacted by the conditions under which the cells were cultivated. The potential additive or synergistic bioactivity of several amphidinols or other co-extracted compounds in the samples tested, in particular against bacteria, warrants in that aspect further scrutiny. The highest biocidal activities were observed for the Fraction-4 samples, returning MBCs of 8 and 128 µg⋅mL − 1 against S. aureus , and E. faecalis , respectively, and a MFC of 1 µg⋅mL − 1 against C. albicans . These extracts all contained varying amounts of the amphidinols AM-22, AM-C and AM-B. Martinez et al. [ 2019 ] showed purified AM-22 to be effective against C. albicans and A. fumigatus at 64 µg⋅mL − 1 . Given that amphidinols seem bioactive against several fungi at doses inferior to 64 µg⋅mL − 1 , the conduct of potentiation or synergism experiments is warranted, as is the acquisition of data on their pharmaco-kinetics. Such trials could consider the use of individual or several purified amphidinols applied together or with other known bioactive compounds, such as miconazole, which is biocidal against C. albicans, S. aureus and E. faecalis between 0.78–6.25 µg⋅mL − 1 [Isham et al. 2010; Nenoff et al. 2017 ]. In addition, and noteworthily, Fraction5 extracts showed substantial bacteriostatic activity against E. faecalis , which may be associated with m/z 1320, which was the most abundant compound in these extracts, and would deserve purification and structure elucidation. A mode of action not associated with penicillin binding proteins Experimental data obtained using AM-3 has suggested the mechanism of action of amphidinols to be linked with the capacity of their polyol chain to penetrate membrane lipidic bilayers in the presence of sterol, causing the death of eukaryotic cells such as yeasts as well as hemolysis [Satake et al 2017 ; Wakamyia et al. 2020]. Here, the Fraction-4 samples of A. carterae , which were the most bioactive against the three pathogens used in this study, were tested using the Bocillin-FL assay for the presence of molecules containing ring structures similar to the beta-lactam ring of penicillin. Such structures are able to acylate and covalently bind to the active serine residue at the catalytic site of specific penicillin binding proteins present in the cell wall of bacteria, leading to its disruption and cell death [Djoric’ et al. 2020, Arbeloa et al. 2004 ]. No concentration-dependent decrease of fluorescence in both S. aureus and E. faecalis was observed, excluding the presence of metabolites in the extracts targeting PBPs. Further studies are hence necessary to better characterise the mode of action of amphidinols on relevant bacterial pathogens. Declarations Author’s contribution : Conceptualisation by MEB, GTAF and NT. Methodological design by MEB, DF, EM and FC. Data curation by EM and MEB. Analyses by MEB, FC, EM, OPT and NT. Writing, review and editing were performed by MEB, DF, FC, EM, GTAF, OPT and NT. Declaration of Competing Interest : The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding: The authors acknowledge financial support from the VES4US project funded by the European Uninion’s Horizon 2020 research and innovation programme under grant agreement No 801338. 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Wellkamp M, Garcis-Camacho F, Duran-Riveroll LM, Tebben J, Tillmann U, Krock B (2020) LC-MS/MS Method Development for the Discovery and Identification of Amphidinols Produced by Amphidinium. Mar. Drugs 18: 497. doi: 10.3390/md18100497. World Health Organization (WHO) (2021) Global Antimicrobial Resistance and Use Surveillance System (GLASS) Report 2021 ; WHO: Geneva, Switzerland. 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. 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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-3048270","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":226346225,"identity":"9297bde1-e6d5-4a20-8143-3544a4eba945","order_by":0,"name":"Maria Elena Barone","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACdgYGZgiL+QCQkJAhrIUZroUtAaSFhxQtPAZgkqAOfmbeA8wFNffkzNl7Pr+6UWPBw8B++OgGfFokm/kSmGccKza27Dm7zTrnGNBhPGlpN/BpMTjMY8DMw5aQuOFG7jbjHDagFgkeM7xa7MFa/gG13H/zzDjnHxFagOoNmHnbQLbwMD/ObSNCiwTQlsMz+xKMDc6kmTHn9knwsBHyC397j+Hjgm8JcgbHDz/+nPOtTo6f/fAxvFpA4ACUZpMAk4SUIwPmD6SoHgWjYBSMgpEDANdKQFCtxkp+AAAAAElFTkSuQmCC","orcid":"","institution":"Atlantic Technological University - Sligo","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Elena","lastName":"Barone","suffix":""},{"id":226346226,"identity":"259b82bb-0abc-4692-903d-4a336597479d","order_by":1,"name":"Maria Elena Barone","email":"","orcid":"https://orcid.org/0000-0001-8240-9464","institution":"Institute of Technology Sligo: Atlantic Technological University - Sligo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Elena","lastName":"Barone","suffix":""},{"id":226346227,"identity":"3afb4cc3-3032-47a0-95a2-1a04355436fc","order_by":2,"name":"Elliot Murphy","email":"","orcid":"","institution":"National University of Ireland - Galway Martin Ryan Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elliot","middleName":"","lastName":"Murphy","suffix":""},{"id":226346228,"identity":"b32ad360-f484-4042-88a4-ca47a46b9bb9","order_by":3,"name":"David Fierli","email":"","orcid":"","institution":"Institute of Technology Sligo: Atlantic Technological University - Sligo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Fierli","suffix":""},{"id":226346229,"identity":"d342687a-d9bd-4985-a440-3348fc00f78e","order_by":4,"name":"Floriana Campanile","email":"","orcid":"","institution":"Universita degli Studi di Catania","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Floriana","middleName":"","lastName":"Campanile","suffix":""},{"id":226346230,"identity":"9420a55c-c467-4d2a-9c1d-357a71eb35dc","order_by":5,"name":"Gerard T. A. Fleming","email":"","orcid":"","institution":"NUIG: University of Galway","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gerard","middleName":"T. A.","lastName":"Fleming","suffix":""},{"id":226346231,"identity":"a2751829-820f-43b7-a063-f796529612de","order_by":6,"name":"Olivier P. Thomas","email":"","orcid":"","institution":"National University of Ireland - Galway Martin Ryan Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"P.","lastName":"Thomas","suffix":""},{"id":226346232,"identity":"67b65007-8e17-4f51-a4c8-7e7543ab87c6","order_by":7,"name":"Nicolas Touzet","email":"","orcid":"","institution":"Institute of Technology Sligo: Atlantic Technological University - Sligo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Touzet","suffix":""}],"badges":[],"createdAt":"2023-06-11 05:34:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3048270/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3048270/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":41867926,"identity":"d718f92e-eb56-4f4e-9b98-e131c28af723","added_by":"auto","created_at":"2023-08-21 13:17:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49657,"visible":true,"origin":"","legend":"\u003cp\u003eAbundance (peak areas) of the major amphidinol derivatives identified in Fraction 4 and Fraction 5 (A) and their individual relative proportions (B). The dash line separates Fraction 4 (left) from Fraction 5 (right) samples.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3048270/v1/f0a351ae47eae027e87503a3.png"},{"id":41867925,"identity":"32a03283-816c-4c5e-9fce-582726d70190","added_by":"auto","created_at":"2023-08-21 13:17:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27450,"visible":true,"origin":"","legend":"\u003cp\u003eCID spectra of potentially new compounds found in A. carterae strain LACW11: A) AM-1320 t\u003csub\u003eR\u003c/sub\u003e 206.8, B) AM-C t\u003csub\u003eR\u003c/sub\u003e 228.1, C) AM-1454 t\u003csub\u003eR\u003c/sub\u003e 219.3.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3048270/v1/4bfe3a4b4dd06ed86cf2bad3.png"},{"id":41867929,"identity":"5b630665-20b8-4c27-84ac-f217fe03eeb8","added_by":"auto","created_at":"2023-08-21 13:17:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1434784,"visible":true,"origin":"","legend":"\u003cp\u003eHeat Map of the MICs and MB-FCs obtained for the \u003cem\u003eA. carterae\u003c/em\u003e fractions analysed against three pathogens (gradient of activity from red (low) to green (high).\u0026nbsp; The activities are expressed in µg×mL\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3048270/v1/c6e0e64d990fdfddd56f94af.png"},{"id":41867927,"identity":"6a9d9ab3-cbf1-4dcd-8a2b-9b2a3fa74ed1","added_by":"auto","created_at":"2023-08-21 13:17:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91068,"visible":true,"origin":"","legend":"\u003cp\u003eCompetition assays for crude extract binding to PBPs in E. faecalis ATCC 29212 using Bocillin FL.\u0026nbsp; C+ free control treated without exposure to crude extract; 1) F4-H\u003csub\u003e3\u003c/sub\u003eCO\u003csub\u003e2\u003c/sub\u003e, 18.8 µg×mL\u003csup\u003e-1\u003c/sup\u003e 2) F4-H\u003csub\u003e3\u003c/sub\u003eCO\u003csub\u003e2 \u003c/sub\u003e37.6 µg×mL\u003csup\u003e-1\u003c/sup\u003e, 3) F4-H\u003csub\u003e3\u003c/sub\u003eCO\u003csub\u003e2 \u003c/sub\u003e75.3 µg×mL\u003csup\u003e-1\u003c/sup\u003e, 4) F4-H\u003csub\u003e3\u003c/sub\u003eCO\u003csub\u003e2\u003c/sub\u003e 150.6 µg×mL\u003csup\u003e-1\u003c/sup\u003e 5) F4-Salinity 32 µg×mL\u003csup\u003e-1\u003c/sup\u003e, 6) F4-Salinity 64 µg×mL\u003csup\u003e-1\u003c/sup\u003e, 7) F4-Salinity 128 µg×mL\u003csup\u003e-1\u003c/sup\u003e, 8) F4-Salinity 256 µg×mL\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp; No gradual decrease of band fluorescence was observed compared to the control.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3048270/v1/b34904536323bfa8f74a1fd7.png"},{"id":41869179,"identity":"8e90f6e2-8d3e-4f6c-bcfd-cb7f795738a4","added_by":"auto","created_at":"2023-08-21 13:25:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":669053,"visible":true,"origin":"","legend":"\u003cp\u003ePCA analysis indicating the relationship between individual compounds, extracts and bioactivities. ∆ represents Fraction-3 extracts, \u003cstrong\u003e▢ \u003c/strong\u003erepresents Fraction 4 extracts and 〇 represents Fraction-5 extracts.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3048270/v1/60629d411e01b575156ff53f.png"},{"id":48931763,"identity":"02621a08-917d-4e96-b16d-894d89c644cb","added_by":"auto","created_at":"2023-12-28 19:22:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2736346,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3048270/v1/9938706e-ef99-4a1c-b95f-56e9d9ff74b7.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBioactivity of amphidinol-like extracts of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAmphidinium carterae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e grown under varying cultivation conditions\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Key points","content":"\u003cul\u003e\n \u003cli\u003eAmphidinols were most prevalent in SPE-derived/Fraction-4 samples and mainly included AM-B, AM-C, AM-22 and AM-A, as determined by LC-HRMS/MS.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul\u003e\n \u003cli\u003eAntimicrobial activities were observed for some fractions against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e and were the highest in Fraction-4 samples (eluted in H\u003csub\u003e2\u003c/sub\u003eO/MeOH 25:75).\u003c/li\u003e\n \u003cli\u003eThe content in amphidinols was enhanced on average 7-fold for the carbonate supplementation treatment compared to the control.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eMicroalgae can biosynthesise a wide diversity of metabolites, which include for example antioxidants, pigments, fatty acids, vitamins, proteins or carbohydrates. Those have applications in industrial sectors associated with the nutraceutical, bioenergy, pharmacological or cosmetics fields [Pisoschi et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Montes D\u0026rsquo;Oca et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Saide et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Yarkent et al. 2015; Khan et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e]. The cultivation of marine microalgae offers some advantages over terrestrial plant crops in that their physico-chemical environment can be carefully controlled in photobioreactors, that they do not compete for arable lands or that they can contribute to safeguarding freshwater resources [Saide et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e]. The metabolic plasticity of microalgae can also be exploited to trigger the accumulation by cells of high-value compounds. For example, some species can overproduce lipids or carotenoids under conditions of high salinity, high illumination exposure or nutrient limitation [Sun et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Illman et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Khotimchencko et al. 2005; Renaud et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wensel et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e]. In addition, two-stage cultivation modes have increasingly been considered to produce biomass under favorable conditions then to trigger, later in the growth of the culture using a stressor, the production of sought-after compounds [Sun et al \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicroalgae are therefore viewed as promising sources of metabolites. Several natural products of microalgal origins such as macrolides (e.g. amphidinolide Q), alkaloids (e.g., eucapsitrione), aromatic compounds (e.g., cyanobacterin), pigments (e.g., fucoxanthin), fatty acids (e.g., EPA), phenolic derivatives (e.g., cyanobacterial 4-40-hydroxybiphenyl), polyketides (e.g., amphidinin B) or terpenoids have shown promising bioactivity against a wide range of pathogens [Liu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Swain et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Karpiński et al. 2019; Desbois et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kubota et al. 2006; Venugopal et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong microalgae, some marine dinoflagellates have been shown to produce bioactive polyhydroxylated polyketides known as amphidinols [Satake et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1991\u003c/span\u003e]. These complex polyketides have been extracted from several species within the genus \u003cem\u003eAmphidinium\u003c/em\u003e (e.g., \u003cem\u003eA. carterae\u003c/em\u003e, \u003cem\u003eA. gibbosum\u003c/em\u003e, \u003cem\u003eA. massartii\u003c/em\u003e, \u003cem\u003eA. operculatum\u003c/em\u003e) [Wellkamp et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. In excess of 60 derivatives have been isolated from \u003cem\u003eAmphidium\u003c/em\u003e sp. and classified in different groups such as amphidolactones, lasonolide, iriomoteolides, amphirions, colopsinols, amphezonol, luteophanol, karatungiols, amphidinolides, amphidinols, amphidirins or lingshuiol [Takahashi et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, McConnell et al. 1994, Song et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Blunt et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Kumagai et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Kumagai et al. 2014, Akakabe et al. 2014, Kobayashi et al. 2003, Kobayashi et al. 2006, Kubota et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Washida et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Kobayashi et al. 2008, Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e]. Several purified compounds such as karatungiol A and lutheophanol D have shown potent bioactivity against bacterial and fungal pathogens, including for instance \u003cem\u003eStaphylococcus\u003c/em\u003e sp., \u003cem\u003eMicrococcus\u003c/em\u003e sp., \u003cem\u003eAspergillus niger\u003c/em\u003e or \u003cem\u003eCandida albicans\u003c/em\u003e, as well as several human tumoral cell lines [Barone-Murphy et al. 2021; Martinez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wellkamp et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kubota et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kobayashi et al. 2006].\u003c/p\u003e \u003cp\u003ePrevious work carried out with extracts of CCAP 1102/8 \u003cem\u003eAmphidinium carterae\u003c/em\u003e isolated from the west of Ireland showed the presence of AM-A, AM-B, AM-22, and a new derivative later identified as AM-C [Murphy et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e]. Fractions obtained by solid phase extraction enriched in AM-A and AM-C exhibited bactericidal effects on the Gram-positive pathogens \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eE. faecalis.\u003c/em\u003e Here, the objective of this study was to cultivate CCAP 1102/8 \u003cem\u003eA. carterae\u003c/em\u003e was under varying conditions using a two-stage batch regime to assess potential variation in its amphidinol profile determined by LC-HRMS, and changes in the bioactivity of the corresponding fraction against Gram-positive bacteria and the fungus \u003cem\u003eCandida albicans\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicroalgal cultivation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFive photobioreactor (GroTech) were prepared using Guillard f/2 medium (final volume of 6.3 L) with an initial seeding of 0.5 mg/mL (wet weight) of the marine dinoflagellate CCAP 1102/8 \u003cem\u003eAmphidinium carterae\u003c/em\u003e. The reactors were incubated for 20 days at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003eo\u003c/sup\u003eC under ca. 60\u0026ndash;80 \u0026micro;mol m\u003csup\u003e2\u003c/sup\u003e s-\u003csup\u003e1\u003c/sup\u003e illumination provided by white light LED panels using a 14:10 light:dark photoperiod. The culture was aerated (210 mL/min) through a sterile 0.22 \u0026micro;m-pore size syringe filter. On day 20, after the culture had reached the late exponential growth phase, the cultivation conditions were modulated in terms of exposure to blue light, hydrogen peroxide, sodium carbonate and sodium chloride (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To do so, the reactors were supplemented with 630 mL of filter-sterilised seawater supplemented where appropriate with the relevant chemicals.\u003c/p\u003e \u003cp\u003eOn day 30, the microalgal biomass was collected from each reactor after centrifugation (2000 \u003cem\u003erpm\u003c/em\u003e, 5 min) and desalted with ammonium formate (0.5 M). The samples were then freeze-dried overnight (Scanvac. MillRock) and stored at -20\u003csup\u003eo\u003c/sup\u003eC until further processing.\u003c/p\u003e \u003c/div\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\u003eSecond stage cultivation conditions applied on day-20 for CCAP 1102/8 \u003cem\u003eAmphidinium carterae\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbbreviation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCondition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlue Light\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLED 100 \u0026micro;mol m-\u003csup\u003e2\u003c/sup\u003e s-\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHumphrey et al. 1983\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh Salinity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.8 g/L of NaCl \u003c/p\u003e \u003cp\u003e(from 30 up to 55 mS/cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBoukhris et al. 2017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaHCO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSodium Carbonate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5 mmol l\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKitaya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydrogen Peroxide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5 mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBarone et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eseawater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en/a\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=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSolid Phase Extraction (SPE)\u003c/h2\u003e \u003cp\u003eExtracts were prepared for each \u003cem\u003eA. carterae\u003c/em\u003e reactor, from which 500 mg of dried biomass was extracted with 60 mL (3 x 20 mL) of methanol aided by sonication (Elma P Sonic) (3 x 15 min freq.\u0026nbsp;37% power 90%) followed by centrifugation (3 x 10 min at 4500 rpm). Each crude extract was filtered (VWR Qualitative Retention 40 \u0026micro;m), transferred to pre-weighed vials and dried at 40\u0026deg;C under N\u003csub\u003e2\u003c/sub\u003e stream. The extracts were resuspended in 10 mL of MeOH (purity?) and 500 mg of C\u003csub\u003e18\u003c/sub\u003e powder (POLYGOPREP 50\u0026ndash;60 C\u003csub\u003e18\u003c/sub\u003e) was added to each. The extracts were then partially dried under reduced pressure. A C\u003csub\u003e18\u003c/sub\u003e SPE cartridge was conditioned with MeOH (2 x 6 mL) followed by H\u003csub\u003e2\u003c/sub\u003eO (2 x 6 mL). Once conditioned, the dried samples bound to C\u003csub\u003e18\u003c/sub\u003e was loaded onto the column. Six fractions (12 mL) were then recovered by eluting solvent mixtures of decreasing polarity: (A) 100% H\u003csub\u003e2\u003c/sub\u003eO, (B) H\u003csub\u003e2\u003c/sub\u003eO:MeOH (75:25 \u003cem\u003ev/v\u003c/em\u003e), (C) H\u003csub\u003e2\u003c/sub\u003eO:MeOH (50:50 \u003cem\u003ev/v\u003c/em\u003e), (D) H\u003csub\u003e2\u003c/sub\u003eO:MeOH (25:75 \u003cem\u003ev/v\u003c/em\u003e), (E) MeOH 100% and (F) MeOH:CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (50:50 \u003cem\u003ev/v\u003c/em\u003e). The fractions were dried under reduced pressure and weighed prior to storage for further chemical and antimicrobial activity analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLiquid Chromatography Mass Spectrometry (LC-HRMS)\u003c/h2\u003e \u003cp\u003eApproximately 0.2 mg of dried fractions was resuspended in the corresponding fraction solvent at a concentration of 1 mg\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e prior to chemical profiling.\u003c/p\u003e \u003cp\u003eSamples were run in positive MS (200\u0026ndash;3000 \u003cem\u003em/z\u003c/em\u003e) and MS/MS modes. High resolution mass spectra data were obtained with an Agilent 6540 qTof mass spectrometer UHPLC-DAD-HRMS. MS/MS data used a cone voltage of 40 V, and a collision energy of 75 V. The cone and desolvation gas flows were set at 300 and 12 L/min, respectively, and the source temperature was 300 C. A binary gradient elution was used, with phase A consisting of water and phase B of acetonitrile in water (both containing 6.7 mM ammonium formate). The column used was a 50 mm \u0026times; 2.1 mm i.d., 1.7 \u0026micro;m, Acquity UPLC BEH C\u003csub\u003e18\u003c/sub\u003e (Waters). The gradient was from 30\u0026ndash;90% B over 11 min at 0.4 mL/min, held for 1 min, and returned to the initial conditions over 1 min and held for 2 min to equilibrate the system. The injection volume was 5 \u0026micro;L and the column and sample temperatures were 40\u0026deg;C and 10\u0026deg;C, respectively. HRMS spectra were obtained using the LCMS conditions described by Welkamp et al. [2020]. All fractions were analysed by UHPLC-HRMS/MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial strains\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMicrobiological assays were carried out using the Gram-positive strains \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923, \u003cem\u003eE. faecalis\u003c/em\u003e ATCC 29212 and \u003cem\u003eCandida albicans\u003c/em\u003e ATCC 90028. The strains were grown in selective agar media (Bile Aescuilin Azide (BEA) for \u003cem\u003eE. faecalis\u003c/em\u003e, Mannitol Salt Agar (MSA) for \u003cem\u003eS. aureus\u003c/em\u003e) then incubated at 37\u0026deg;C overnight prior to carrying out the bioassays. Sabouraud Dextrose Agar (SDA) and overnight incubation at 35\u003csup\u003eo\u003c/sup\u003eC were used for \u003cem\u003eC. albicans\u003c/em\u003e. All media were manufactured by Oxoid (Basingstoke, UK).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Minimum Inhibitory Concentrations (MIC)\u003c/h2\u003e \u003cp\u003eThe susceptibility tests were carried out using the broth microdilution assay with kanamycin as positive bacterial break point control according to the CLSI guidelines [Clinical \u0026amp; Laboratory Standard Institute, 2020]. \u003cem\u003eC. albicans\u003c/em\u003e ATCC 90028 was analysed according to the CLSI M27-A2 guidelines using Amphotericin B as control.\u003c/p\u003e \u003cp\u003eA single colony of the ATCC strains was inoculated in 5 mL of BHI (Brain Heart Infusion broth) for bacterial strains, and RPMI-1640 for \u003cem\u003eC. albicans\u003c/em\u003e, for 4 to 6 hours, to achieve the exponential phase. Then, 10\u003csup\u003e5\u003c/sup\u003e CFU/mL was inoculated to in 96-well plate in 0.1 mL of sterile Muller-Hinton broth (MHB).\u003c/p\u003e \u003cp\u003eAll of the extracts were re-suspended in acetone:deionised water (10:90 v/v). Prior to bacterial inoculation, scalar dilutions (1:2) of the extracts were performed from the 1st to the 11th wells of a row of a 96-well plate with a final volume of 100 \u0026micro;L of MHB. The 12th well was used as a growth control. Control wells of MHB and solvents were also included. Triplicate wells were used for all the samples and the plates were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 14\u0026ndash;18 h. Wells inoculated with C. \u003cem\u003ealbicans\u003c/em\u003e were incubated at 35\u003csup\u003eo\u003c/sup\u003eC. The MIC values were determined after 24 h and confirmed after 48 h. Nitro blue-tetrazolium (1 mg/mL) was then added to each well and incubated for 30 minutes to detect the MIC points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Minimum Bactericidal and Fungicidal Concentration\u003c/h2\u003e \u003cp\u003eA volume of 50 \u0026micro;L from all the wells above the MIC values were inoculated in Muller-Hinton agar media. The plates were further incubated for 24 h and 48 h for bacteria and yeast, respectively. The minimum bactericidal/fungicidal concentrations were then identified as the lowest concentration of extract returning no growth. All analyses were performed in triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePenicillin-binding Protein (PBP) competition assay\u003c/h2\u003e \u003cp\u003eThe potential binding of the compound at different MBC concentrations (0.5X, 1X, 2X) to Penicillin Binding Proteins (PBPs) was evaluated in a competition assay with BOCILLIN\u0026trade; FL (Invitrogen\u0026trade;), a fluorescent reporter molecule analog to Penicillin V. The membrane fractions for the detection of PBPs were prepared based on Djoric\u0026rsquo; et al. (2020) and Arbeloa et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) methodologies. Protein samples were incubated at room temperature for 10 min with 5 \u0026micro;g/mL (7.5 \u0026micro;M) of BocillinTM FL (Invitrogen\u0026trade;), followed by SDS-PAGE on a Criterion\u0026trade; TGX\u0026trade; (Tris-Glycine eXtended) 10% polyacrylamide gel. Electrophoresis was carried out for 2 h at a constant voltage of 100V. The fluorescent Bocillin covalently bound to the PBPs was detected with excitation at 488 nm and emission at 520 nm (Typhoon FLA 9500; filter Alexa 488; PMT 1000; pixel 50 \u0026micro;m).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData treatment\u003c/h2\u003e \u003cp\u003eData collected was analysed using IBM SPSS Statistics 26 package 2. Principal Component Analysis (PCA) was carried out to assess the relationship between peak heights and bioactivity using a correlation matrix in a Varimax rotation mode.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLiquid chromatography and mass spectrometry analysis\u003c/h2\u003e \u003cp\u003eThe fractions were first analysed by UPLC-DAD-HRMS/MS for the presence of amphidinol-like compounds for each of the five growth conditions. Amphidinol type compounds were detected through their characteristic fragmentation patterns in Fractions-4 (H\u003csub\u003e2\u003c/sub\u003eO/MeOH 25:75) and \u0026minus;\u0026thinsp;5 (MeOH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These compounds were below the level of detection for fractions 1, 2, 3 and 6. Retention times for the compounds ranged from 3.4\u0026ndash;5.6 min with \u003cem\u003em/z\u003c/em\u003e of 1361, 1463, 1667, 1343, 1320 and 1454 during a 11-min UHPLC run. Compounds with \u003cem\u003em/z\u003c/em\u003e 1361 and 1463 shared identical fragmentation patterns with AM-A and AM-B, respectively, and were concluded to be both compounds from analysis of data previously published by Welkamp et al. [2020]. Both compounds shared key fragments at \u003cem\u003em/z\u003c/em\u003e 1085 (C-29/C-30) and 687 (C-29/C-30 and C-1/C-1\u0026rsquo;) as well as a characteristic loss of water molecules. AM-22 was also identified in Fraction 4 through the \u003cem\u003em/z\u003c/em\u003e 1667 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e. Further, the recently characterised AM-C previously reported as N16 as mentioned in Murphy et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e] and Welkamp et al. [2020] was detected with the \u003cem\u003em/z\u003c/em\u003e 1343 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e. The fragmentation pattern of AM-C and its \u003cem\u003em/z\u003c/em\u003e 1343 revealed key fragments at \u003cem\u003em/z\u003c/em\u003e 1085 and 945. A comparative study was performed on Fractions 4 and 5 for each growth condition and the control sample. Ionisation potential was assumed to be shared between analogues so that the relative integration of the amphidinol derivatives can be assessed for each fraction. AM-B appeared as the major analogue in Fraction-4 while a derivative of AM-A with \u003cem\u003em/z\u003c/em\u003e 1320 and a very close fragmentation pattern was found to be the major amphidinol in Fraction-5. In Fraction 4, the control treatment exhibited the lowest relative abundance in targeted amphidinols, while the highest abundance was for the NaHCO\u003csub\u003e3\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatments. Overall, the highest abundance in AMs was observed in Fraction-5 extracts, in particular for the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NaCl treatments, where the unknown amphidinol derivative at \u003cem\u003em/z\u003c/em\u003e 1320 was very prominent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe unknown derivative AM-1320 (with a sodium adduct at \u003cem\u003em/z\u003c/em\u003e 1342) exhibited the highest concentration of all analogues present. The analogue presented a key fragment at \u003cem\u003em/z\u003c/em\u003e 1084 instead of 1085 for AM-A and -B, indicating the possible substitution of a hydroxyl group for an NH\u003csub\u003e2\u003c/sub\u003e between C-44 and C-65 as the 945 fragment is still present (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). An unreported \u003cem\u003em/z\u003c/em\u003e 1454.84 was also identified with a similar elution time to AM-A. They both shared similar fragments. This may be a new derivative analogue coeluting with AM-A but showing a distinct fragmentation pattern as AM-B (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioactivity: determination of Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal-Fungicidal Concentrations (MB-FCs)\u003c/h2\u003e \u003cp\u003eThe bioactivity of the six fractions generated \u003cem\u003evia\u003c/em\u003e solid phase extraction from the five reactors was assessed against three pathogens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The bioactivities varied for MBCs between 8 and 512 \u0026micro;g\u0026sdot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eS. aureus\u003c/em\u003e and between 128 and 4096 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eE. faecalis\u003c/em\u003e, while the MFCs varied between 16 and 512 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eC. albicans\u003c/em\u003e (5 to 6 scalar dilutions overall). Fractions-1 and \u0026minus;\u0026thinsp;2 did not return appreciable antimicrobial activity against the pathogens tested. The Fraction-6 samples gave very weak MICs against \u003cem\u003eE. faecalis\u003c/em\u003e. For Fraction-3, only the samples from the NaHCO\u003csub\u003e3\u003c/sub\u003e and BL treatments returned bactericidal and fungicidal activities against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, respectively. The Fraction-5 extracts inhibited \u003cem\u003eE. faecalis\u003c/em\u003e, with a MIC value of 16 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, MBCs could not be determined for any of the extracts analysed, indicating a bacteriostatic effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe main antimicrobial activity was observed in Fraction-4, which returned significant MICs and MB-FCs against each microbial pathogen. \u003cem\u003eS. aureus\u003c/em\u003e reacted the most to the Fraction-4 sample of the NaHCO\u003csub\u003e3\u003c/sub\u003e treatment (MBC of 8 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003cem\u003eC. albicans\u003c/em\u003e appeared more susceptible to the Fraction-4 from the blue light treatment (MFC of 1 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003cem\u003eE. faecalis\u003c/em\u003e was less sensitive to Fraction-4 than the other two pathogens (MBC\u0026thinsp;\u0026gt;\u0026thinsp;128 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePenicillin-binding Protein (PBP) competition assay\u003c/h2\u003e \u003cp\u003eFraction-4 extracts gave the highest bioactivities against the bacterial pathogens tested and were selected for analysis by the PBP competition assay. The extracts used for PBP labeling with Bocillin FL showed no demonstrable binding affinity for PBPs with the extracts tested against ATCC 29212 \u003cem\u003eE. faecalis\u003c/em\u003e and ATCC 25923 \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains, suggesting that no compound in these fractions showed affinity for PBPs at the tested concentrations ranging from 0.5X to 4X MBC values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal Component Analysis (PCA)\u003c/h2\u003e \u003cp\u003eThis analysis focused on the most bioactive fractions (Fractions3, 4 and 5) containing amphidinol-like compounds to delineate patterns of bioactivity in the extracts tested against the three pathogens used (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Fraction-5 were predominantly grouped together along the positive domain of Component 1 (PC1) while Fraction-3 extracts clustered along the negative domain. Fraction-4 extracts were distributed between both the negative and positive domains of component 2 (PC2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSome amphidinols identified in the fraction by LC-HRMS were clearly associated with Fraction-3 (AM-1348, AM-1371), Fraction-4 (AM-B, AM-C, AM-22, AM-1454, AM-1225, AM-A) and Fraction-5 (AM-C, AM-1320, AM-1325). Noticeably, the bioactivity vectors were mostly oriented toward the positive domain of PC2 and Fraction-4 extracts (except that of MIC for \u003cem\u003eE. faecalis\u003c/em\u003e along the positive domain of PC1).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicroalgae have attracted interests due to their capacity to produce high-value compounds with potential applications in the nutraceutical and cosmetics sectors [Fabris et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e]. They have also been increasingly considered as potential sources of new antimicrobial compounds, the development of which is urgent to alleviate the emerging antibiotic resistance of various pathogens in health settings worldwide [WHO 2021]. Multiple bioprospecting studies for new microalgal species and strains have been conducted and various extracts and compounds tested against a range of pathogens, including bacteria and fungi [McGee et al., 2020; Desbois et al.2008; Falaise et al. 2019]. Of interest here, polyketide compounds of the amphidinol type, which are biosynthesised by \u003cem\u003eAmphidinium\u003c/em\u003e spp., have shown promising bioactivity against such pathogens Amphidinol-containing fractions of a strain of \u003cem\u003eA. carterae\u003c/em\u003e (sub-clade III) previously isolated from Ireland were shown to exhibit antimicrobial activity against Gram-positive bacteria [Kubota et al. 2006, Martinez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Barone et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e]. Here, this dinoflagellate was grown under varying conditions to assess whether or not they could influence the amphidinol-like compound profile and the antimicrobial properties of extracts.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eVariations in amphidinol profile\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAmphidinium carterae\u003c/em\u003e strain CCAP 1102/8 was grown via a two-stage process. LC-MS analysis was carried out on SPE-derived fractions in which variations in the concentration of several amphidinol-like compounds were observed. The extracts which indicated the highest concentrations of amphidinols were obtained from the biomass retrieved from cultures exposed to stressors. However, when harvested, these cultures returned lesser amounts of biomass compared to the control treatment. This is in agreement with previous studies carried out on a variety of microalgal species, which have indicated that the modulation of cultivation conditions can often increase the intra-cellular yields of particular metabolites but hamper growth [Liyanaarachchi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Pan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Humphrey et al. 1983; Das et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Boukhirs et al. 2017, Qiao et al. 2020]. For instance, relevant to \u003cem\u003eAmphidinium\u003c/em\u003e sp., Tsirigoti et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e] demonstrated the benefits of applying low temperature to increase the production of DHA, an ω-3 fatty acid which has previously shown antimicrobial activity against \u003cem\u003eS. aureus\u003c/em\u003e and MRSA strains [Desbois et al. 2006].\u003c/p\u003e \u003cp\u003eSeveral amphidinols with similar and characteristic fragmentation patterns were detected by LC-HRMS/MS analysis, with variations in their concentration in Fractions-4 and \u0026minus;\u0026thinsp;5. In Fraction-4, the control treatment exhibited the lowest relative concentration of the amphidinols, while the highest amounts were obtained for the NaHCO\u003csub\u003e3\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e samples. AM-B, AM-C, AM-22 and AM-A were prominent in Fraction-4. Their abundance in the extracts of the NaHCO\u003csub\u003e3\u003c/sub\u003e, Salinity, Blue Light and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatments were on average 2- to 4-fold higher than the control, respectively. AM-1320 followed by AM-C were the major identified amphidinol in Fraction-5. The concentration in AM-C was the greatest for the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, with a 2.1-fold enhancement compared to the control treatment. Future work should consider the extent by which further modulation in supplement concentrations, light intensity or photoperiod patterns could affect the profile and abundance of amphidinols. Likewise, different time points could be analysed during the second stage of cultivation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial activity of amphidinol-containing fractions\u003c/h2\u003e \u003cp\u003eSeveral amphidinol-like compounds have been isolated from \u003cem\u003eAmphidium\u003c/em\u003e species [Satake et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1991\u003c/span\u003e, Kobuta et al. 2004, Kobayashi et al. 2005, Welkemp et al. 2020, Martinez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Barone et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e]. Some purified compounds such as Amphidinol-22, Amphidinol-A or Amphidinol-20 have shown different bioactivities against various pathogens and cancer cell lines [Martinez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Welkamp et al. 2020]. Crude methanolic extracts of \u003cem\u003eA. carterae\u003c/em\u003e biomass and several purified amphidinols have previously shown antifungal activities, with MICs ranging from 560 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 9 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eagainst \u003cem\u003eAspergillus\u003c/em\u003e spp. or \u003cem\u003eCandida albicans\u003c/em\u003e [Echigoya et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Martinez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nuzzo et al.; Cutignano et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e]. Some antibacterial properties have also previously been attributed to amphidinol-like molecules such as Lutheophanol D, Amphidinin C, Amphidinin E, Amphidinin F and Amphidinin G extracted from \u003cem\u003eA. carterae\u003c/em\u003e against \u003cem\u003eMicrococcus lentus\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e [Kubota et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Kubota et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e]. Amphidinols have not been tested as extensively against bacteria. Recent work showed however that methanolic extracts from \u003cem\u003eA. carterae\u003c/em\u003e (strain CCMP1314 from the USA, sub-clade III) containing AM-22 elicited antimicrobial activity against \u003cem\u003eE. coli\u003c/em\u003e, Methicillin-Resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA), Methicillin-Susceptible \u003cem\u003eS. aureus\u003c/em\u003e (MSSA) and \u003cem\u003eM. tuberculosis\u003c/em\u003e [Martinez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e]. This antibacterial potential was also supported with our CCAP 1102/8 \u003cem\u003eAmphidinium carterae\u003c/em\u003e strain from the West of Ireland [Barone et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e]. The present study corroborates these findings against Gram-positive bacteria and the fungus \u003cem\u003eC. albicans\u003c/em\u003e, further detailing how the bioactivity and amphidinol signature of extracts could be impacted by the conditions under which the cells were cultivated. The potential additive or synergistic bioactivity of several amphidinols or other co-extracted compounds in the samples tested, in particular against bacteria, warrants in that aspect further scrutiny.\u003c/p\u003e \u003cp\u003eThe highest biocidal activities were observed for the Fraction-4 samples, returning MBCs of 8 and 128 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e against \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e, respectively, and a MFC of 1 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eagainst \u003cem\u003eC. albicans\u003c/em\u003e. These extracts all contained varying amounts of the amphidinols AM-22, AM-C and AM-B. Martinez et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e] showed purified AM-22 to be effective against \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eA. fumigatus\u003c/em\u003e at 64 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Given that amphidinols seem bioactive against several fungi at doses inferior to 64 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the conduct of potentiation or synergism experiments is warranted, as is the acquisition of data on their pharmaco-kinetics. Such trials could consider the use of individual or several purified amphidinols applied together or with other known bioactive compounds, such as miconazole, which is biocidal against \u003cem\u003eC. albicans, S. aureus\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e between 0.78\u0026ndash;6.25 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [Isham et al. 2010; Nenoff et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, and noteworthily, Fraction5 extracts showed substantial bacteriostatic activity against \u003cem\u003eE. faecalis\u003c/em\u003e, which may be associated with m/z 1320, which was the most abundant compound in these extracts, and would deserve purification and structure elucidation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eA mode of action not associated with penicillin binding proteins\u003c/h2\u003e \u003cp\u003eExperimental data obtained using AM-3 has suggested the mechanism of action of amphidinols to be linked with the capacity of their polyol chain to penetrate membrane lipidic bilayers in the presence of sterol, causing the death of eukaryotic cells such as yeasts as well as hemolysis [Satake et al \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wakamyia et al. 2020]. Here, the Fraction-4 samples of \u003cem\u003eA. carterae\u003c/em\u003e, which were the most bioactive against the three pathogens used in this study, were tested using the Bocillin-FL assay for the presence of molecules containing ring structures similar to the beta-lactam ring of penicillin. Such structures are able to acylate and covalently bind to the active serine residue at the catalytic site of specific penicillin binding proteins present in the cell wall of bacteria, leading to its disruption and cell death [Djoric\u0026rsquo; et al. 2020, Arbeloa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e]. No concentration-dependent decrease of fluorescence in both \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e was observed, excluding the presence of metabolites in the extracts targeting PBPs. Further studies are hence necessary to better characterise the mode of action of amphidinols on relevant bacterial pathogens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e: Conceptualisation by MEB, GTAF and NT. \u0026nbsp; Methodological design by MEB, DF, EM and FC. \u0026nbsp;Data curation by EM and MEB. \u0026nbsp;Analyses by MEB, FC, EM, OPT and NT. \u0026nbsp;Writing, review and editing were performed by MEB, DF, FC, EM, GTAF, OPT and NT. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors acknowledge financial support from the VES4US project funded by the European Uninion\u0026rsquo;s Horizon 2020 research and innovation programme under grant agreement \u0026nbsp; No 801338. \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e This article does not contain any studies with human participants performed by any of the authors. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: The data that has been used is confidential.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkakabe M,\u003cem\u003e\u0026nbsp;\u003c/em\u003eKumagai K,\u003cem\u003e\u0026nbsp;\u003c/em\u003eTsuda M, Konishi Y,\u003cem\u003e\u0026nbsp;\u003c/em\u003eTominaga A,\u003cem\u003e\u0026nbsp;\u003c/em\u003eKaneno D,\u003cem\u003e\u0026nbsp;\u003c/em\u003eFukushi E, Kawabata\u003cem\u003e\u0026nbsp;\u003c/em\u003eJ, Masuda A, Tsuda M (2016) Iriomoteolides-10a and 12a, Cytotoxic Macrolides from Marine Dinoflagellate \u003cem\u003eAmphidinium\u0026nbsp;\u003c/em\u003eSpecies. 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Algal Res. 53:102017.\u003c/li\u003e\n \u003cli\u003eVenugopal VC, Thakur A, Chennabasappa LK, Mishra G, Singh K, Rathee P, Ranjan A (2020) Phycocyanin Extracted from \u003cem\u003eOscillatoria minima\u003c/em\u003e Shows Antimicrobial, Algicidal, and Antiradical Activities: In \u003cem\u003esilico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e Analysis. Antiinflamm. Antiallergy Agents Med Chem. 19(3):240-253.\u003c/li\u003e\n \u003cli\u003eYarkent \u0026Ccedil;, Gurlek C, Oncel SS (2020) Potential of microalgal compounds in trending natural cosmetics: A review. Sustain. Chem. Pharm. 17:1000304 (2352-5541).\u003c/li\u003e\n \u003cli\u003eWakamiyaY, Ebine M, Matsumori N, Oishi T (2020) Total Synthesis of Amphidinol 3: A General Strategy for Synthesizing Amphidinol Analogs and Structure-Activity Relationship Study. Journal of the American Chemical Society. J. Am. Chem. Soc\u003cem\u003e.\u0026nbsp;\u003c/em\u003e142: 3472-3478.\u003c/li\u003e\n \u003cli\u003eWashida K, Koyama T, Yamada K, Kita M, Uemura D (2006) Karatungiols A and B, two novel antimicrobial polyol compounds, from the symbiotic marine dinoflagellate Amphidinium sp., Tetrahedron Lett. 47 (15): 2521-2525.\u003c/li\u003e\n \u003cli\u003eWensel P, Helms G, Hiscox B, Davis WC, Kirchhoff H, Bule M, Yu L, Chen S (2014) Isolation, characterization, and validation of oleaginous, multi-trophic, and haloalkaline-tolerant microalgae for two-stage cultivation. Algal Res. 4:2\u0026ndash;11.\u003c/li\u003e\n \u003cli\u003eWellkamp M, Garcis-Camacho F, Duran-Riveroll LM, Tebben J, Tillmann U, Krock B (2020) LC-MS/MS Method Development for the Discovery and Identification of Amphidinols Produced by Amphidinium. Mar. Drugs 18: 497. doi: 10.3390/md18100497.\u003c/li\u003e\n \u003cli\u003eWorld Health Organization (WHO) (2021) \u003cem\u003eGlobal Antimicrobial Resistance and Use Surveillance System (GLASS) Report 2021\u003c/em\u003e; WHO: Geneva, Switzerland.\u003c/li\u003e\n\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":true,"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":"microalgae, MNPs, antimicrobials, amphidinols, LC/HRMS, Amphidinium carterae, Staphylo-coccus aureus, Enterococcus faecalis, Candida albicans.","lastPublishedDoi":"10.21203/rs.3.rs-3048270/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3048270/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroalgae have attracted interests from the bioenergy, bioremediation, cosmetics and pharmaceutical sectors due to produce biofuels, pigments, omega-3 fatty acids, antioxidants or antimicrobial compounds. \u0026nbsp;Among others, amphidinol-like compounds biosynthesised by dinoflagellate species of the genus \u003cem\u003eAmphidinium\u003c/em\u003e have shown promising antimicrobial properties.\u003c/p\u003e\n\u003cp\u003eHere, a two-stage cultivation regime was used to grow \u003cem\u003eA.carterae\u003c/em\u003e (subclade III), which was exposed to different conditions, including an illumination regime switch to LED blue light, a salinity enhancement treatment, sodium carbonate or hydrogen peroxide supplementation.\u0026nbsp; A solid phase extraction of the dried biomass was carried out and returned 6 fractions for each treatment, which were analysed by LC-HRMS.\u003c/p\u003e\n\u003cp\u003eAmphidinol-like compounds AM-B, AM-C, AM-22 and AM-A were detected through their fragmentation patterns, mostly in Fraction-4 samples.\u0026nbsp; AM-B was the major analogue in Fraction-4 (\u003cem\u003em/z\u003c/em\u003e 1463.69, 42.3\u003csup\u003e+\u003c/sup\u003e/\u003csub\u003e-\u003c/sub\u003e16.5 % of amphidinol composition), followed by AM-C (\u003cem\u003em/z\u003c/em\u003e 1343.84, 21.4\u003csup\u003e+\u003c/sup\u003e/\u003csub\u003e-\u003c/sub\u003e6.6 %). \u0026nbsp;In Fraction-5, along with an unidentified analog with m/z AM-1320, AM-C was the most prevalent amphidinol (15.5\u003csup\u003e+\u003c/sup\u003e/\u003csub\u003e-\u003c/sub\u003e3.0 %) which was on average as abundant as in Fraction-4.\u0026nbsp; This returned the highest antimicrobial activity against the pathogens \u003cem\u003eS.aureus\u003c/em\u003e, \u003cem\u003eE.faecalis\u003c/em\u003e and \u003cem\u003eC.albicans\u003c/em\u003e, with MBC ranging from 1 to 512 µg×mL\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp; Further analysis using the Bocillin competition assay showed that the antibacterial activity was not associated with the presence of compounds targeting penicillin binding proteins (PBPs) in the cell wall of bacteria.\u003c/p\u003e\n\u003cp\u003eResults indicate that the modulation of both amphidinol profile and fraction bioactivity can be induced by adjusting the cultivation parameters used to grow two-stage batch cultures of \u003cem\u003eA. carterae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Bioactivity of amphidinol-like extracts of Amphidinium carterae grown under varying cultivation conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-21 13:17:27","doi":"10.21203/rs.3.rs-3048270/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":"132df2b5-e5c9-472a-8407-622b5514e71c","owner":[],"postedDate":"August 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-28T19:14:30+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-21 13:17:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3048270","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3048270","identity":"rs-3048270","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}