Introduction

Present in all environments (industrial, medical, marine...), bacteria develop very quickly and manage to live even in extreme conditions by exploiting the resources available around them but also thanks to their ability to organize in biofilm. This last capability is a huge issue in health, agro-industry and other productive sectors using metal alloys for example, such as oil and gas industries [1] where microbial-induced corrosion is responsible for huge economic losses [2]. In addition to this, about 60% of bacterial infections are due to microbial biofilms that are insensitive to 100% of the chemical antibiotics currently used [3,4].

Indeed, the microbial adhesion to a surface (metal, plastic...) leads to the formation of a very resistant microbial biofilm, hence the need to develop antibacterial strategies to prevent their adhesion or inhibit their growth. Different factors influence bacterial adhesion, namely, the bacterium itself (chemical composition, surface...) [5], the surface of the material (chemical nature, geometry, roughness) [6,7] and the environment (temperature, presence of organic molecules) [8]. Actions could be carried out separately or simultaneously on those three families of factors.

 Faced with this significant resistance, researchers are increasingly focusing on antibacterial properties of plant-based compounds. Indeed, plants known to have developed protective mechanisms against bacteria are, composed of antimicrobial molecules (polyphenols, terpenes, alkaloids...) [9–11]. Marine resources such as macro-algae are also a source of interesting constituents with reported biological activities such as antiviral, antifungal or antibacterial activities [12–16]. Brown algae were reported to contain diverse bioactive components that play a protective role against marine herbivorous organisms [17,18].

Phlorotannins were described as the principal bioactive compound with antibacterial activity in ten Sargassum-belonging brown algae [19,20], nevertheless, other sargassum-containing secondary metabolites such as quinones from S. paradoxum, S. fallax and S. sagamianum [21–23]. Triterpenes and triterpenoid derivatives from S. wightii [24], S. henslowianum [25]. Saponins from S. aquifolium [26],flavonoids from S. crassifolium and S.oligocystum [27,28]and phenolic non identified compounds from S. mangarevense [29] were described to exhibit antibacterial activity.

Sargassum fluitans III, S. natans I and S. natans VIII are the main holopelagic brown seaweed of which thousands of tons wash up every year on the coasts of the Caribbean islands [30]. These algae strandings caused several damages : ecological (disturbance of the ecosystems), economical (material corrosion, perturbations of touristic activities), but also represents a danger to local populations’ health once its decomposition process begins (inhalation of toxic gases) [31]. In order to deal with this issue, the valorization of these algae is strongly encouraged. We recently reported the results of a study examining extraction and electrochemical analysis of natural compounds extracted from Sargassum fluitans III. The objective of this research was the development of new organic bio-sourced corrosion inhibitors, ecofriendly and biodegradable. The results obtained showed that the extract of Sargassum fluitans III could serve as an effective inhibitor for the C38 steel in acidic media [32]. The current study represents a continuation of this previous research, exploring additional activities that Sargassum extracts can exhibit. Thus, the aim of the present paper is to emphasize for the first time, the antibacterial activity of Sargassum fluitans III extract and its secondary metabolites.

Materials and Methods

Sampling

Sargassum collection and preparation

The Sargassum is defined as brown macrophytes of the fucaceae family, which grow in tropical waters. They are primarily benthic, but some, such as Sargassum natans (I and VIII) and Sargassum fluitans III, involved in sargassum rafts that wash ashore on the coasts of the Caribbean, are called ‘holopelagic’ because they proliferate on the water surface without attaching to a hard substrate. They gather in the form of rafts or sargassum patches stretching for several kilometers [33,34]. These three species have similar properties but are distinguished by somewhat different morphologies, as shown in Figure 1.

Figure 1: Morphological differences of the three pelagic Sargassum species.

The brown macroalgae S. fluitans III, S. natans I and S. natans VIII types were collected by hand picking method in the south-east coastline of Martinique (14° 28 ‘40.4”N; 61° 01’ 45.1”W). They were immediately and thoroughly washed using distilled water to remove salt and superfluous matter and selected to keep only mature, disease free, fresh algae. It was then sorted and identified based on their morphology differences [35] left to dry at room temperature and conserved in vacuum-packed bags until use.

Ethanolic and secondary metabolites extraction

Dried algae were crushed with a Pulverisette 14 rotor crusher. 20g of the grinding product was then used in a reflux system for 3 hours with 400 mL of a green solvent composed of water/ethanol (30/70, 50/50 or 30/70 v/v%). The extracted solution was then filtered (12 µm pore filter paper) under vacuum pump and evaporated using a rotary evaporator (Heidolph®). Similarly, the families of extracted secondary metabolites were obtained by modified extraction protocols initially established by Botosa and Bruneton [11,36]. The alkaloids were extracted using a previously described experimental procedure [37], while flavonoids [38,39], saponins [40], triterpenes [41], anthocyanins [42,43], coumarins [44,45], quinones [46], and tannins [47] were extracted using specific protocols.

The extracted fractions from different families were collected and weighed. Depending on their solubility, the solids obtained were subsequently solubilized, in water or ethanol for microbiological analysis. The yield of the various extractions carried out was calculated as following:

Where m and m° correspond respectively to the mass of dry plants before and after extraction.

Bacterial strains

The bacterial strains Shewanella fodinae (Gram negative) [48] was isolated from an electroactive bioanode from a mangrove sediment microbial fuel cell. Vibrio alginolyticus (Gram negative) [49], Staphylococcus warneri (Gram positive) [50], Staphylococcus capitis (Gram positive) [51], Micrococcus yunnanensis (Gram positive) [52,53] and Alteromonas macleodii (Gram negative) [54,55] were isolated from a biofilm formed on DC01 Carbon steel and 304L stainless steel coupons exposed to marine corrosion in the presence of Sargassum. The strains were identified and were frozen at -80°C in Brain and Heart Infusion (BHI) containing 10% glycerol. Before use, bacterial strains were thawed and maintained in a Marine Broth medium (MB) at 27°C for 48h, tested regarding purity before their use.

Antibacterial analysis

Evaluation of antibacterial activity by the disk diffusion method

1 mL containing 10⁸ CFU/mL of fresh bacterial suspension of each bacterium was streaked uniformly onto individual plates containing Marine Agar (MA). In parallel to this, a 6mm diameter sterile paper disk was soaked in an increasing volume of ethanolic extract supplemented with 5% DMSO and left to dry for 3h at room temperature under a sterile atmosphere. Then, disks were placed on the inoculated plates and incubated at 27 °C for 48 hours. The bioactivity was determined by the observation of clearance zones and their diameters were measured in millimeters, including the disk. A 5% DMSO disk was used as negative control and Amoxicillin and Benzathine benzylpenicillin as well as CuSO₄ (100 μg/mL) containing 5% DMSO were used as positive controls.

Determination of the Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) of extracts was determined in triplicate, using 96-wells microdilution method [56]. In brief, 100 µL of a twofold serial dilution of extract ranging from 8mg/mL to 100µg/mL were prepared in 96-wells plate, as following, 100 µL of sterilized MB were pipetted onto all the wells from column 1 to column 12, then, 100 µL of the deserved extract was added to the well 1, mixed well by sucking up and down, next, 100 µL were pipetted from well 1 and transferred onto the well 2. This operation was repeated until column 10 where pipetted 100 µL was discarded. After that, 100 µL of an overnight culture containing 10⁶ CFU/mL were added in every well and incubated for 48h at 24 °C, under shaking. A growth control and a sterility control well were also prepared for every bacterial strain. The MIC was determined as the lowest concentration of extract exhibiting no visible growth [57] and confirmed by absorbance at 600nm using a microplate reader. Thus, the MIC of an extract corresponds to an inhibition of bacterial growth greater than 50%. The percentage of inhibition of bacterial growth was calculated as following:

Where A_c is the optical density (OD) of the sample at the concentration of S. fluitans III extract and A₀ is the OD of the negative control (sample without Sargassum extract).

In order to assess the sensitivity of bacteria used, Amoxicillin and Benzathine benzylpenicillin at final concentrations ranging from 25 mg/mL to 0.05 mg/mL as well as the CuSO₄ at a concentration of 100 µg/mL were used.

Determination of the Minimum Bactericidal Concentration

Minimal bactericidal concentration (MBC) was recorded as the lowest extract concentration killing 99.9% of bacterial inocula after 24h of incubation at 27 °C. MBC was performed on all extracts. 10 µL were taken from the MIC experiment (MIC value) and the two wells around and were spread on MA plates [58]. Once plates were seeded, they were incubated for 48 hours at 27 °C. The number of colonies was counted and the concentration of extracts giving less than 10 colonies was considered as MBC [59].

Results

Extraction and Characterization of Sargassum species

Extractions were conducted on three morphotypes of Sargassum (S. fluitans III, S. natans I and VIII) through reflux, employing water/ethanol as an economical and environmentally friendly solvent. The extraction yields obtained for different ratios, (30:70), (50:50) and (70:30) (water:ethanol), were 10.20%, 15.80% and 17.08% respectively. The IR analysis of the extracts obtained for the three morphotypes revealed the presence of identical peaks, as illustrated in Figure 2. Most of the observed IR bands are indicative of the presence of various families within the total extract, specifically: 3347–3320 cm^-1, intermolecular O-H elongation; 2981–2881 cm^-1, symmetrical C-H elongation in an aromatic ring; 1646–1644 cm^-1, phenyl-type C=C elongation; 1407–1379 cm^-1, phenolic ring stretching from in-plane OH deformation; 1087– 1043 cm^-1, cyclic ether C-O elongation; 879–638 cm^-1, out-of-plane aromatic ring C-H deformation. Additionally, a band around 1650 cm^-1 is characteristic of quinone derivatives. The IR spectra also exhibit the distinctive absorption bands of an alcoholic hydroxyl function (3450 cm^-1), methyl radicals (2960 and 1400 cm^-1). The chemical composition remains generally consistent, albeit with slight variations, likely attributed to the extraction solvent ratio. The accumulation of Sargassum along the Caribbean Sea coastline was reported to be predominantly comprised of Sargassum fluitans III [60,61]. Considering the chemical composition and the high yield using (70:30) water/ethanol together with these findings, our research was centered on the Sargassum fluitans III extracted with a 70:30.

Figure 2: IR spectra of Sargassum fluitans III extract.

Secondary metabolites composition of Sargassum fluitans III extracts

In order to identify the family of compounds responsible for the antibacterial activity, the different families present in the extract of S. fluitans III are isolated, identified and tested. Phytochemical tests were conducted on the S. fluitans III extract to identify the several families present in the crude extract, following Grenand’s method [62]. Table 1 summarizes the percentage distribution of these families in the total extract.

The phytochemical results revealed the presence of diverse compound families, including coumarins, anthocyanins, quinones, flavonoids, saponins, tannins, and triterpenes. An extraction, as descripted in the material section, was performed for each family to determine their respective ratios. As can be seen from the Table 1, the total relative percentage is 93%, not reaching 100%, which may be attributed to losses during extraction or the presence of unidentified families.

Table 1: Analysis of phytochemical screening tests of Sargassum fluitans III.

Antibacterial analysis of Sargassum fluitans III

Screening of antimicrobial activity

To determine the most effective extraction ratio of S. fluitans III, we evaluated the antibacterial activity of three extraction ratios [(30:70), (50:50), and (70:30)] on Shewanella fodinae using the disk diffusion method. CuSO4 was used as a positive control, showing an average inhibition diameter of 13 mm. The results indicate that higher ethanol content in the solvent corresponds to better antibacterial activity. Specifically, extraction ratios (70:30) and (50:50) demonstrated diameters of less than 10 mm, indicating lower antibacterial activity, regardless of concentration. Conversely, the (30:70) extract exhibited the strongest inhibitory activity, with diameters greater than 10 mm and approaching the value of the positive control’s inhibition diameter, even at a concentration of 1 µL of crude extract. Additionally, 5% DMSO did not inhibit bacterial growth, confirming the antibacterial activity specially to the sargassum extract.

Figure 3: Antibacterial activity of Sargassum fluitans III assessed by disk diffusion method. Averages of triplicates obtained for each extraction ratio.

To determine the range of minimal inhibitory concentrations, the disk diffusion method was employed with varying amounts of extract. Two concentration ranges were utilized: the first range representing a lower extract amount, below 1 mg, and the second range for a higher extract amount, equal to or above 1 mg. As depicted in Figure 3, with the exception of V. alginolyticus and Alteromonas, the growth of the other bacteria used in this study was inhibited starting from 1 mg of S. fluitans III. However, significant inhibition of A. macleodii and V. alginolyticus occurred when 4 mg and 8 mg of extract were used, respectively. Therefore, concentrations up to 4 mg/mL were utilized to determine the minimal inhibitory concentration in liquid medium.

Minimal Inhibitory Concentration of ethanolic and secondary metabolites extracts

The antibacterial activity of the ethanolic extract (30:70) of S. fluitans  III and its secondary metabolites against S. fodinae, V. alginolyticus, and A. macleodii as Gram-negative bacteria, and S. warneri, S. capitis, and M. yunnanensis as Gram-positive bacteria, was determined using minimal inhibitory concentrations (MIC) in liquid media, with concentrations ranging from 0 to 4 mg/mL.

Table 2: Minimum bactericidal concentrations (MBCs), Minimum inhibitory concentrations (MICs) and MBC/MIC ratio of S. fluitans III on the different bacterial strains tested.

The results were compared to the activity of Amoxicillin and Benzathine benzylpenicillin against the same bacteria. As shown in Table 2, for all bacterial strains, the ethanolic extract exhibited an antibacterial effect in a dose-dependent manner. Most strains showed almost complete inhibition of growth in the presence of the crude extract (>90%) for concentrations under 2 mg/mL. Assessment of antibacterial effects revealed a MIC of 0.25 mg/mL for S. warneri and V. alginolyticus, and S. fodinae, indicating strong antibacterial activity [63,64]. Whereas a moderate activity was observed against S. capitis and A. macleodii with MICs of 1 mg/mL, and finally, the weakest activity of S. fluitans was recorded against M. yunnanensis with the highest MIC of the study at 4 mg/mL.

The comparison of the ethanolic extract of S. fluitans III with antibiotics revealed that the extract generally exhibits higher efficacy against the tested bacteria. Except for S. fodinae, which showed greater susceptibility to Amoxicillin, the MICs of S. fluitans III against all other bacteria were lower than those observed for antibiotics (Table 2).

Furthermore, the antibacterial activity of alkaloids, flavonoids, quinones, coumarins, anthocyanins, saponins, and triterpenes from S. fluitans III was tested at concentrations up to 4 mg/mL. Among these secondary metabolites, flavonoids, saponins, and triterpenes showed no antibacterial activity at the concentrations used against V. alginolyticus, A. macleodii, S. capitis, and M. yunnanensis. None of the tested concentrations allowed for an inhibition greater than 50%. In contrast, alkaloids and coumarins exhibited strong activity, with MICs ranging from (0.01-0.5 mg/mL) and (0.01-2 mg/mL), respectively, surpassing the efficacy of the ethanolic extract against almost all tested bacteria. Tannins and anthocyanins showed activity solely against S. fodinae and Staphylococci-belonging bacteria, with higher efficacy against S. warneri.

Comparing the efficacy of the secondary metabolites, alkaloids demonstrated stronger activity against Gram-negative bacteria (0.01-0.05 mg/mL) than Gram-positive bacteria (0.1 mg/mL), while coumarins from S. fluitans III exhibited broadspectrum inhibition, with similar efficacy against Gram-positive and Gram-negative bacteria. Quinones were ten-fold more active against Gram-negative bacteria (0.01-0.05 mg/mL) than Grampositive bacteria (0.1-0.5 mg/mL). These results suggest that alkaloids, coumarins, and quinones extracted from Sargassum may have broad-spectrum inhibition, whereas tannins and anthocyanins exhibit more restricted activity.

Determination of the minimal bactericidal concentration of effective extracts

The antibacterial activity was assessed using the minimal bactericidal concentration (MBC) to minimal inhibitory concentration (MIC) ratio of a given extract against a particular bacterium. A bactericidal effect was defined when the MBC/ MIC ratio was equal to or less than 4, while a bacteriostatic effect was indicated when the ratio exceeded 4 [65]. Analysis of MBC revealed that S. fluitans III inhibited the growth of V. alginolyticus, A. macleodii, S. capitis, and M. yunnanensis in a bacteriostatic manner, while bactericidal activity was observed only against S. fodinae and S. warneri.

Considering the secondary metabolites, alkaloids, coumarins, and anthocyanins exhibited MBC values within the tested concentration range. Other extracts also demonstrated notable activities, such as tannins and quinones. Coumarins displayed inhibition against all tested bacteria, with bactericidal activity against A. macleodii and M. yunnanensis, and bacteriostatic activity against the other bacteria. Alkaloids exhibited bactericidal action solely against S. capitis. Flavonoids, saponins, and triterpenes showed bactericidal activity against S. capitis and M. yunnanensis, while quinones demonstrated only bacteriostatic action.

Furthermore, the results for tannins and anthocyanins indicated bactericidal action against M. yunnanensis within the tested concentration range. These findings underscore that S. fluitans III contains metabolites with potent bacterial inhibition, with coumarins and alkaloids showing promising activity.

Discussion

In addition to their ecological role, brown algae, particularly Sargassum, represent a valuable resource due to their secondary metabolites, which have been reported to possess antibacterial activity across various Sargassum species. However, the antibacterial potential of S. fluitans III has not been explored to date. Our study aims to assess, for the first time, the antibacterial activity of ethanolic and secondary metabolite extracts from S. fluitans III against six bacterial species isolated from marine environments.

In our investigation, specific solvent extractions revealed the presence of secondary metabolites, including alkaloids, flavonoids, anthocyanins, coumarins, saponins, tannins, and triterpenes, constituting up to 16% of the dry mass. Previous studies have highlighted the high phenol content in the Sargassum genus [66], particularly in tannins, which can reach up to 1% of the dry mass. We observed a higher tannin content of 2.4% in the algae used in our study, indicating variability in biochemical composition influenced by environmental factors such as salinity, temperature, UV exposure, season, geographical location of harvesting, and processing methods [67,68]. Furthermore, solvent polarity and temperature play crucial roles in determining crude composition, with solvent polarity affecting compound solubility and temperature impacting stability [69], consequently affecting bioactive potential.

The IR spectroscopy analysis revealed the presence of various compound families within the total extract, as indicated by most of the observed IR bands. This result is consistent with a previous study that emphasized the presence of the same chemical families among the other species, albeit with a significant difference in their quantity [70].

The screening of antibacterial activity of various crude extracts of S. fluitans revealed antibacterial activity across all ratios, with the 30:70 ratio demonstrating higher effectiveness compared to 50:50 and 70:30. This indicates that the total crude extract of S. fluitans contains molecules with antibacterial properties. In a prior study, the crude extract of S. natans exhibited potent antibacterial activity [71], with a larger clearance zone (26 mm radius) compared to our study. This variance could be attributed to biochemical differences resulting from the choice of extraction solvent. In contrast to our methodology, the aforementioned study utilized petroleum ether as the extraction solvent. Therefore, while all Sargassum algae in our study were harvested and processed identically, variations in the bioactivity of our crude extracts may stem from differences in biochemical composition due to varying ethanol ratios used for extraction.