Fatty acid profile and antimicrobial activity of chloroform extracts from the red microalga Porphyridium purpureum against pathogenic intestinal bacteria

Microalgae such as Porphyridium purpureum have been investigated due to their unique biochemical composition and potential biological activities. Antimicrobial resistance is a major public health concern, particularly affecting enteric bacteria of the intestinal microbiota. Antimicrobial activity was evaluated by determining the minimum inhibitory concentration (MIC) against pathogenic intestinal bacteria, including Escherichia coli, Enterococcus faecalis, and Salmonella typhimurium. Fatty acid profile was performed using gas chromatography–mass spectrometry (GC–MS). Cytotoxicity of the lipid extract of P. purpureum was assessed in Vero cells, and the selectivity index (SI) was calculated as the ratio between cytotoxicity and antibacterial activity. The isolated lipid extract and exopolysaccharides from P. purpureum showed no direct antimicrobial activity, however, the lipid extract exhibited a synergistic effect when combined with ciprofloxacin against S. typhimurium and E. coli. Fatty acid analysis revealed the presence of major compounds such as methyl palmitate, methyl arachidonate, and methyl linoleate. The extract presented an IC 50 = 1149.21 μg/mL in Vero cells and a selectivity index >10, indicating low cytotoxicity. The observed synergistic activity was restricted to the ciprofloxacin combinations evaluated against Gram-negative bacteria. The fatty acids identified in the extract, such as methyl palmitate, methyl arachidonate, and methyl linoleate, may contribute to this differential activity by targeting membrane integrity. The lipid extract of P. purpureum did not exhibit antibacterial activity when tested alone but demonstrated a potential modulatory effect in combination with ciprofloxacin against Gram-negative bacteria. These findings suggest that the extract may serve as a source of compounds with antibiotic adjuvant potential and warrant further studies to identify the active constituents and elucidate the mechanisms underlying the observed synergistic effect.

The intestinal microbiota is a complex ecosystem harboring thousand of commensal and symbiotic bacteria that contribute to host homeostasis, intestinal barrier integrity, immunity, metabolism, digestion, and defense against pathogens (McInnes et al., 2020). Alterations in this microbiota due to medication use, inadequate dietary habits, smoking, genetic factors, or colonization by pathogenic strains, can compromise this balance and trigger dysbiosis, which is the imbalance of the intestinal microbiota (Afzaal et al., 2022; Matzaras et al., 2022). Therefore, dysbiosis associated with the inappropriate use of antibiotics may favor the selection of resistant microorganisms and expand the reservoir of resistance genes within the intestinal microbiota, contributing to the spread of antimicrobial resistance (Deshpande et al., 2025).

Opportunistic and pathogenic bacteria, such as Escherichia coli, Salmonella spp., and Staphylococcus aureus, can cause infections ranging from gastrointestinal disorders to severe systemic conditions like sepsis (Viana et al., 2025). The treatment of these infections becomes more challenging when these microorganisms exhibit multidrug resistance. Furthermore, biofilm formation and horizontal gene transfer facilitate the dissemination of this resistance (Khan et al., 2021; Matzaras et al., 2022; McInnes et al., 2020).

Additionally, the inappropriate and excessive use of antibiotics compromises not only pathogenic microorganisms but also the commensal microbiota, contributing to intestinal imbalance and exerting selective pressure that favors the spread of resistant bacteria and the emergence of resistance mechanisms (Cusumano et al., 2025). Consequently, the intestinal microbiota can act as a reservoir for genes associated with bacterial resistance (Deshpande et al., 2025). Therefore, it is fundamental to develop alternative therapeutic strategies capable of selectively controlling pathogenic microorganisms while minimizing impacts on the resident commensal microbiota (Ali and Tuama, 2025).

Antimicrobial resistance (AMR) is one of the most critical global public health challenges of the 21° century. According to the Global Burden of Bacterial Antimicrobial Resistance (Gram) report, AMR was associated with approximately 4.95 million deaths in 2019, of which 1.27 million were directly attributed to resistant pathogens (Murray et al., 2022). Furthermore, projections indicate that in the absence of effective measures to mitigate its impacts, mortality associated with bacterial resistance could reach approximately 10 million annual deaths by 2050 (Naghavi et al., 2024). Conversely, there has been a deceleration in the discovery and development of new antibiotics in recent decades, as many compounds currently under development still show restricted activity against Gram-negative bacteria and do not present mechanisms of action distinct from existing ones. This limits therapeutic progress and may contribute to cross-resistance, also impacting the selectivity between commensal and pathogenic microorganisms (Chen et al., 2023; WHO Bacterial Priority Pathogens List 2024, 2024).

In this scenario, natural products stand out as sources for the bioprospecting of new antimicrobial agents (Bharathi and Lee, 2024), as they may offer mechanisms of action distinct from those observed in antibiotics used in clinical practice, expanding therapeutic possibilities against antimicrobial resistance (Bhowmick et al., 2020). Among the sources of natural products, aquatic organisms such as microalgae have been investigated due to their metabolic potential. These organisms are exposed to diverse environmental conditions, such as variations in salinity, luminosity, and nutrient availability, which can stimulate the biosynthesis of primary metabolites with biological properties (Atanasov et al., 2021; Srinivasan et al., 2021).

Among aquatic organisms, the red microalga Porphyridium purpureum (Rhodophyta) stands out for its metabolic diversity, as it can develop in both marine and freshwater environments. This represents an adaptive advantage and expands its potential for cultivation and biotechnological application (Bayu et al., 2023; Li et al., 2021). This species is also widely recognized for producing sulfated exopolysaccharides, phycobiliproteins, and fatty acids (Bayu et al., 2023). Moreover, the controlled cultivation of P. purpureum allows for the sustainable production of biomass rich in bioactive compounds, whose antiviral, antioxidant, and anti-inflammatory properties have already been described in the literature (Agustina et al., 2020; Hayes et al., 2023; Liu et al., 2025; Risjani et al., 2021; Talyshinsky et al., 2002).

Among the various metabolites produced by microalgae, lipids belonging to the primary metabolism, especially long-chain fatty acids, have been frequently associated with antimicrobial activity. In P. purpureum, polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA) and eicosapentaenoic acid (EPA) are notable. These can exert antimicrobial effects through interaction with the bacterial cytoplasmic membrane, promoting changes in its permeability and structural integrity (Cepas et al., 2021; Domingues et al., 2022).

In addition to membrane destabilization, fatty acids can interfere with essential cellular processes, such as the electron transport chain and bacterial enzymatic metabolism, resulting in structural damage and cell lysis (Desbois and Smith, 2010; Yoon et al., 2018). In this context, characterizing the lipid profile of extracts obtained from P. purpureum becomes relevant to understanding the potential role of these metabolites in antimicrobial activity against intestinal pathogens. To obtain these compounds, organic solvents such as chloroform are widely used in lipid extraction protocols due to their high efficiency in solubilizing non-polar molecules, such as fatty acids and other lipids present in microalgae (Wang et al., 2025).

The present study aimed to characterize the lipid profile of the chloroform extract and evaluate the extraction of exopolysaccharides from the red microalga P. purpureum. Furthermore, it sought to investigate the antimicrobial activity of both fractions against pathogenic intestinal bacteria, assessing their potential as sources of bioactive compounds for the development of alternative strategies to control bacterial infections.

Reagents and equipment

All reagents and equipment used in the experiments are described in this section. Boron trifluoride solution (14%) was purchased from Sigma-Aldrich®. Dimethyl sulfoxide (DMSO PA) was obtained from Dinâmica. Mueller–Hinton broth/agar was supplied by HiMedia. Ciprofloxacin (Sigma-Aldrich) and ampicillin sodium ultrapure (USB) were used as reference antibiotics. Chloroform was purchased from Labsynth. The following analytical reagents were used: 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP) (Sigma-Aldrich) and 2′,7′-dichlorofluorescein (DCF) (Sigma-Aldrich).

The equipment used included a rotary evaporator (Fisatom, São Paulo, Brazil), a microplate reader (Infinite® 200 PRO, Tecan). Gas chromatography coupled with mass spectrometry (GC–MS) analyses were performed using a Shimadzu GC-2010 Plus gas chromatograph coupled to a QP2010 SE mass spectrometer. Ultrasonic treatments were carried out using an Elmasonic® E 30H ultrasonic bath (Elma).

Cultivation of P. purpureum

The cultivation and biomass preparation of P. purpureum were carried out in collaboration with AlgaSul® Microalgae Biotechnology (Rio Grande, RS, Brazil). The microalga was cultivated in a 15 L bubble column photobioreactor inside a cultivation room under controlled temperature of 25 °C and artificial illumination with intensity of 150 µmol photons m -2 cm -1. Cultures were grown in artificial seawater (32 g L -1) enriched with F/2 medium in a batch system (Guillard, 1975). The cultures were maintained for approximately 9 days under a photoperiod of 12 h of light and 12 h of dark. Growth was monitored by optical density, while pH was maintained throughout the cultivation period. Biomass was harvested when the culture reached absorbance of 0.6, corresponding to a biomass concentration of 0.39 g. L -1 according to the standard calibration curve provided by the company. Porphyridium purpureum biomass was obtained from a single cultivation batch. The harvested biomass was used for lipid extraction and subsequent analyses.

Total lipids of P. purpureum

Lipid extracts were obtained from the dry biomass using chloroform as the solvent at a 1:6 ratio (biomass (g)/solvent (mL)). The extraction was carried out by an exhaustive percolation method, consisting of three 20-min cycles under magnetic stirring (adapted from D’Oca et al., 2011). After the third extraction cycle, the extract was filtered, and the chloroform was removed under reduced pressure using a rotary evaporator until a dried extract was obtained. The dried extract was then resuspended in 50% dimethyl sulfoxide (DMSO) to obtain a final concentration of 40 mg/mL for antimicrobial activity assays. Lipid yield was determined by comparing the mass of the extract to the initial dry mass of the plant material (w/w).

Fatty acid profile by gas chromatography mass spectrometry (GC-MS) of fatty acids

For the chemical characterization of fatty acids in P. purpureum, the extract was derivatized, that way 3 mL boron trifluoride (14%) were added in the extract, and the mixture was heated at 70 °C for 20 min. After heating, the solution was washed in a separatory funnel with 15 mL of hexane and 20 mL of distilled water, separating the organic and aqueous phases. The organic phase was dried again in a rotary evaporator, and the residues were resuspended with absolute ether. Subsequently, the ether was evaporated, and the sample was solubilized in hexane for analysis on a gas chromatograph (Metcalfe and Schmitz, 1961).

Fatty acid methyl esters (FAMEs) were analyzed using a Shimadzu GC-2010 Plus coupled to a QP2010 SE mass spectrometer. Separation was performed on a capillary column (5% dimethyl polysiloxane/95% diphenyl). The injector was set at 250 °C, operating in split mode (1:100). The oven program followed Borges et al. (2016). Helium (99.999%) was used as carrier gas at 1.3 mL/min. Fatty acids were identified by comparing mass spectra and retention times with the NIST Mass Spectral Library 20 (2020).

Exopolysaccharide (EPS) extraction

Exopolysaccharides (EPS) were extracted following Cristofoli et al. (2023) methodology: 1 mL of P. purpureum culture was mixed with 2 mL of cold absolute ethanol (1:2) and subjected to ultrasonic treatment for 20 min at 20 °C. The mixture was then refrigerated overnight to reduce EPS solubility and filtered to collect the precipitate. The precipitate was washed twice with Milli-Q water and recovered by centrifugation at 1000 rpm for 20 min. After the second cycle, the supernatant was discarded, and the pellet was dried in a silica desiccator. Finally, the material was resuspended in Milli-Q water to a final concentration of 40 mg/mL and stored at 10 °C, protected from light. EPS yield was calculated from the dry EPS mass and expressed as milligrams of EPS per liter of culture (mg/L) according to the following equation: E P S y i e l d m g / L = D r y w e i g h t o f E P S g C u l t i v a t i o n v o l u m e L X 1000 (Cristofoli et al., 2023).

Fourier-Transform Infrared spectroscopy (FTIR) analysis

The P. purpureum exopolysaccharides were prepared following the protocol described for Cristofoli et al. (2023). The samples were analyzed using a Fourier-Transform Infrared (FTIR) spectrometer in transmittance mode, equipped with an Attenuated Total Reflection (ATR). Spectra were acquired over a range of 400–4,000 cm -1 with a resolution of 4 cm -1. Functional groups were identified by correlating the resulting spectra with specialized literature and previous studies on the genus Porphyridium sp.

Preparation of bacterial inoculum

The bacteria used in the antimicrobial evaluation were obtained from the strain bank of the Núcleo de Desenvolvimento de Novos Fármacos (NUDEFA) at the Federal University of Rio Grande (FURG). The species tested included potential intestinal pathogens, represented by the reference strains E. coli (ATCC 25922), Salmonella enterica serovar Typhimurium (ATCC 14028), and Enterococcus faecalis (ATCC 51299). The strains were cultured on Mueller-Hinton (MH) agar and were incubated at 37 °C for 24 h.

Bacterial suspensions were prepared in test tubes, into which the bacteria were inoculated in 0.85% saline solution. The bacterial inoculum was adjusted to an optical density (OD 600) corresponding to the McFarland 0.5 scale (between 0.080 and 0.100, approximately equivalent to 1 × 10 8 cells/mL). Finally, the bacterial suspension was diluted in MH medium at a ratio of 1:100 (approximately 5 × 10 5 cells/mL), ensuring standardization of cell concentration for subsequent assays (CLSI, 2021).

Minimum inhibitory concentration (MIC)

The Minimum Inhibitory Concentration (MIC) was determined by the broth microdilution method following the guidelines of the CLSI M100 document (CLSI, 2021) using sterile 96-well microplates. Briefly, 50 μL of Mueller–Hinton broth was added to each well, followed by 50 μL of P. purpureum extract in the first well, which was serially diluted to obtain final concentrations ranging from 800 to 6.25 μg/mL. Subsequently, 50 μL of bacterial inoculum was added to each well. The final concentration of DMSO in the first well was 0.5% (v/v), decreasing proportionally in the subsequent serial dilutions.

The following controls were included: bacterial growth control, sterility control (Mueller–Hinton broth only), vehicle control (0.5% DMSO), extract control, and antibiotic control (ciprofloxacin). Plates were incubated at 35 °C ± 2 °C for 24 h, followed by the addition of 30 μL of 0.02% resazurin. After 2 h, color change was assessed, with pink indicating bacterial growth and blue indicating growth inhibition. MIC was defined as the lowest concentration that completely inhibited visible bacterial growth. The antimicrobial assays were performed in triplicate on three different experimental days using the same extract.

Fractional inhibitory concentration index (FICI)

Interactions between the extract and antibiotic were evaluated using the checkerboard method, as described by Bellio et al. (2021). The fractional inhibitory concentration (FIC) was calculated by comparing the MIC in combination with the MIC of each compound alone, and the fractional inhibitory concentration index (FICI) was obtained as the sum of both FICs.

Assays were performed in 96 well microplates with serial dilutions (1:2) of ciprofloxacin (32–0.25 μg/mL) against E. coli and S. enterica Typhimurium and ampicillin for E. faecalis, according to Clinical and Laboratory Standards Institute (2020) guidelines, and P. purpureum extract (800–6.25 μg/mL) was added to the wells, followed by bacterial inoculum. The final concentration of DMSO in the first well was 0.5%, decreasing proportionally in the subsequent dilutions.

The following controls were included: bacterial growth control, medium sterility control, vehicle control (0.5% DMSO), extract control, and antibiotic control (ciprofloxacin or ampicillin, depending on the bacterial strain). Plates were incubated at 35 °C ± 2 °C for 24 h, after which 30 μL of 0.02% resazurin was added. The experiments were performed in triplicate on three different experimental days using the same lipid extract. FICI values were interpreted as follows: ≤0.5 (synergy), >0.5–4 (indifference), and >4 (antagonism), according to Bellio et al. (2021).

Cytotoxicity activity

The cytotoxicity of P. purpureum extract against the Vero cell line was assessed according to Ansar Ahmed et al. (1994). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic solution (penicillin–streptomycin). A volume of 200 µL of cell suspension (3.4 × 10 5 cells/mL) were seeded into 96 well plates. After 24 h at 37 °C, cells were treated with extract concentrations ranging from 1600 to 200 μg/mL for an additional 24 h. The final concentration of DMSO in the first well was 1.0%, decreasing proportionally in the subsequent serial dilutions. The following controls were included: untreated cell control, vehicle control (1% DMSO), and extract-treated cells.

Then, 30 µL of 0.02% resazurin diluted in DMEM were added, and fluorescence was measured at 620 nm after 6 h. The IC 50 was calculated using the formula I C 50 = 1 − E x p o s e d O D U n e x p o s e d O D × 100 % to determine the concentration required to reduce cell viability by 50%. The assay was performed in triplicate, and IC 50 values were expressed as mean ± standard deviation (SD). The selectivity index (SI) was subsequently calculated as the ratio between IC 50 and the MIC obtained in the antimicrobial assays, as presented in the equation: S I = I C 50 M I C. According to Awouafack et al. (2013), SI values above 10 have been proposed as an indicator of a favorable in vitro safety margin.

Total lipids yield from P. purpureum

The biomass obtained from the cultivation of P. purpureum, using the extraction process by the percolation exhaustion method, resulted in a total lipid yield of 16% (w/w) relative to the dry biomass. This value reflects the recovery efficiency of compounds from the cultivated microalgal material.

Characterization of fatty acids present in P. purpureum

GC–MS analysis of the derivatized chloroform extract enabled the identification of seven fatty acid methyl esters (FAMEs), which represent the fatty acids originally present in the lipid extract after derivatization (Table 1). The major FAMEs detected were methyl palmitate (42.05%), methyl arachidonate (22.73%), methyl linoleate (17.91%), methyl eicosapentaenoate (8.64%), methyl oleate (4.35%), methyl α-linolenate (2.30%), and methyl stearate (2.02%).

Fourier-Transform Infrared spectroscopy (FTIR) of Porphyridium purpureum exopolysaccharides (EPS)

The extraction of exopolysaccharides from P. purpureum resulted in an EPS yield of 841.7 mg/L of culture, calculated from the dry weight of the EPS obtained after extraction.

The exopolysaccharides of P. purpureum were analyzed over a spectral range of 400–4,000 cm -1 with a resolution of 4 cm -1. The spectrum exhibited characteristic absorption bands at 3,340 and 1664 cm -1, corresponding to O–H stretching and carboxyl group vibrations indicating the presence of acidic functional groups and associated organic compounds. The resulting spectra from the biomass analysis are shown in Figure 1.

Minimum inhibitory concentration (MIC) determined by broth microdilution

The antimicrobial activity of the chloroform extract and the exopolysaccharides (EPS) from P. purpureum was evaluated using the broth microdilution method to determine the Minimum Inhibitory Concentration (MIC), with concentrations ranging from 800 to 6.25 μg/mL. No inhibitory effect was observed for either the extract or the EPS against S. enterica serovar Typhimurium, E. coli, and E. faecalis strains at any of the evaluated concentrations.

Evaluation of the synergism of P. purpureum extract and antibiotics

The combination of P. purpureum extract with ciprofloxacin was evaluated using the Checkerboard method to determine the Fractional Inhibitory Concentration Index (FICI), where FICI values ≤0.5 indicate a synergistic effect (Bellio et al., 2021). At a concentration of 6.25 μg/mL, the extract reduced the MIC of ciprofloxacin from 4 to 1 μg/mL against Salmonella Typhimurium (FICI = 0.26), while a concentration of 12.5 μg/mL reduced the MIC from 4 to 2 μg/mL against E. coli (FICI = 0.50), indicating synergism between the extract and ciprofloxacin. Conversely, for E. faecalis, the P. purpureum extract showed no synergism when associated with the antibiotic ampicillin, indicating an indifferent effect (FICI = 2).

Despite the reduction in MIC observed for the extract antibiotic combination, the susceptibility profile of the bacteria, according to CLSI criteria (2021), did not change categories, remaining resistant for S. Typhimurium and intermediate for E. coli. However, the association of the extract with ciprofloxacin resulted in bacterial inhibition at lower extract concentrations, decreasing from >800 μg/ mL (MIC of the extract alone) to 6.25 μg/mL against E. coli and to 12.5 μg/mL against S. Typhimurium when combined with ciprofloxacin (Table 2).

Cytotoxicity assessment

The in vitro cytotoxicity of the P. purpureum extract was evaluated in Vero cells using a colorimetric assay with resazurin as an indicator. The cells were exposed for 24 h to different extract concentrations, ranging from 1600 to 200 μg/mL. The chloroform extract exhibited cytotoxic activity, with a calculated IC 50 value of 1149.21 μg/mL (0.1005 ± 0.0298).

The selectivity of the extract was evaluated by the Selectivity Index (SI), determined by the ratio between the IC 50 in Vero cells and the MIC obtained from the combinations that showed synergism with antibiotics, as shown in Table 3. The SI is an in vitro screening parameter that reflects the selectivity of the extract and provides an initial indication of its biological safety in relation to antimicrobial activity. Thus, the higher its value, the greater the activity against the microorganism and the lower the cytotoxicity toward Vero cells.

Characterization of fatty acids present in the lipids of P. purpureum

It is important to note that the GC-MS analysis was performed after derivatization, therefore, the compounds detected in this study correspond to fatty acid methyl esters (FAMEs) derived from the fatty acids originally present in the extract.

Therefore, the lipid fraction composition identified in this study is similar to chemical characterizations of P. purpureum presented in other studies, particularly regarding the predominance of primary metabolites such as palmitic acid (PA, C16:0) and arachidonic acid (ARA, C20:4n-6) (Su et al., 2016; Li et al., 2019; Kavitha et al., 2016; Ardiles et al., 2020; Castro-Varela, Sáez, and Gómez, 2021). Due to the presence of multiple double bonds along their hydrocarbon chains, which reduce intermolecular interactions between adjacent chains, PUFAs like ARA are essential structural components of cellular and thylakoid membranes, enhancing membrane fluidity. In contrast, saturated fatty acids (SFAs) are synthesized within the chloroplast, serving both as critical energy reserves and as precursors for the synthesis of other cellular fatty acids (Ji et al., 2021; Li et al., 2024).

Comparatively, a study by Lu et al. (2020) demonstrated that total lipid production in P. purpureum reached its highest percentage (approximately 10.65%) when cultivated in saline water (34 g/L) at a pH around 8.0, which aligns with the cultivation parameters employed in the present study. An alkaline pH is considered optimal for the activity of enzymes involved in the conversion of long-chain fatty acids, such as PA and ARA, thereby increasing the content of these compounds when the microalga is exposed to these conditions (Cohen, Vonshak, and Richmond, 1988). Figure 2 illustrates a comparison between the fatty acids identified in this research and other chemical characterization studies of P. purpureum.

ARA stands out in P. purpureum samples, a species recognized for its exceptional capacity to accumulate this compound (Khozin-Goldberg, Iskandarov, and Cohen, 2011). This characteristic stems from the predominance of the omega-6 biosynthetic pathway in this rhodophyte, in contrast to green microalgae, which primarily utilize (ALA) as a precursor for long-chain PUFAs. Furthermore, ARA exhibits high plasticity in P. purpureum and it is more susceptible to environmental variations than other fatty acids (Ji et al., 2021; Li et al., 2024).

Fourier-Transform Infrared spectroscopy (FTIR) of Porphyridium purpureum exopolysaccharides (EPS)

The results obtained in this study agree with literature employing FTIR for the characterization of compounds in Porphyridium species. The FTIR spectrum of the P. purpureum exopolysaccharide displayed characteristic bands of various functional groups. The band at 3,340 cm -1 was attributed to O–H stretching vibrations, associated with hydroxyl groups, as reported by Karbowiak et al. (2011) and Cristofoli et al. (2023).

The band at 670 cm -1 is likely associated with alkylene C-H bond, agreement with Cristofoli et al. (2023) and Jayaraman et al. (2016). Meanwhile, the band at 1644 cm -1 is consistent with carbonyl (C=O) stretching within the 1500–1710 cm -1 region, or possibly C=C bonds, suggesting the presence of carboxylic acids, ketones, or amides, as discussed by Fernando et al. (2017). Finally, the band at 1108 cm -1 corresponds to the deformation of CH 2 and CH 3 groups, potentially including contributions from C–O or S=O bonds, according to Ziadi et al. (2018) and Mandal et al. (2011).

Antimicrobial activity of P. purpureum extract

The antimicrobial activity of P. purpureum is widely attributed to the presence of sulfated exopolysaccharides (EPS) produced by this genus (Bayu et al., 2023; Gargouch et al., 2021; Ben Hlima et al., 2021). In contrast, we conducted antimicrobial assays using EPS extracted from P. purpureum at concentrations ranging from 800 μg/mL to 6.25 μg/mL against E. coli, S. Typhimurium, and E. faecalis, however, no growth inhibition was observed for any of the tested microorganisms.

Although studies evaluating the antimicrobial activity of its lipophilic extract are scarce, it is well-established that the compounds identified in this research possess promising antimicrobial potential (Casillas-Vargas et al., 2021). The antimicrobial mechanisms of fatty acids primarily include: the disruption of plasma membrane stability, facilitating ion influx that alters intra- and extracellular charges, thereby impairing the selective permeability of both prokaryotes and eukaryotes (Wu et al., 2006; Desbois and Smith, 2010; Yoon et al., 2018). Regarding their chemical nature, polyunsaturated fatty acids (PUFAs) appear to exhibit greater antimicrobial potency compared to saturated fatty acids (SFAs) such as palmitic and oleic acids. This can be attributed to their higher fluidity and, consequently, enhanced interaction with the plasma membrane and penetration into the cellular interior (Casillas-Vargas et al., 2021; Wu et al., 2006).

Although the chloroform extract alone did not inhibit bacterial growth, its combination with ciprofloxacin reduced the MIC against E. coli and S. Typhimurium, indicating a synergistic interaction. These findings suggest that the extract may act as an antibiotic adjuvant rather than as a direct antimicrobial agent. One possible explanation is that lipid constituents of the extract may increase bacterial membrane permeability, thereby facilitating ciprofloxacin uptake. However, this mechanism was not directly investigated in the present study and remains to be confirmed.

An in vitro antimicrobial assay combining ARA with the aminoglycoside gentamicin against Staphylococcus aureus resulted in a significant reduction of the antibiotic’s minimum inhibitory concentration (MIC) from 37 ± 22 μg/mL to 1.7 ± 0.5 μg/mL. Conversely, PA tested in the same study failed to exhibit synergistic effects when combined with the same drug against S. aureus. In that same study, LA, which possesses two fewer double bonds than ARA, showed synergism with gentamicin, although to a lesser extent than ARA (Beavers et al., 2022). Another study evaluating the synergistic potential of neomycin combined with PUFAs against S. aureus reported FICI values of 0.38 and 0.19 for EPA (C20:5) and ALA (C18:3), respectively. Although EPA possesses a higher degree of unsaturation than ALA, the latter is characterized by a lower molecular weight (Desbois and Lawlor, 2013).

This synergistic effect can be attributed to the ability of specific PUFAs to induce disruptions in the plasma membrane, thereby facilitating drug penetration into the intracellular environment and potentiating its activity. The efficacy of these compounds tends to be dependent on their structural characteristics, such as the degree of unsaturation and molecular size (Casillas-Vargas et al., 2021). Furthermore, the thick peptidoglycan cell wall characteristic of Gram-positive bacteria, such as S. aureus, does not appear to hinder the interaction between PUFAs and the microorganism’s cytoplasmic membrane.

In Checkerboard Assay involving PUFAs and SFAs against Gram-negative bacteria, although scarce, the interaction between levofloxacin—a fluoroquinolone, similar to the ciprofloxacin tested in this work and medium-chain fatty acids (MCFAs), such as capric acid (CA, C10:0) and geranic acid (GA, C10:2), is noteworthy (Liu et al., 2022; Alkhawaja et al., 2023). Both levofloxacin-CA and levofloxacin-GA combinations exhibited FICI values of 0.022 against Pseudomonas aeruginosa, 0.125 against Klebsiella pneumoniae, and 0.260 against E. coli. Despite their structural differences, the shorter chain length of these compounds compared to the fatty acids identified in the present study appears to enhance the intracellular concentration of the antimicrobial agent.

In contrast to previous reports, our results demonstrate for the first time that the chloroform extract of P. purpureum, predominantly composed of PUFAs, potentiated the activity of ciprofloxacin against E. coli and S. Typhimurium. In contrast, no synergistic effect was observed for E. faecalis when the extract was combined with ampicillin. However, this difference should be interpreted with caution because different antibiotics were evaluated for Gram-negative bacteria (ciprofloxacin) and E. faecalis (ampicillin). Therefore, the absence of synergism cannot be attributed just to differences in Gram-positive cell envelope structure but may also reflect antibiotic specific interactions with the lipid extract. Further studies using the same antibiotic across different bacterial species are required to clarify this issue.

Nevertheless, the fatty acid composition of the extract may have contributed to the synergistic interaction observed with ciprofloxacin. In particular, fatty acids such as palmitic acid (PA, C16:0) and linoleic acid (LA, C18:2), together with other constituents of the lipid fraction, may facilitate antibiotic activity by altering membrane properties, although the individual contribution of each compound remains to be determined. Therefore, the observed synergistic effect is likely the result of the combined action of multiple lipid constituents rather than a single fatty acid, highlighting the need for future studies using isolated compounds and mechanistic assays to identify the molecules responsible for this activity. Additionally, future investigations should include a broader panel of clinical isolates to further validate the antimicrobial potential of the P. purpureum lipid extract.

Cytotoxicity assessment of P. purpureum extract

No studies have been found evaluating Porphyridium sp. Extracts in cell lines such as fibroblasts; instead, existing research has focused on tumor lineages (Gardeva et al., 2009; Silva-Núñez et al., 2023) or isolated compounds from the genus, such as polysaccharides (Huheihel et al., 2002). There is also a gap in studies evaluating the selectivity index, which is an important parameter for the initial assessment of the relationship between cytotoxicity and antimicrobial activity in vitro (Awouafack et al., 2013).

In our study, the P. purpureum extract demonstrated greater activity against bacteria than against mammalian cells, as shown by the selectivity index. Eukaryotic cell membranes contain cholesterol, which assists in the selective permeability of the lipid bilayer (Subczynski et al., 2017). In contrast, prokaryotic cells include a peptidoglycan layer and, in the case of Gram-negative bacteria, an outer membrane containing lipopolysaccharides and porins that regulate molecular entry (Maher and Hassan, 2023). Although the outer membrane acts as a selective barrier, its composition and charge may favor interactions with bioactive compounds. Furthermore, the lower structural and metabolic complexity of bacteria may increase their susceptibility to these compounds, which may have contributed to the observed selectivity (Cos et al., 2006; Maher and Hassan, 2023; Matei and Ioana Visan, 2025).

In our study, GC–MS analysis of the fatty acid methyl esters (FAMEs) derived from the P. purpureum chloroform extract revealed a fatty acid profile characterized by methyl palmitate, methyl arachidonate, and methyl linoleate, indicating that palmitic, arachidonic, and linoleic acids are major constituents of the original lipid extract. However, the extract alone did not demonstrate antimicrobial activity against the tested bacterial strains at the evaluated concentrations. When combined with ciprofloxacin, the extract reduced the MIC of the antibiotic against the Gram-negative bacteria S. Typhimurium and E. coli, indicating a possible modulatory effect that enhances the activity of ciprofloxacin. In contrast, no synergistic effect was observed against the Gram-positive bacterium E. faecalis, suggesting that the modulatory activity may depend on the specific bacteria antibiotic combination evaluated. Because different antibiotics were used for Gram-negative bacteria and E. faecalis, further studies are required before attributing these differences to bacterial cell envelope characteristics alone. The extract presented an IC 50 of 1149.21 μg/mL in Vero cells, resulting in selectivity indices that reinforce its potential as an adjuvant agent. Our results suggest that while the extract is not effective as a standalone antimicrobial agent, it can act as a modulator of ciprofloxacin activity, especially against Gram-negative bacteria.

Finally, these results indicate the potential of P. purpureum extract as a therapeutic adjuvant, highlighting the importance of future studies focused on the isolation and individual evaluation of the identified fatty acids. Such studies are necessary to elucidate which compounds are responsible for the observed synergistic effect and to expand the prospects for application in the discovery of new antimicrobial agents.