Antimicrobial photodynamic activity of toluidine blue encapsulated in mesoporous silica nanoparticles against Pseudomonas aeruginosa and Staphylococcus aureus

In the present study, the antimicrobial and antibiofilm efficacy of toluidine blue (TB) encapsulated in mesoporous silica nanoparticles (MSN) was investigated against Pseudomonas aeruginosa and Staphylococcus aureus treated with antimicrobial photodynamic therapy (aPDT) using a red diode laser 670?nm wavelength, 97.65?J cm^?2 radiant exposure, 5?min). Physico-chemical techniques (UV-visible (UV-vis) absorption, photoluminescence emission, excitation, and FTIR) and high-resolution transmission electron microscopy (HR-TEM) were employed to characterize the conjugate of TB encapsulated in MSN (TB MSN). TB MSN showed maximum antimicrobial activities corresponding to 5.03 and 5.56 log CFU ml^?1 reductions against P. aeruginosa and S. aureus, respectively, whereas samples treated with TB alone showed 2.36 and 2.66 log CFU ml^?1 reductions. Anti-biofilm studies confirmed that TB MSN effectively inhibits biofilm formation and production of extracellular polymeric substances by P. aeruginosa and S. aureus.     

Bacteriocin-like inhibitory substances from <Emphasis Type="Italic">Campylobacter</Emphasis> spp.

Twenty-five Campylobacter isolates were screened for production of antimicrobial substances using a deferred antagonism assay. Sixteen isolates showed activity against either Staphylococcus aureus, Salmonella enterica serovar Enteritidis or Candida albicans. The inhibitory activity was sensitive to treatment with pronase E, trypsin and pepsin, suggesting that the antimicrobial compound(s) are proteinaceous. Activity spectra of isolates included S. aureus, Micrococcus luteus, Streptococcus sp., Bacillus subtilis, a drug-resistant clinical isolate of S. aureus and one isolate of C. albicans. Producing isolates showed cross-immunity and inhibitory activity was only observed on solid media. The findings of this study suggest that Campylobacter produces proteinaceous inhibitory substances.     

93 Spectroscopic investigation of methylene blue binding to DNA

The interaction of methylene blue (MB) with DNA has been investigated by UV absorption spectra, Fluorescence spectra and UV-melting method. Analysis of the results of the melting experiments shows that melting temperature (T _m) of the complexes increases with the [total ligand]: DNA ratio (r) at two concentrations of Na⁺ (2?mM Na⁺ and 20?mM Na⁺) providing support for conclusion that MB is a stabilizer of DNA helix structure. By contrast, the shapes of dependences of width of transition (ΔT) on r at low and high [Na⁺] are different which points to the existence of different types of binding modes of MB with DNA. UV-spectroscopy experiments and fluorescence spectra indicated that the binding modes of MB with DNA depended on r. At high r (r?>?0.25), remarkable hypochromic effect with no shift of λ _max in the absorption spectra of MB was observed. The fluorescence of MB was quenched which indicated that MB was bound to phosphate groups of DNA by electrostatic interaction. At low r ratios (r?<?0.2), the absorption spectra of MB upon increasing the concentration of DNA showed gradually decrease in the peak intensities with a red shift. This phenomenon is usually associated with molecular intercalation into the base stack of the ds-DNA. Using the Scatchard’s model, the complex formation constants for MB with DNA were determined: the binding constant K?≈?6.5?×?10⁵ and binding site size n?≈?4. Obtained data are not typical for intercalation model of ligands to DNA. Moreover, comparison between these data and our early experimental results of interaction of ethidium bromide with DNA made it possible to suggest that this binding type of MB is, more probably, semi-intercalation mode (Vardevanyan et al., 2003). This conclusion is in accordance with the analysis of the model structures of MB–DNA complexes which clearly shows the importance of solvent contributions in suggested structural form (Tong et al., 2010).     

Virulence Factors and Anti Fungal Sensitivity Pattern of<Emphasis Type="Italic"> Candida</Emphasis> Sp. Isolated from HIV and TB Patients

The study comprised of 60 Candida spp., 50 isolates from HIV and TB positive individuals (immunocompromised) and 10 isolates from non-HIV and -TB patients (immunocompetent). Among the 60 Candidal isolates, 83.3% were identified as C. albicans, 11.6% as C. glabrata and rest 5% as C. krusei. There is no study in production pattern of extracellular enzymes of Candida spp. isolated from HIV and TB patients in comparison with non-HIV and -TB patients in India. The comparison of phospholipase activities showed that there was a significant difference between the groups at (P = 0.001). The non-HIV and -TB groups of C. glabrata and C. krusei did not show detectable phospholipase activity when compared to the HIV and TB groups. The mean difference in the phospholipase activities of these two groups was significant (P = <0.001). Candida spp. of both the groups do not possess the ability to hydrolyze gelatin. All the strains possessed the ability to show alpha haemolysis. Even though it had shown alpha haemolysis, the significant difference in haemolytic activity was observed only in C. albicans (P = <0.001). None of the isolates from the two groups possessed the ability to hydrolyze gelatin. In the resistance profile of Candida spp., C. albicans of HIV and TB groups had shown resistance to fluconazole, Itraconazole, ketaconazole, nystatin but showed 100% sensitivity towards amphotericin-B. The isolates of C. krusei and C. glabrata showed no resistance to any of the drugs tested. In the case of, non-HIV and -TB patients the resistance pattern was low.     

Photodynamic antimicrobial chemotherapy by liposome-encapsulated water-soluble photosensitizers

Photodynamic antimicrobial chemotherapy is an alternative method for killing bacterial cells in view of the increasing problem of multi-antibiotic resistance. We examined the effect of three water-soluble photosensitizers (PhS): methylene blue (MB), neutral red (NR) and rose bengal (RB) on Gram-positive and Gram-negative bacteria. We compared the efficacy of PhS in their free form and encapsulated in liposomal formulations against various bacterial strains, and determined conditions for the effective use of encapsulated PhS. We found that all three PhS were able to eradicate the Gram-positive microbes Staphylococcus aureus and Sarcina lutea; and MB and RB were effective against St. epidermidis. In the case of the Gram-negative species, MB and RB were cytotoxic against the Shigella flexneri, NR-inactivated Escherichia coli and Salmonella para B, and BR was effective in killing Pseudomonas aeruginosa. None of the examined PhS showed activity against Klebsiella pneumoniae. MB and NR enclosed in liposomes gave a stronger antimicrobial effect than free PhS for all tested prokaryotes, whereas encapsulation of RB led to no increase in its activity. We suggest that encapsulation of PhS can increase the photoinactivation of bacteria.     

Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile

Background

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections.

Methods

PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29.

Results

Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm²) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT.

Conclusion

This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon.     

In vitro antimicrobial activity of light-activated phthalocyanines

Background

Photodynamic antimicrobial therapy (PACT) is proposed as a topical, non-invasive approach suitable for treatment of locally occurring infection. Research of photosensitizers, (PS) as well as their development, is aimed at finding effective antimicrobial substances which would have a broad-spectrum potency. The aim of this paper is to evaluate the antimicrobial effect of phthalocyanine (Pc) derivatives.

Methods

Fifteen different Pc compounds were investigated. Their photokilling activity was tested on Staphylococcus aureus, Escherichia coli and Candida albicans. After treating of microbial cells with Pc at the concentrations: 1 mg/l, 2 mg/l, 4 mg/l, 8 mg/l for 30 minutes, the cultures were irradiated with low-power laser light at a wavelength of 670 nm (20 J/cm², 40 J/cm²). The effectiveness of photoinactivation was evaluated based on the decrease in number (log₁₀) of viable bacteria.

Results

Eight Pc compounds tested showed antibacterial effects against S. aureus, but only four were effective against E. coli and two against C. albicans. The most effective photosensitizers were amphiphilic sulphonated zinc Pc compounds [(3-diethylammonium)-propylsulphonamide citrate (Pc3) and cationic tetramethylenepyridinium chloride of hydroxyaluminum Pc (Pc7)].

Conclusions

The most efficient phthalocyanines (Pc3, Pc7) cause a significant decrease in viable counts of all tested microbes.     

Encapsulation of methylene blue in polyacrylamide nanoparticle platforms protects its photodynamic effectiveness

The ability to prevent methylene blue (MB), a photosensitizer, from being reduced by plasma reductases will greatly improve its efficacy in photodynamic therapy (PDT) applications. We have developed a delivery approach for PDT by encapsulating MB using nanoparticle platforms (NPs). The 30-nm polyacrylamide-based NPs provide protection for the embedded MB against reduction by diaphorase enzymes. Furthermore, our data shows the matrix-protected MB efficiently induces photodynamic damage to tumor cells. The unprecedented results demonstrate the significant in vitro photodynamic effectiveness of MB when encapsulated within NPs, which promises to open new opportunities for MB in its in vivo and clinical studies.     

Control of apple blue mold by <Emphasis Type="Italic">Pichia pastoris</Emphasis> recombinant strains expressing cecropin A

Recombinant Pichia pastoris yeasts expressing cecropin A (GS115/CEC), was evaluated for the control of the blue mold of apple caused by Penicillium expansum due to cecropin A peptide’s effective antimicrobial effects on P. expansum spores by the thiazolyl blue (MTT) assay. Then, the protein concentration was determined and it was expressed at high levels up to 14.2 mg/L in the culture medium. Meanwhile, the population growth was assayed in vivo. The population growth of recombinant strain GS115/CEC was higher than that of non-transformed strain GS115 in red Fuji apples wounds. Recombinant yeast strains GS115/CEC significantly inhibited growth of germinated P. expansum spores in vitro and inhibited decay development caused by P. expansum in apple fruits in vivo when compared with apple fruits inoculated with sterile water or the yeast strain GS115/pPIC (plasmid pPIC9k transformed in GS115). This study demonstrated the potential of expression of the antifungal peptide in yeast for the control of postharvest blue mold infections on pome fruits.     

Different photodynamic action of proflavine and methylene blue on bacteriophage

Summary The irradiation with visible light (Li) of temperate Serratiaphage that is maximally sensitized with either proflavine (PF) or methylene blue (MB) induces—apart from lethal lesions—mutations which are phenotypically expressed as clear (c) or lightly turbid (l) plaques. The mutagenicity of the MB+Li and PF+Li treatment differs in several respects: (i) Up to an inactivation of 6 to 8 lethal hits MB+Li is a much more potent mutagen than PF+Li. (ii) at low levels of survival the dose curve of mutation frequency with MB+Li reaches a peak and then decreases drastically while the mutation frequency after PF+Li continues to increase in proportion to lethal hits induced. (iii) Mapping of 100 MB+Li and 77 PF+Li induced c or l mutants indicates significant difference in the electiveness of the four genomic regions of c or l mutants.     

Photoantimicrobials as a potential local approach to geriatric UTIs

Aims: To test the efficacy of acceptable photoantimicrobial agents against bacterial pathogens implicated in complicated urinary tract infection (UTI) in comparison with conventionally employed antibacterials. Methods and Results: Toluidine blue (TB), methylene blue (MB), 5‐aminolaevulinic acid (ALA), trimethoprim and levofloxacin were employed in the study against the typical UTI‐implicated pathogens Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Proteus mirabilis. Standard bacterial cell culture was used to assay the activity both in the dark and under 660‐nm LED‐illuminated conditions. TB and MB were highly photoactive across the range and exhibited rapid kill rates, their effects being assayed after 20‐min illumination, rather than the 18‐h incubation employed with the other compounds. Trimethoprim was inactive against all bacteria except Pr. mirabilis, while levofloxacin maintained highly bactericidal activity throughout. ALA required high concentrations for effective action but, for porphyrin production in situ, also required an 18‐h incubation. Conclusions: TB and MB were highly and rapidly photobactericidal in comparison with the remaining agents tested. Significance and Impact of the Study: Ubiquitous catheterization of geriatric patients offers a portal for light delivery to the urinary tract. The photoantimicrobial approach thus offers considerable potential.     

Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form

Aims: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. Methods and Results: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 μg ml⁻¹) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast’s transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 μg ml⁻¹. This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. Conclusion: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. Significance and Impact of the Study: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.     

The influence of post-filtration washing on the in vitro assay ofCandida albicans adherence to human buccal epithelial cells

A simple in vitro assay technique was used to determine the effect of post-filtration washing on the adherence ofC. albicans (NCPF 3736) to human buccal epithelial cells (BEC). Washing was carried out with a range of volumes of phosphate buffered saline (PBS), viz. 0, 5, 10 and 20 ml, at a standard flow rate. Both the number ofC. albicans adherent to BEC and the percentage of BEC with adherentC. albicans were significantly decreased (p<0.001 for each of these measures) after washing with 5 ml PBS. Further increases in the volume of PBS did not significantly decrease either measure of adherence. These data indicate that only a small volume of PBS, 5 ml, is required to achieve the removal of non-adherentC. albicans from the surface of BEC. The result of the adherence assay is not significantly affected by increasing the volume of PBS used. It is concluded that considerable savings in time may be made through using only a small (5 ml) volume of washing buffer at a standard flow rate.Abbreviations BEC buccal epithelial cells - PBS phosphate buffered saline - MEM Eagle's minimum essential medium - NCPF National collection of pathogenic fungi     

Nitrate uptake and extracellular alkalinization by the green alga Hydrodictyon reticulatum in blue and red light

Nitrate uptake and the medium alkalinization related to it were studied with nets of the coenocytic, giant cell, green alga Hydrodictyon reticulatum. A comparison of red, blue and white light irradiation showed no special control of nitrate uptake and of the corresponding alkalinization of the external medium by light quality, but rather a response as expected for the photosynthetic apparatus. In the dark, nitrate uptake rates amounted to one-fifth of those in saturating white light. This is in contrast to the chlorococcal microalga Monoraphidium braunii, where blue light specifically switched on nitrate uptake-dependent alkalinization and where uptake and reduction of nitrate strongly depended on blue light; the rates in pure red light and in the dark being very low. The stoichiometric ratio between nitrate taken up and extracellular alkalinization was close to 1 (0.86) in air with CO2 but close to 2 (1.84) in N2 for nitrate pre-loaded cells. In the absence of any carbon source, a high proportion of the absorbed and reduced nitrogen is released, most of it as ammonium which causes the excess alkalinization and some as nitrite, which lowers the ratio. Nitrite and ammonium release rates under anaerobic, CO2-free conditions were also independent of red or blue light and continued for several hours when the medium was buffered at pH 6. The data indicate that nitrate uptake, but less its reduction, is regulated differently in vacuolate, coenocytic algae from microalgae. In Hydrodictyon, nitrate uptake and reduction seems to be controlled by energy supply; in various microalgae, in addition, it is controlled specifically by blue light.     

Comparison of Riboflavin and Toluidine Blue O as Photosensitizers for Photoactivated Disinfection on Endodontic and Periodontal Pathogens In Vitro

Photoactivated disinfection has a strong local antimicrobial effect. In the field of dentistry it is an emerging adjunct to mechanical debridement during endodontic and periodontal treatment. In the present study, we investigate the effect of photoactivated disinfection using riboflavin as a photosensitizer and blue LED light for activation, and compare it to photoactivated disinfection with the widely used combination of toluidine blue O and red light. Riboflavin is highly biocompatible and can be activated with LED lamps at hand in the dental office. To date, no reports are available on the antimicrobial effect of photoactivated disinfection using riboflavin/blue light on oral microorganisms. Planktonic cultures of eight organisms frequently isolated from periodontal and/or endodontic lesions (Aggregatibacter actinomycetemcomitans, Candida albicans, Enterococcus faecalis, Escherischia coli, Lactobacillus paracasei, Porphyromonas gingivalis, Prevotella intermedia and Propionibacterium acnes) were subjected to photoactivated disinfection with riboflavin/blue light and toluidine blue O/red light, and survival rates were determined by CFU counts. Within the limited irradiation time of one minute, photoactivated disinfection with riboflavin/blue light only resulted in minor reductions in CFU counts, whereas full kills were achieved for all organisms when using toluidine blue O/red light. The black pigmented anaerobes P. gingivalis and P. intermedia were eradicated completely by riboflavin/blue light, but also by blue light treatment alone, suggesting that endogenous chromophores acted as photosensitizers in these bacteria. On the basis of our results, riboflavin cannot be recommended as a photosensitizer used for photoactivated disinfection of periodontal or endodontic infections.     

Photodynamic therapy for American cutaneous leishmaniasis: the efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

The aim of this study was to investigate the effectiveness of Photodynamic Therapy (PDT) using Methylene Blue (MB) as the photosensitizing compound and a Light-Emitting Diode (LED) in American cutaneous leishmaniasis (ACL). Hamsters were experimentally infected with Leishmania (Leishmania) amazonensis. After the development of the lesions in the footpad, the animals were treated with MB three times a week for 3 months. Ten minutes after each application of MB, the lesions were irradiated with LED for 1 h. The lesions were evaluated weekly by the measurement of the hamster footpad thickness. At the end of the treatment the parasitic load was quantified in the regional lymph node of the hamsters. The treatment promoted a decrease in the thickness of infected footpad (P = 0.0001) and reduction in the parasitic load in the regional lymph node (P = 0.0007) of the animals from group treated with MB + LED. PDT using MB + LED in ACL caused by L. amazonensis shows a strong photodynamic effect. This therapy is very promising, once it is an inexpensive system and the own patient can apply it in their wound and in their house without the need of technical assistance.     

Design of a Simple Model of <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms Formed under Conditions of Flow: Development,Architecture, and Drug Resistance

Candida albicans biofilms on most medical devices are exposed to a flow of body fluids that provide water and nutrients to the fungal cells. While C. albicans biofilms grown in vitro under static conditions have been exhaustively studied, the same is not true for biofilms developed under continuous flow of replenishing nutrients. Here, we describe a simple flow biofilm (FB) model that can be built easily with materials commonly available in most microbiological laboratories. We demonstrate that C. albicans biofilms formed using this flow system show increased architectural complexity compared to biofilms grown under static conditions. C. albicans biofilms under continuous medium flow grow rapidly, and by 8 h show characteristics similar to 24 h statically grown biofilms. Biomass measurements and microscopic observations further revealed that after 24 h of incubation, FB was more than twofold thicker than biofilms grown under static conditions. Microscopic analyses revealed that the surface of these biofilms was extremely compact and wrinkled, unlike the open hyphal layer typically seen in 24 h static biofilms. Results of antifungal drug susceptibility tests showed that C. albicans cells in FB exhibited increased resistance to most clinically used antifungal agents.     

Improved DNA‐FISH for cytometric detection of Candida spp

Aims: We developed improved methods for DNA‐based fluorescence in situ hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via flow cytometry. Methods and Results: Two previously reported C. albicans‐targeted DNA probes were evaluated against whole cells of C. albicans and related Candida species using a rapid, high‐temperature hybridization protocol. One probe (CalB2208) was shown for the first time to be suitable as a FISH probe. Although cell labelling for both probes was relatively bright, we were able to substantially improve our results by altering fixation and hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered formalin and ethanol was most effective. Probe intensity was improved as much as ten‐fold through the use of unlabelled helper probes, and buffer containing 0·9 mol l^?1 NaCl plus 10% formamide yielded the best hybridizations for both probe/helper cocktails. Although optimal labelling occurred with longer hybridizations, we found that C. albicans could be completely differentiated from the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest probe (Calb‐1249) and that a formal washing step was not required. Specificities of probe/helper cocktails under optimal conditions were determined using a panel of target and nontarget cell types, including four strains of Candida dubliniensis. Calb‐1249 cross‐reacted slightly with Candida parapsilosis and strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found that CalB2208 was exclusive for C. albicans. The molecular basis of this specificity was confirmed by DNA sequencing. Conclusions: We describe DNA probe‐based approaches for rapid and bright labelling of Candida spp. and for specific labelling of C. albicans without cross‐reaction with C. dubliniensis. Our work improves upon previously described methods. Significance and Impact of the Study: The methods described here for rapid FISH‐based detection of Candida spp. may have applications in both clinical and food microbiology.     

Isolation and characterization of polyphenol oxidase- and peroxidase-producing <Emphasis Type="Italic">Bacillus</Emphasis> strains from fully fermented tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Sixteen aerobic endospore-forming Bacillus spp. were isolated from fully fermented tea leaf samples from 10 tea factories in Lahijan and Langrod cities (Gillan province, Iran). Bacillus spp. isolates were characterized using phenotypic characteristics, antibiotic susceptibility and cellular fatty acid (CFA) patterns. Based on the data obtained, five isolates of tea Bacillus spp. (TB): TB2, TB4, TB6, TB10 and TB12 belonged to the species B. subtilis. Two isolates, TB1 and TB14 were recognized as B. licheniformis. Two Bacillus spp. isolates, TB9 and TB 16 were identified as B. sphaericus. Two isolates, TB5 and TB13 were shown to be B. pumilus. Two isolates, TB7 and TB15 belonged to B. cereus. Amongst the isolates, Bacillus sp. TB3, Bacillus sp. TB8 and Bacillus sp. TB11 showed different phenotypic traits, distinct antibiotic sensitivity and fatty acid profiles, and they may represent novel species. The isolates showed polyphenol oxidase (tyrosinase) and peroxidase activities. The highest polyphenol oxidase and peroxidase activities were observed for Bacillus sp. TB3 and B. licheniformis TB14, respectively, where values of 5.48 and 3.73 units mL⁻¹ were observed.     

Antimicrobial activity of the acidophilic eukaryotic microalga Coccomyxa onubensis

Extremophilic microalgae are unexplored as a source of pharmaceuticals despite the fact that its biomass can be produced at large scale with low risk of contamination. A significant amount of antimicrobial activity was produced by extracts obtained from the eukaryotic acidophilic microalgae Coccomyxa onubensis in non‐polar solvents, such as hexane, diethyl ether, and chloroform or in weakly polar solvents, such as dichloromethane, against Gram‐negative and Gram‐positive bacteria, and also the yeast Candida albicans. The most effective activity was shown by chloroform extract against Escherichia coli S, Salmonella enterica, and Proteus mirabilis; hexane extract against P. mirabilis, Sa. enterica, and Ca. albicans; dichloromethane extract against Sa. enterica or diethyl ether extract against E. coli S and the Gram‐positive Staphylococcus aureus MB. The lowest minimum inhibitory concentration values were recorded against E. coli S (305 μg mL ^?1) and P. mirabilis (153 μg mL ^?1) (using chloroform extract) and against P. mirabilis (106 μg mL^?1) (using hexane extract). Fatty acids, but not carotenoids, seem to be involved in the antimicrobial activity of this microalga. However, further biochemical and biotechnological studies must be conducted in order to characterize and purify the bioactive principles from Co. onubensis for assessing its potential as a pharmaceutical source and feasibility of production.