Anti-Infective Potential of Marine Invertebrates and Seaweeds from the Brazilian Coast

This manuscript describes the evaluation of anti-infective potential in vitro of organic extracts from nine sponges, one ascidian, two octocorals, one bryozoan, and 27 seaweed species collected along the Brazilian coast. Antimicrobial activity was tested against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231) by the disk diffusion method. Antiprotozoal activity was evaluated against Leishmania braziliensis (MHOM/BR/96/LSC96-H3) promastigotes and Trypanosoma cruzi (MHOM/BR/00/Y) epimastigotes by MTT assay. Activity against intracellular amastigotes of T. cruzi and L. brasiliensis in murine macrophages was also evaluated. Antiviral activity was tested against Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the plaque number reduction assay (IC50). Cytotoxicity on VERO cells was evaluated by the MTT assay (CC50). The results were expressed as SI = CC50/IC50. The most promising antimicrobial results were obtained against S. aureus and C. albicans with Dragmacidon reticulatum. Among the seaweeds, only Osmundaria obtusiloba showed moderate activity against P. aeruginosa. Concerning antiprotozoal activity, Bugula neritina, Carijoa riseii, Dragmaxia anomala and Haliclona (Halichoclona) sp. showed the most interesting results, mainly against extracellular promastigote forms of L. braziliensis (66, 35.9, 97.2, and 43.6% inhibition, respectively). Moreover, six species of seaweeds Anadyomene saldanhae, Caulerpa cupressoides, Canistrocarpus cervicornis, Dictyota sp., Ochtodes secundiramea, and Padina sp. showed promising results against L. braziliensis (87.9, 51.7, 85.9, 93.3, 99.7, and 80.9% inhibition, respectively), and only Dictyota sp. was effective against T. cruzi (60.4% inhibition). Finally, the antiherpes activity was also evaluated, with Haliclona (Halichoclona) sp. and Petromica citrina showing the best results (SI = 11.9 and SI > 5, respectively). All the active extracts deserve special attention in further studies to chemically characterize the bioactive compounds, and to perform more refined biological assays.

Brazil is a continental country, with 8,500 km of Atlantic coastline that supports an exclusive and rich diversity of endemic marine fauna and flora that can offer rich rewards for the chemical study of marine natural products in the search for novel bioactive secondary metabolites with potential medicinal properties. However, so far only a few classes of Brazilian marine organisms have been investigated for their chemical and pharmacological properties [15][16][17][18][19][20][21][22][23][24][25]. We therefore believe that the identification of Brazilian organisms with significant biotechnological potential for use in drugs is an important goal [18].
Some studies regarding bioprospection of Brazilian marine organisms have been reported. In 2002, Monks and co-workers performed the first biological screening with marine sponges collected from the Santa Catarina coast, in the south of Brazil. Several activities, such as cytotoxic, antichemotactic and antimicrobial properties were detected for the organic and aqueous extracts of 10 marine sponges [19].
Silva et al. [20] evaluated the in vitro antiherpes (HSV-1, KOS strain), anti-adenovirus (human AdV serotype 5) and anti-rotavirus (simian RV SA11) activities of extracts from 27 different marine sponges (Porifera) collected from the Brazilian coast. The results showed that the aqueous extracts from Cliona sp., Agelas sp., Tethya sp., Axinella aff. corrugata, Polymastia janeirensis and Protosuberites sp. were highly promising and deserve special attention in further studies. Furthermore, Frota-Jr and co-authors reported the antitumor activity of the marine sponge P. janeirensis in human U138MG glioma cell line [21,22].
Jimenez and colleagues performed the first ascidian antitumor screening with organisms from the Northeast coast of Brazil. The results suggest these are a rich source of natural compounds with cytotoxic properties [23].
Recently, Soares and colleagues [25] evaluated the antiviral activity of extracts from 36 species of seaweeds from seven locations of the Brazilian coastline against HSV-1 and HSV-2 strains. The results obtained reinforce the role of seaweeds as an important source of compounds with for the development of new drugs against herpes.
The marine biodiversity loss that has been observed worldwide [26], but especially in Brazil, is driving an unprecedented loss of biotechnological potential related with these organisms [27,28]. In attention to the human constant need for new drugs and therapies in the present work, we performed an anti-infective (antibacterial, antifungal, antiprotozoal and antiviral) screening of 95 different extracts and fractions from 13 marine invertebrates collected from the southern Brazilian coast, and 27 seaweeds from the northeastern Brazilian coast.

Results and Discussion
This paper describes the in vitro antimicrobial, antiprotozoal and antiviral evaluation of organic extracts and fractions from 13 marine invertebrate species (nine sponges, one ascidian, two octocorals, and one bryozoans (Table 1), and 27 seaweeds species [sixteen Rhodophyta (59.2%), seven Phaeophyceae (26%), and five Chlorophyta (14.8%) ( Table 2). A total of 95 extracts and fractions (65 from marine invertebrates and 30 from seaweeds) were assayed. The results showed that 53 samples (56%) exhibited some anti-infective activity against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli and Candida albicans (antimicrobial), Leishmania braziliensis and Trypanosoma cruzi (anti-protozoal), as well as against HSV-1 replication (antiviral).   # All seaweeds were collected in the intertidal zone. a,b same species, but collected in different locales.
The most interesting antimicrobial results were obtained with the sponge D. reticulatum that showed significant growth inhibition (13-16 mm) against S. aureus and C. albicans. A similar result was proved in another screening carried out with marine organisms from the southeastern Brazilian coast where the sponge D. reticulatum showed a weak antimicrobial activity against S. aureus and C. albicans [24]. As far as we are aware, this is the first report of this biological activity for D. reticulatum, D. anomala, and Trachycladus sp.
Furthermore, a weak antimicrobial activity against S. aureus, E. faecalis, and E. coli was also detected in the present study for Haliclona (Halichoclona) sp. and Petromica citrina (9-12 mm inhibition zone), and T. ignis (6-8 mm inhibition zone). In the same way, we verified a weak antimicrobial activity against S. aureus and C. albicans of the n-hexane extract from the octocoral Leptogorgia punicea. Table 3. Antibacterial and antifungal screening of marine invertebrates by disc diffusion method. As far as we aware, this is the first report for antimicrobial activity for this gorgonian species. On the other hand, the extracts from B. neritina, C. riseii, C. celata, D. granulatum, G. sepia and P. janeirensis did not show antimicrobial activity against the assayed microorganism strains.

S. aureus E. faecalis E. coli P. aeruginosa C. albicans
Recently it was reported that aqueous extract of P. citrina (collected in Rio de Janeiro State, southeast of Brazil) showed a large spectrum of activity against clinical strains and resistant-bacteria including S. aureus, S. epidermidis, E. coli, E. faecalis, M. fortuitum and N. gonorrhoeae. All these activities were related to the presence of halistanol trisulphate A in this marine sponge [29,30]. Moreover, the antifungal activity for this substance, isolated from Petromica ciocalyptoides was also reported [31].
Another study led by Monks et al. [19] concerning the antimicrobial activity against E. coli, S. aureus, S. epidermis, B. subtilis, and M. luteus strains of southern Brazilian sponges, including Guitarra sp., T. ignis, Haliclona aff. tubifera, demonstrated that H. aff. tubifera showed moderate activity against E. coli and weak activity against S. aureus, S. epidermis, and M. luteus. Our results are in agreement with those obtained by these authors, although we used different libraries of microbial strains and extracts.
Additionally, these extracts and fractions were assayed on L. brasiliensis amastigotes in bone marrow macrophages from mice, and only the sponge Haliclona (Halichoclona) sp. and the octocoral C. riisei were active (Table 5). Based on these preliminary results, the E1 extract from C. riisei was fractionated by chromatographic techniques leading to the isolation of an active pregnane steroid [32]. Finally, the extracts from C. celata, P. citrina, P. janeirensis and Trachycladus sp. were not active against L. braziliensis or T. cruzi. In this work, the antiviral activity against Herpes Simplex Virus type 1 (HSV-1, KOS strain) was also evaluated. Before the evaluation of the antiviral activity, the cytotoxic effects of the selected samples were investigated on VERO cells by MTT assay, and for each tested sample, a CC 50 value was calculated. Of the 95 extracts and fractions tested, only the E3F2 fractions from the sponges Haliclona (Halichoclona) sp. and P. citrina showed antiviral activity (SI = 11.92 and, SI > 5, respectively).
In 2006, Silva and co-workers [20] performed an in vitro study on the antiherpes, anti-adenovirus and anti-rotavirus activities of marine sponges collected from the Brazilian coast, including Haliclona sp., Polymastia janeirensis and T. ignis. Of these, only the organic extract (methanol/toluene, 3:1 v/v) from P. janeirensis showed antiherpetic activity [20].

Species Extracts Bacterial and fungal strains S. aureus E. faecalis E. coli P. aeruginosa C. albicans
As far as we are aware, there are no reports in the literature on the antiprotozoal activity for four of these seaweeds species (A. saldanhae, C. cupressoides a , Padina sp., and O. secundiramea). Concerning the activity of FS extract from the red seaweed L. dendroidea (= formerly Laurencia obtusa), only a weak (14.6%) antileishmanial activity against the promastigote forms of L. braziliensis was observed (Table 7). Furthermore, two species of seaweeds, A. saldanhae (SI = 12.3) and Padina sp. (SI = 7.5), were effective against L. brasiliensis amastigotes. Additionally, C. cervicornis, C. cupressoides a , Dictyota sp., and O. secundiramea were strongly cytotoxic for bone marrow macrophages (Table 8).  Previous studies performed by Veiga-Santos et al. [5] and Machado et al. [37] showed that lipophilic extracts from L. dendroidea collected from the southeastern coast of Brazil strongly inhibited the growth of T. cruzi and L. amazonensis. These results are not completely in agreement with our findings for L. dendroidea and this discrepancy may be due to the different geographic regions where this species was collected, as well as the seawater conditions.
Another study led by Santos and colleagues [4] found that lipophilic extracts from the brown seaweed C. cervicornis collected from the northeastern coast of Brazil also strongly inhibited the growth of L. amazonensis. From this species, a 4-acetoxydolastane diterpene was isolated, which demonstrated dose-dependent activity during 72 h of treatment, exhibiting IC 50 values of 2.0, 12.0 and 4.0 μg/mL for promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis, respectively.
Concerning antiviral activity, none of the species tested displayed any anti-HSV-1 activity. Although Soares and colleagues [25] reported the anti-HSV-1 activity for the red alga L. dendroidea collected from the coast of Rio de Janeiro, in our work this specie showed high cytotoxicity against VERO cells and none antiviral activity was detected.
To summarize, the present work reports the antimicrobial, antiprotozoal and antiviral evaluation of organic extracts from nine sponges, two octocorals, one ascidian, one bryozoan, and 27 seaweeds species, collected along the Brazilian coastline. Of a total of 95 extracts and fractions, 53 (56%) showed some anti-infective activity against S. aureus, E. faecalis, P. aeruginosa, E. coli, C. albicans, L. braziliensis, T. cruzi, and HSV-1.
Clearly, the marine invertebrates and seaweeds from the Brazilian coast could play an important part in the future control of the global infectious-disease burden. Although substantial progress has been made in identifying new biotechnological potential from these organisms, further chemical analysis and biological studies are required for investigating the mechanism of action, the chemical content as well as the potential use of these marine organisms extracts in the prevention of pathologies.
Seaweeds specimens (Rhodophyta, Pheophyceae, and Chlorophyta) were collected in the midlittoral zone of the southern and northeastern Brazilian coast, in August/October 2011 ( Table 2). The epiphytic organisms from the seaweeds were manually cleaned immediately after collection, and air dried. The voucher specimens were deposited at the Herbarium of the Department of Botany at Universidade Federal de Santa Catarina, Brazil.

Preparation of the Extracts
Organic extracts from marine invertebrates were prepared according to a standard procedure (Figure 1). Organic extracts from marine seaweeds were obtained using two distinct methods: CH 2 Cl 2 /MeOH (2:1) for dried seaweeds (DS extracts), and Me 2 CO for fresh seaweeds (FS extracts).
The antibacterial and antifungal activities were evaluated by the disk diffusion method as previously described by de Oliveira et al. [41], with minor modifications. Briefly, filter paper disks (5 mm) were impregnated with 20 μl of the extracts or fractions solutions (100 mg/mL) and then placed on Muller-Hinton agar plates (HIMEDIA ® ), which were inoculated with the microorganisms according to the standard protocol described by the Clinical Laboratory Standard Institute [42]. The plates were incubated at 35 °C (± 1°C), and after 18 h, the diameters of the inhibition zones were measured. Filter-paper disks containing DMSO were used as negative control and no inhibition was observed. Standard antibiotic disks were selected according to the sensitivity of the microorganism tested: ampicillin (10 μg), oxacillin (1 μg), ceftazidime (30 μg) and fluconazole (25 μg) [43].

Antileishmanial and Antitrypanosomal Activities
Leishmania braziliensis (MHOM/BR/96/LSC96-H3) promastigotes were grown at 26 °C in Schneider's medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 5% heat inactivated fetal bovine serum (FBS) and 2% urine. Trypanosoma cruzi (MHOM/BR/00/Y) epimastigotes were grown at 26 °C in Liver Infusion Tryptose (LIT) medium containing 10% FBS. Both parasite cultures were grown in 10 U/mL penicillin and 10 μg/mL of streptomycin (Gibco ® ). For the growth inhibition assays, L. braziliensis promastigotes or T. cruzi epimastigotes in the exponential phase of growth were harvested and washed twice in phosphate-buffered saline (PBS) by centrifugation at 1,500 × g. for 10 min. The parasites were counted in a Neubauer hemocytometer and seeded in 96-well microplates at 5.4 × 10 6 (T. cruzi) or 3 × 10 6 parasites/mL (L. braziliensis) in a final volume of 180 μL in LIT or Schneider´s medium, respectively. Parasites were incubated for 48 h at 26 °C in the presence of 20 μL of the samples (final concentration = 50 μg/mL). The standard drugs amphotericin B (Sigma) at 0.1 μM and benznidazole (Sigma) at 30 μM were used as positive controls and 1% DMSO was used as negative control. Parasite survival was assessed by the MTT assay [44]. The assays were carried out in triplicate, and the results were expressed as percentage of parasite growth inhibition.

Activity against Intracellular Amastigotes of T. cruzi and L. braziliensis in Murine Macrophages
In this work, only the samples that showed parasite growth inhibition higher than 40% against the extracellular forms were analyzed through this methodology. Murine (Balb/C) bone marrow derived macrophages were differentiated for 7 days in 6 well plates, with Dulbecco's Modified Eagle Medium (DMEM-Gibco) supplemented with HEPES (25 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), FBS (10%) and 25% (v/v) supernatant of the murine fibroblast cell line L929 at 37 °C and 5% CO 2 , as described by Marim and co-workers [45], with minor modifications. Adherent cells were washed with PBS, trypsinized, counted in a Neubauer hemocytometer and concentration adjusted to 4.10 5 cells/mL. Cell viability was assessed using Trypan Blue (0.04%). Next, 100 μl of cell suspension were seeded in 96 well plates and cultivated for 24 h at 37 °C. Thereafter, macrophages were infected with L. braziliensis axenic amastigotes (10 parasites/cell) for 3 h, at 34 °C and 5% CO 2 or with VERO cell derived T. cruzi trypomastigotes (5 parasites/cell) for 4 h, at 37 °C and 5% CO 2 . Non-internalized parasites were removed by washing with PBS. After 24 h of incubation, 20 μL of the samples was added to the infected cell monolayers starting from 50 μg/mL and incubated for 48 h in 5% CO 2 (34 °C for L. braziliensis and 37 °C for T. cruzi). The cells were washed with PBS, methanol fixed and Giemsa stained. The percentage of infected cells and the number of intracellular amastigotes were assessed using an Olympus IX70 optical inverted microscope, randomly counting 100 cells/well at a magnification of 400×. The reduction of the parasitic index was calculated as described elsewhere [46], and the 50% inhibitory concentration was calculated by linear least squares regression, using the software GraphPad Prism 5.0. Amphotericin B (0.2 μM) and benznidazole (15 μM) were used as positive controls. DMSO 1% was used as negative control. The experiments were carried out in triplicate and repeated at least twice.

Cytotoxic Activity against J774.G8 Macrophage Cell Line
Murine J774.G8 phagocytic cells were seeded in 96 well plates with DMEM supplemented with HEPES (25 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and FBS (10%), and incubated for 72 h with the samples starting from 500 μg/mL. The assays were carried out in triplicate and cell viability was determined as described above for VERO cells. The CC 50 was calculated by minimum square linear regression with the software GraphPad Prism 5.0.

Cytotoxicity Assay
Confluent VERO cells were exposed to different concentrations of the samples for 72 h. After incubation, cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide] assay [44]. The assays were carried out in triplicate, and the results were expressed as the CC 50 , which was defined as the concentration that reduced cell viability by 50%, when compared to the untreated controls.

Viral plaque Number Reduction Assay
This assay followed the procedures described by Kuo et al. [48], with minor modifications. Approximately 100 PFU of HSV-1 was adsorbed for 1 h at 37 °C on confluent VERO cells. Cultures were then overlaid with MEM containing 1.5% carboxymethylcellulose (CMC; Sigma) with or without different concentrations of the samples. After 72 h, the cells were fixed and stained with naphtol blueblack (Sigma), and the plaques were counted. The assays were carried out in triplicate, and the results were expressed as the IC 50 , which was defined as the concentration that reduced the number of viral plaques formed by 50%, when compared to the untreated controls. Acyclovir (Sigma) was used as a positive control.
Moreover, this work also shows the importance of bioprospecting studies highlighting the importance of marine biodiversity as sources of potential natural compounds with pharmacological properties or biotechnological potential that could be used in the development of new drugs. All the active extracts deserve special attention in further studies to chemically characterize the bioactive compounds as well as more refined biological assays.