Acessibilidade / Reportar erro

Marine organisms as source of extracts to disrupt bacterial communication: bioguided isolation and identification of quorum sensing inhibitors from Ircinia felix

Abstract

In this study, 39 extracts from marine organisms were evaluated as quorum sensing inhibitors, collected in the Colombian Caribbean Sea and the Brazilian Coast including 26 sponges, seven soft corals, five algae and one zooanthid. The results showed that crude extracts from the soft coral Eunicea laciniata, and the sponges Svenzea tubulosa, Ircinia felix and Neopetrosia carbonaria were the most promising source of quorum sensing inhibitors compounds without affecting bacterial growth, unlike the raw extracts of Agelas citrina, Agelas tubulata, Iotrochota arenosa, Topsentia ophiraphidites, Niphates caycedoi, Cliona tenuis, Ptilocaulis walpersi, Petrosia pellasarca, and the algae Laurencia catarinensis and Laurencia obtusa, which displayed potent antibacterial activity against the biosensors employed. The crude extract from the sponge I. felix was fractionated, obtaining furanosesterterpenes which were identified and evaluated as quorum sensing inhibitors, showing a moderate activity without affecting the biosensor's growth.

Keywords
Quorum sensing; Marine products; Ircinia felix ; Furanosesterterpenes; Biofilm

Introduction

Due to the great chemical variability provided by biological diversity existing in the marine environment, research in marine natural products has received great interest (Blunt et al., 2010Blunt, J.W., Copp, B.R., Munro, M.H.G., Northcote, P.T., Prinsep, M.R., 2010. Marine natural products. Nat. Prod. Rep. 27, 165–237.). Many of these molecules are responsible for stability and adaptability of marine organisms to a changing environment; besides they are involved in the symbiotic relationship between marine macro-organisms and associated microorganisms that has been thoroughly documented. This entire chemical signaling makes this multiorganismal consortium behave as a biochemical and physiological unit fundamental to the equilibrium of the whole ecosystem (Harvell et al., 2007Harvell, D., Jordán-Dahlgren, E., Merkel, S., Rosenberg, E., Raymundo, L., Smith, G., Weil, E., 2007. Coral disease environmental drivers, and the balance between coral and microbial associates. Oceanography 20, 172–195.).

Structure and relative density of microbial populations are associated with macro-organisms' change in time and location. This alteration directly influences the organism health as well as the production of bioactive substances. Therefore, multiorganism consortium could regulate settlement of exogenous bacteria using compounds to avoid a biofilm with an undesirable composition (Teplitski and Ritchie, 2009Teplitski, M., Ritchie, K., 2009. How feasible is the biological control of coral diseases? Trends Ecol. Evol. 24, 378–385.). Coordination between macro-organisms and associated bacteria is done, as well as between bacteria, by means of communication systems regulated by chemical molecules. In bacteria, this phenomenon is known as quorum sensing (QS), and signals [N-acyl homoserine lactones (AHL) for Gram negative bacteria] involved in this mechanism are also responsible for inter-kingdom communication (Lowery et al., 2008Lowery, C.A., Dickerson, T.J., Janda, K.D., 2008. Interspecies and interkingdom communication mediated by bacterial quorum sensing. Chem. Soc. Rev. 37, 1337–1346.).

Due to QS systems, bacteria behave in a very different way when they are organized as a complex community because in such a way, they coordinate different phenotypes that provide the community with more resistance and adaptability to the environment, like antibiotic production, sporulation, DNA exchange, expression and secretion of virulence factors, bioluminescence, pigment production, and remarkable biofilm maturation (Ng and Bassler, 2009Ng, W.L., Bassler, B.L., 2009. Bacterial quorum-sensing network architectures. Annu. Rev. Genet. 17, 197–222.; Greenberg, 2003Greenberg, E.P., 2003. Bacterial communication and group behavior. J. Clin. Invest. 112, 1288–1290.; Boyer and Wisniewski-Dyé, 2009Boyer, M., Wisniewski-Dyé, F., 2009. Cell–cell signalling in bacteria: not simply a matter of quorum. FEMS Microbiol. Ecol. 21, 1–19.).

Bacterial biofilms are sessile communities of differentiated cells associated with a surface, submerged in an extracellular matrix of excreted polysaccharides (also termed exopolysaccharides, EPS), proteins and DNA. Specific structure and composition of biofilms vary with the resident species and environmental conditions (Dickschat, 2010Dickschat, J.S., 2010. Quorum sensing and bacterial biofilms. Nat. Prod. Rep. 27, 343–369.). Biofilm maturation is also controlled by QS that regulates many phenotypes as EPS production, adhesines and other fundamental requirements for the consolidation of a strong matrix (Dickschat, 2010Dickschat, J.S., 2010. Quorum sensing and bacterial biofilms. Nat. Prod. Rep. 27, 343–369.). Since biofilms are known to be important cues for settlement of larvae of many marine invertebrates (Dobretsov et al., 2009Dobretsov, S., Teplitski, M., Paul, V., 2009. Mini-review: quorum sensing in the marine environment and its relationship to biofouling. Biofouling 25, 413–427.), the control of biofilm formation on the surface becomes a target for biofouling control. Furthermore, biofilm has been disrupted over unanimated surfaces using quorum sensing inhibitors (QSI), leading to the reduction of macrofouling (Dobretsov et al., 2009Dobretsov, S., Teplitski, M., Paul, V., 2009. Mini-review: quorum sensing in the marine environment and its relationship to biofouling. Biofouling 25, 413–427.).

One of the most promising alternatives for biofilm control is the use of marine natural products, which are extracted from some invertebrate organisms with a macro-fouling free surface that could be an indicator of the production of compounds with settlement deterrence properties (Qian et al., 2007Qian, P.Y., Lau, S.C.K., Dahms, H.U., Dobretsov, S., Harder, T., 2007. Marine biofilms as mediators of colonization by marine macroorganisms: implications for antifouling and aquaculture. Mar. Biotechnol. 12, 399–410.; Meseguer et al., 2004Meseguer, D., Kiil, S., Dam-Johansen, K., 2004. Antifouling technology – past, present and future steps towards efficient and environmentally friendly antifouling coatings. Progr. Org. Coat. 50, 75–104.). Following the necessity of antifouling compounds without toxic effects on colonizing species, quorum sensing disruption has become a promising alternative. QSI may act in four different ways: first, inhibiting the signal molecules biosynthesis (i.e. AHL); second, inducing degradation of the signal molecules; third, blocking specific bind sites of AHL to LuxR type proteins; and finally, by inhibition of DNA transcription (Dobretsov et al., 2009Dobretsov, S., Teplitski, M., Paul, V., 2009. Mini-review: quorum sensing in the marine environment and its relationship to biofouling. Biofouling 25, 413–427.). A quorum sensing inhibitor suppresses specific genetic expression of bacteria without causing death. Since survivable mechanisms of bacteria are not induced by QSI compound, bacterial resistance is not developed; thus it has also been considered for years by the pharmaceutical industry as a promising strategy for the design and development of antipathogenic compounds, useful in controlling microbial chronic infections, due to the observed microbial development of resistance to antibiotics (Rasmussen and Givskov, 2006Rasmussen, T.B., Givskov, M., 2006. Quorum sensing inhibitors as anti-pathogenic drugs. Int. J. Med. Microbiol. 296, 149–161.). These increasing efforts focused on new alternatives intended to prevent and treat severe infections in order to minimize the total antimicrobial exposure, and includes several QS-modulating therapies such as macrolide antibiotics, QS vaccines, and competitive QS inhibitors that have been proved to be helpful in diminishing the translation of QS-directed toxins or by prematurely activating the QS response to alert the immune system to bacteria hiding in a low cell density (Martin et al., 2008Martin, C.A., Hoven, A.D., Cook, A.M., 2008. Therapeutic frontiers: preventing and treating infectious diseases by inhibiting bacterial quorum sensing. Eur. J. Clin. Microbiol. 27, 635–642.).

This study is aimed to extend knowledge about the Colombian and Brazilian marine biodiversity and to evaluate the QSI activity of marine organisms' extracts, which have not been widely studied around the world and, as far as we are aware, never done before at the South-Eastern Caribbean Sea and South Brazilian Coast. We present evaluations of the QSI activity of 39 marine organisms' extracts from the Colombian Caribbean Sea and the Brazilian Coast (Santa Catarina State), using Chromobacterium violaceum ATCC 31532 and Escherichia coli pSB401 as biosensors coupled with Pseudomonas putida IsoF by disc diffusion assays. Besides, we evaluated a furanosesterterpene enriched-fraction from the active sponge Ircinia felix, as an approach to identify some of the possible active compounds.

Materials and methods

Organisms and extract preparation

Organisms were collected by scuba diving in Santa Marta bay and Islas del Rosario at the Colombian Caribbean Sea, and in Santa Catarina State on the Brazilian Atlantic Coast (Table 1; authorization code: DTC-C-33 11/7 of UAESPNN; permission No. 4 of 10/02/2010). Organisms were dried and kept frozen until extraction. Dried organisms were then cut into small pieces and extracted three times with dichloromethane:methanol (1:1); solvent was then evaporated until the crude extracts were obtained, and stored at 4 °C before using. The extracts of Svenzea tubulosa and Neopetrosia carbonaria were partitioned using Kupchan methodology (Anta et al., 2002Anta, C., Gonzáles, N., Santafé, G., Rodríguez, J., Jiménez, C., 2002. New Xenia diterpenoids from the Indonesian soft coral Xenia sp. J. Nat. Prod. 65, 766–768.) and the fractions soluble in dichloromethane (DCM), butanol (WB) and water (WW) were tested.

Table 1
QSI and antibacterial results of marine extracts from Colombian Caribbean Sea and Brazilian South Coast.

Extraction and isolation of furanosesterterpenes from I. felix

The crude extract (35.3 g) of I. felix, obtained as described above from 562.2 g of fresh material, was partitioned with a mixture of DCM:H2O (1:1, v/v), yielding the dichloromethane fraction that was concentrated under reduced pressure. This fraction (4.5 g) was submitted to vacuum column chromatography over silica gel (Merck 60) using a gradient increasing in polarity, starting with 100% benzene to ethyl acetate:methanol (1:1, v/v). Seven fractions (IFF1-IFF7) were yielded and evaluated in the QSI assay. Fraction IFF3 (1.7 g), eluted with benzene:ethyl acetate (7:3, v/v), showed QSI activity and was further chromatographed again in vacuum column chromatography over silica gel (Merck 60); and eluted with hexane:ethyl acetate (7:3, v/v), hexane:ethyl acetate (1:1, v/v) and 100% ethyl acetate, led to nine fractions (Fractions IFF3-1 to IFF3-9). Fraction IFF3-3 (600 mg), eluted with hexane:ethyl acetate (7:3, v/v), containing a mixture of sesterterpenes compounds, showing QSI activity, was acetylated. Briefly, an aliquot (300 mg) of fraction IFF3-3 was submitted to acetylation with 6 ml of a mixture of acetic anhydride:pyridine (1:2, v/v) under agitation for 24 h. The reactions' products were monitored by thin-layer chromatography. The solvent mixture was poured over 10 ml of hydrochloric acid (5%) cold solution and extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtrated and dried under reduced pressure, yielding 332.7 mg of an acetylated furanosesterterpene fraction. This fraction was submitted to high-performance liquid chromatography (HPLC) (for chromatographic conditions, see ‘Preparative HPLC separations' section) to yield compounds 1a and 1b (2.0 mg), 2a and 2b (2.5 mg) and 3 (6.0 mg), identified by 1H and 13C NMR, and by comparison with the literature data (Martínez et al., 1995Martinez, A., Duque, C., Hara, N., Fujimoto, Y., 1995. Variabilin 11-metiloctadecanoate a branched-chain fatty acid ester of furanosesterpene tetronic acid, from sponge Ircinia felix. Nat. Prod. Lett. 6, 281–284., 1997Martínez, A., Duque, C., Sato, N., Fujimoto, Y., 1997. (8Z, 13Z, 20Z)-strobilinin and (7Z, 13Z, 20Z)-felixinin: new furanosesterterpene tetronic acids from marine sponges of the genus Ircinia. Chem. Pharm. Bull. 45, 181–184.).

Fraction I (mixture of the two compounds)

Colorless oil. [α]D25 23.0 (c 0.193, CH3OH). 1a. (7Z,13Z,18R,20Z)-felixinin acetate. 1H NMR (400 MHz, CDCl3) (ppm): 7.35 (1H, m, H-1), 7.21 (1H, s, H-4), 6.27 (1H, s, H-2), 5.15 (1H, t, J = 6.7 Hz, H-7), 5.08 (1H, t, J = 6.8 Hz, H-15), 5.04 (1H, d, J = 10.2 Hz, H-20), 2.82 (1H, m, H-18), 2.43 (2H, t, H-5a and H-5b), 2.35 (3H, s, H-27), 2.22 (2H, c, J = 7.5 Hz, H-6a and H-6b), 1.96–2.06 (m, H-16, H-12 and H-10), 1.82 (3H, s, H-25), 1.65 (3H, s, H-14 and H-9), 1.43–1.33 (m, H-17 and H-11), 1.05 (3H, d, J = 6.8 Hz, H-19). 1b. (8Z,13Z,18R,20Z)-strobilinin acetate. 1H NMR (400 MHz, CDCl3) (ppm): 7.34 (1H, m, H-1), 7.21 (1H, s, H-4), 6.27 (1H, s, H-2), 5.12 (1H, t, J = 6.7 Hz, H-10), 5.08 (1H, t, J = 6.8 Hz, H-15), 5.04 (1H, d, J = 10.2 Hz, H-20), 2.82 (1H, m, H-18), 2.39 (2H, t, H-5a and H-5b), 2.34 (3H, s, H-27), 2.03 (2H, m, H-7), 1.96–2.06 (m, H-16 and H-12), 1.82 (3H, s, H-25), 1.67 (3H, s, H-14 and H-9), 1.62 (m, H-6), 1.43–1.33 (m, H-17 and H-11), 1.05 (3H, d, J = 6.8 Hz, H-19).

Fraction II (mixture of the two compounds)

Colorless oil. [α]D25 23.5 (c 0.20, CH3OH). 2a. (7E,13Z,18R,20Z)-felixinin acetate. 1H NMR (400 MHz, CDCl3) (ppm): 7.34 (1H, m, H-1), 7.20 (1H, s, H-4), 6.27 (1H, s, H-2), 5.16 (1H, m, H-7), 5.08 (m, H-15), 5.05 (1H, d, J = 10.3 Hz, H-20), 2.82 (1H, m, H-18), 2.44 (2H, t, J = 6.5 Hz, H-5a and H-5b), 2.35 (3H, s, H-27), 2.25 (2H, t, J = 6.5 Hz, H-6a and H-6b), 1.95–2.09 (m, H-11 and H-10), 1.88–1.95 (m, H-16, H-12), 1.81 (3H, s, H-25), 1.65 (3H, s, H-14), 1.57 (3H, s, H-9), 1.30–1.50 (m, H-17), 1.06 (3H, d, J = 6.7 Hz, H-19). 2b. (8E,13Z,18R,20Z)-strobilinin acetate. 1H NMR (400 MHz, CDCl3) (ppm): 7.34 (1H, m, H-1), 7.20 (1H, s, H-4), 6.26 (1H, s, H-2), 5.08 (m, H-10), 5.08 (1H, m, H-15 and H-10), 5.04 (1H, d, J = 10.3 Hz, H-20), 2.82 (1H, m, H-18), 2.37 (2H, t, H-5a and H-5b), 2.34 (3H, s, H-27), 1.95–2.09 (m, H-16, H-12, H-7), 1.81 (3H, s, H-25), 1.67 (3H, s, H-14), 1.58 (3H, s, H-9), 1.64 (m, H-6), 1.30–1.50 (m, H-17 and H-11), 1.05 (3H, d, J = 6.7 Hz, H-19).

Fraction III (3)

(7E,12E,18R,20Z)-variabilin acetate, colorless oil. [α]D25 26.1 (c0.167, CH3OH). 1H NMR (400 MHz, CDCl3) (ppm): 7.33 (1H, s, H-1), 7.20 (1H, s, H-4), 6.28 (1H, s, H-2), 5.16 (1H, t, J = 6.9 Hz, H-7), 5.07 (1H, t, J = 6.9 Hz, H-12), 5.04 (1H, d, J = 10.2 Hz, H-20), 2.81 (1H, m, H-18), 2.44 (2H, t, J = 7.4 Hz, H-5a and H-5b), 2.35 (3H, s, H-27), 2.23 (2H, c, J = 14.7 and 7.4 Hz, H-6a and H-6b), 2.05 (2H, m, J = 8.0 and 7.6 Hz, H-11a and H-11b), 1.98 (2H, t, J = 7.8 Hz, H-10a and H-10b), 1.94 (2H, t, J = 7.0 Hz, H-15a and H-15b), 1.82 (3H, s, H-25), 1.58 (3H, s, H-9), 1.55 (3H, s, H-14), 1.34(4H, m, H-16), 1.34 (4H, m, H-17), 1.05 (3H, d, J = 6.7 Hz, H-19). RMN 13C (100 MHz, CDCl3) (ppm): 142.5 (CH, C1), 138.8 (CH, C4), 124.4 (CH, C12), 123.7 (CH, C7), 111.1 (CH, C2), 39.7 (CH2, C10), 39.5 (CH2, C15), 36.6 (CH2, C17), 31.1 (CH, C18), 28.4 (CH2, C6), 26.6 (CH2, C11), 25.7 (CH2, C16), 25.0 (CH2, C5), 20.6 (CH3, C27), 20.5 (CH3, C19), 16.1 (CH3, C9), 15.8 (CH3, C14), 116.7 (CH, C20), 8.4 (CH3, C26).

Preparative HPLC separations

HPLC separations were carried out on a Merck-Hitachi 6000 system equipped with a L-6000A binary pump, Rheodyne injector and a L-4250 UV–Vis detector. The stationary phase employed was Scharlau Science Nucleosil 120C–18column, (300 mm × 8 mm; i.d.; 10 μm), using isocratic MeOH:H2O (95:5) as mobile phase, with a flow rate at 2 ml/min, and detection at 310 nm.

Bacterial strains and culture conditions

Two different biosensors were used to evaluate QSI activity: C. violaceum ATCC 31532 and E. coli pSB401 coupled with P. putida isoF WT as the exogenous source of AHL to induce bioluminescence production. E. coli and P. putidaisoF WT were cultured in Luria–Bertani broth at 37 °C, and those strains were kindly provided by Prof. Dr. Kathrin Riedel (Department of Microbiology, Institute of Plant Biology, University of Zürich). C. violaceum was cultured in CASO Agar® (Merck, Germany) at room temperature.

QSI assays

C. violaceum ATCC 31532 was inoculated overnight at room temperature in CASO broth and 100 μl of bacteria were spread on agar plates. In contrast, E. coli pSB401 and P. putida isoF WT were inoculated separately at 37 °C overnight to get a cellular density equal to 0.5 MacFarland standard. An equal amount of LB medium with agar at 1.6% was prepared and mixed with bacteria cultures with the intention of preparing 0.8% agar plates, already inoculated with both, the biosensor and the AHL producer strain. Sterile paper discs (5 mm diameter) were loaded with the appropriate volume of crude extracts solutions, dissolved in methanol, in order to obtain 100, 200 and 400 μg per disc. The solvent was left to evaporate. Methanol was used as blank control and methanolic fraction from Cecropia pachystachya was used as positive control. Discs were placed on plates previously inoculated with the appropriate biosensor and were incubated for 24 h at 37 °C for the assay with E. coli pSB401 and P. putida isoF WT; and at 28 °C for C. violaceum ATCC 31532 assay. A QSI positive result was the inhibition of phenotype expression (bioluminescence or violacein, respectively) without affecting the growing process of bacteria. The presence of a growth inhibition zone around the discs was taken as antibacterial activity. This procedure was made by a triplicate. The inhibition of quorum sensing in C. violaceum was measured by visual analysis of violacein production, and for the bioassay with E. coli pSB401 and P. putida isoF WT the bioluminescence inhibition was registered by autoradiography.

Results and discussion

Many different studies have demonstrated the ability of natural compounds to block QS systems. Ornamental, medicinal and edible plants have been the most studied sources of QSI compounds (Dickschat, 2010Dickschat, J.S., 2010. Quorum sensing and bacterial biofilms. Nat. Prod. Rep. 27, 343–369.; Dobretsov et al., 2009Dobretsov, S., Teplitski, M., Paul, V., 2009. Mini-review: quorum sensing in the marine environment and its relationship to biofouling. Biofouling 25, 413–427.); however, only a few examples of systematic screening studies have been documented using raw extracts from marine sources. An investigation based on 78 extracts of 25 species of marine organisms collected from Florida waters showed that cyanobacteria have high QS inhibitory potential (Dobretsov et al., 2011Dobretsov, S., Teplitski, M., Bayer, M., Gunasekera, S., Proksch, P., Paul, V., 2011. Inhibition of marine biofouling by bacterial quorum sensing inhibitors. Biofouling 27, 893–905.). Another study used marine crude extracts of organisms from the Great Barrier Reef in Australia. The authors identified in the sponge Luffariella variabilis the manoalide as responsible for the QSI activity of the raw extract. This compound is known as an anti-inflammatory agent, and its structure has a lactonic ring which resembles the structure of AHL, and has been found to be an active QSI compound in the fusion system lasB::gfp (ASV) of Pseudomonas aeruginosa (Skindersoe et al., 2008Skindersoe, M., Ettinger-Epstein, P., Rasmussen, T., Bjarnsholt, T., De Nys, R., Givskov, M., 2008. Quorum sensing antagonism from marine organisms. Mar. Biotechnol. 20, 56–63.). Some other compounds have been identified as QSI from marine sources, and the most representative QSI compounds are the halogenated furanones, isolated from the red alga Delisea pulchra, which have been demonstrated to be one of the most promising QSI compounds and potent antifouling agents (Boyer and Wisniewski-Dyé, 2009Boyer, M., Wisniewski-Dyé, F., 2009. Cell–cell signalling in bacteria: not simply a matter of quorum. FEMS Microbiol. Ecol. 21, 1–19.). On the other hand, three compounds (floridoside, betonicine and isethionic acid) isolated from the red alga Ahnfeltiopsis flabelliformis showed promising QSI activity with the Agrobacterium tumefaciens NTL4 QS system (Kim et al., 2007Kim, J.S., Kim, Y.H., Seo, Y.W., Park, S., 2007. Quorum sensing inhibitors from the red alga Ahnfeltiopsis flabelliformis. Biotechnol. Bioproc. Eng. 30, 308–311.). Another example is the brominated triptamine-like alkaloids that were found in bryozoan Flustra foliacea and were tested using the P. putida (pKR-C12), P. putida (pAS-C8) and E. coli (pSB403) QS systems as biosensors (Peters et al., 2003Peters, L., König, G.M., Wright, A.D., Pukall, R., Stackebrandt, E., Eberl, L., 2003. Secondary metabolites of Flustra foliacea and their influence on bacteria. Appl. Environ. Microbiol. 69, 3469–3475.).

Extracts tested in the present study were obtained from marine organisms selected specially because of their clean surface; such organisms have microorganism communities in the surfaces which may have an ecological control that avoid settlement of macro-organisms. This control can be done by chemical compounds produced by the marine organism (the host) or by associated microorganisms as bacteria, or by both, the holobiont. As previously mentioned, in this ecological control, QS plays a major role due to regulation of biofilm maturation, causing a selective settlement of bacteria, larvae or other eukaryotic microorganisms (Boyer and Wisniewski-Dyé, 2009Boyer, M., Wisniewski-Dyé, F., 2009. Cell–cell signalling in bacteria: not simply a matter of quorum. FEMS Microbiol. Ecol. 21, 1–19.).

Disc diffusion assays were used to test QSI and antibacterial activity of 39 marine organisms extracts. Table 1 shows the results using different concentrations of extracts with both biosensors. A promising extract as source of QSI compounds must show a QS inhibition without affecting the bacterial growing process to avoid later development of bacterial resistance.

Using E. coli pSB401 coupled with P. putida as a biosensor, fifteen of the marine extracts (40.5%) gave a positive result inhibiting the quorum sensing system regulated by luxI/luxR, at one or both concentrations. Soft corals extracts were the most active, which proportionally contributed to the highest number of active extracts (4 out of 7 were QSI active), 11 of 25 sponges extracts resulted active as QSI, and Laurencia marine algal extracts did not present QSI activity. Fig. 1shows the results of two of the most active raw extracts in this assay, the raw extracts of octocoral Eunicea laciniata, and the sponge S. tubulosa additionally to the raw extracts of octocoral Pseudopterogorgia elisabethae chemotype 2 (San Andres Island – Colombian Caribbean Sea), and the butanol fraction (WB) of sponge N. carbonaria. A mild QSI activity was shown by crude extracts from the sponges Cliona delitrix, dichloromethane fraction of N. carbonaria and a low activity was shown by crude extracts from the octocorals Eunicea succinea, Eunicea fusca and the sponge Ptilocaulis walpersi.

Fig. 1
Disc diffusion assay showing inhibition of bioluminescence of Escherichia coli pSB401 coupled with P. putida IsoF by marine extracts. A and C are pictures of Petri dishes and sensi-discs. B and D are the X-ray plaques of these petri dishes. 1: Svenzea tubulosa (crude extract), 2: Petrosia pellasarca (crude extract) and 3: Eunicea laciniata(crude extract). The clear zones in autoradiography (B and D) and the absence of growth inhibition zones (A and C) show QS inhibitory activity of the extracts.

When C. violaceum ATCC 31532 was used as a biosensor, nine (23%) of the 39 extracts resulted active. Of these, seven corresponded to sponges, one to an octocoral, and the other to a zoanthid. These results are in contrast with the ones obtained by using the E. coli biosensor because with C. violaceum the most active extract belonged to sponges, 27% of the extracts were active (7 of 26) and so were just 14.3% of the octocoral extracts (1 of 7). Likewise, algae extracts showed no activity with this biosensor. Fig. 2 shows the results for the raw extracts of the sponges S. tubulosa and I. felix in the assay with C. violaceum which, in conjunction with the crude extracts from sponges Neopetrosia subtriangularis and Aka cachacrouense, and as well as extracts from octocoral Erythropodium caribaeorum, dichloromethane extracts of S. tubulosa and N. carbonaria, and butanol fraction of N. carbonaria, showed high bacterial communication disruption (QSI) activity. Extracts from the sponge E. caribaeorum and the zoanthid Palythoa caribaeorum showed moderate activity.

Fig. 2
Disc diffusion assay showing inhibition of violaceina production in C. violaceum ATCC 31532. The picture (A) is dichloromethane fraction from Svenzea tubulosa and (B) is crude extract from Ircinia felix. C1 = 100 μg, C2 = 200 μg, C3 = 300 μg, and C4 = 400 μg per disco. Positive control, PC = 100 μg of methanol fraction from Cecropia pachystachya; negative control, NC = 20 μl of methanol with prior evaporation before to placing. The clear zone surrounding the paper disc shows QS inhibitory activity of the extracts.

With these tests we could also see some extracts with antibacterial activities against three Gram negative bacteria: E. coli, P. putida and C. violaceum. Extracts from the sponges Agelas citrina, Agelas tubulata, Iotrochota arenosa and Topsentia ophiraphiditeswere the most potent antibacterial against E. coli and P. putida IsoF, while extracts from the sponges Niphates caycedoi, Cliona tenuis, P. walpersi, Petrosia pellasarcaand the algae Laurencia catarinensis and Laurencia obtusa showed high antibacterial activity against C. violaceum ATCC 31532. These results could be explained due to the fact that the sponges belonging to the genus Agelas are recognized as a source of bromopyrrolidinic alkaloids (Braekman et al., 1992Braekman, J.C., Daloze, D., Stoller, C., Van Soest, R.W.M., 1992. Chemotaxonomy of Agelas (Porifera: Demospongiae). Biochem. Syst. Ecol. 20, 417–431.) with an important ecological key role as fish feeding deterrents (Assmann et al., 2000Assmann, M., Lichte, E., Pawlik, J.R., Köck, M., 2000. Chemical defenses of the Caribbean sponges Agelas wiedenmayeri and Agelas conifera. Mar. Ecol. Prog. Ser.22, 255–262.). It has also been found that such compounds possess interesting antiviral (Keifer et al., 1991Keifer, P.A., Schwartz, R.E., Koker, M.E.S., Hughes Jr., R.G., Rittschof, D., Rinehart, K.L., 1991. Bioactive bromopyrrole metabolites from the Caribbean sponge Agelas conifera. J. Org. Chem. 56, 2965–2975.), antihistaminic (Cafieri et al., 1997Cafieri, F., Carnuccio, R., Fattorusso, E., Taglialatela-Scafati, O., Vallefuoco, T., 1997. Anti-histaminic activity of bromopyrrole alkaloids isolated from Caribbean Agelas sponges. Bioorg. Med. Chem. Lett. 9, 2283–2288.) and antimicrobial activities (Shen et al., 1998Shen, X., Perry, T.L., Dunbar, C.D., Kelly-Borges, M., Hamann, M.T., 1998. Debromosceptrin, an alkaloid from the Caribbean sponge Agelas conifera. J. Nat. Prod. 61, 1302–1303.). The sponges of the genus Iotrochota are known for producing brominated alkaloids (Carletti et al., 2000Carletti, I., Banaigs, B., Amade, P., 2000. Matemone a new bioactive bromine-containing oxindole alkaloid from the Indian Ocean sponge Iotrochota purpurea. J. Nat. Prod. 63, 981–983.) with mild antibacterial activity, and sulfated pyrrole alkaloids with promising anti-HIV-1 activity (Fan et al., 2010Fan, G., Li, Z., Shen, S., Zeng, Y., Yang, Y., Xu, M., 2010. Baculiferins A-O, O-sulfated pyrrole alkaloids with anti-HIV-1 activity, from the Chinese marine sponge Iotrochota baculifera. Bioorg. Med. Chem. 18, 5466–5474.). The sponges of the genus Topsentia are recognized as source of sulfated steroids with a widespread range of bioactivities, for example, ophirapstanol trisulfate – a sulfate steroid isolated from T. ophiraphidites which has shown activity as an inhibitor in the guanosine diphosphate/G-protein RAS exchange assay (GDPX), which is directly related with anticancer activity (Gunasekera et al., 1994Gunasekera, P.S., Sennett, S.H., Kelly-Borgues, M., Bryant, R.W., 1994. Ophirapstanol trisulfate, a new biological active steroid sulfate from the deep water marine sponge Topsentia ophiraphidites. J. Nat. Prod. 57, 1751–1754.). The sponges of the genus Ptilocaulis have been reported by producing guanidine type alkaloids with antimicrobial activity, among others. In 2003, a cyclic guanidine, with antibacterial activity against Staphylococcus aureus, was identified from the sponge Ptilocaulis spiculifer (Yang et al., 2003Yang, S.W., Chan, T.M., Pomponi, S.A., Chen, G., Wright, A.E., Patel, M., 2003. A new bicyclic guanidine alkaloid, Sch 575948, from a marine Sponge, Ptilocaulis spiculifer. J. Antibiot. 56, 970–972.). Algae of the genus Laurencia have shown sesquiterpenes, diterpenes, triterpenes and acetogenins that usually have one or more halogen atoms. These types of compounds were recognized for various activities, such as cytotoxic and antibacterial (Lhullie et al., 2010Lhullie, C., Falkenberg, M., Ioannou, E., Quesada, A., Papazafiri, P., Antunes-Horta, P., Schenkel, E.P., Vagias, C., Roussis, V., 2010. Cytotoxic halogenated metabolites from the Brazilian red alga Laurencia catarinensis. J. Nat. Prod. 73, 27–32.).

Those results are in agreement with a previous work done in our laboratory, where many of the extracts used in this study were tested against a collection of twelve marine bacteria strains, isolated from colonized surfaces (Mora-Cristancho et al., 2011Mora-Cristancho, J.A., Arevalo-Ferro, C., Ramos, F.A., Tello, E., Duque, C., Lhullier, C., Falkenberg, M., Schenkel, E.P., 2011. Antifouling activities against colonizer marine bacteria of extracts from marine invertebrates collected in the Colombian Caribbean Sea and on the Brazilian Coast (Santa Catarina). Z. Naturforsch. C 66, 515–526.). Additionally, for many of these marine strains, presence of QS systems by identification of its AHL production was demonstrated (Cuadrado, 2010Cuadrado, C.T., (Master's thesis) 2010. Aislamiento de N-acilhomoserinlactonas de algunas bacterias procedentes del Mar Caribe Colombiano, como evidencia de la existencia de circuitos de quorum sensing. Universidad Nacional de Colombia, Bogotá, 270 pp.). Therefore, it could be a challenge to evaluate the properties of these QSI active extracts in these marine bacteria involved in the biofouling process of QS systems.

The results of the QSI test with both biosensors employed showed that crude extracts of the sponges S. tubulosa, I. felix, and the butanol fraction of N. carbonaria are the most promising as a source of QSI active compounds. Indeed, these extracts did not show antibacterial activity but they inhibit QS system in both biosensors. These results could be an indicator of a safe use as antifoulants. These organisms could also represent a promising source of molecules that could act as antipathogenic compounds able to combat against antibiotic resistant bacterial strains that represent a serious threat to human health.

In this sense, bioguided isolation was conducted with the crude extract of I. felix. Throughout different chromatographic steps, we obtained a furanosesterterpenes-enriched fraction (IFF3-3) that presented a moderate QSI activity. Due to the known instability of these compounds, this fraction was acetylated and submitted to a preparative HPLC to yield three sub-fractions (Fraction I–III). Fraction I showed a mixture of (7Z,13Z,18R,20Z)-felixinin acetate (1a) and (8Z,13Z,18R,20Z)-strobilinin acetate (1b). Very similarly, fraction II also presented a mixture of these furanosesterterpenes, but with a distinct configuration: (7E,13Z,18R,20Z)-felixinin acetate (2a) and (8E,13Z,18R,20Z)-strobilinin acetate (2b). In fraction III, the compound (7E,12E,18R,20Z)-variabilin acetate (3) was isolated. All these five compounds, which present similar structure to AHL (Dobretsov et al., 2009Dobretsov, S., Teplitski, M., Paul, V., 2009. Mini-review: quorum sensing in the marine environment and its relationship to biofouling. Biofouling 25, 413–427.), have already been reported for I. felix (Martínez et al., 1995Martinez, A., Duque, C., Hara, N., Fujimoto, Y., 1995. Variabilin 11-metiloctadecanoate a branched-chain fatty acid ester of furanosesterpene tetronic acid, from sponge Ircinia felix. Nat. Prod. Lett. 6, 281–284., 1997Martínez, A., Duque, C., Sato, N., Fujimoto, Y., 1997. (8Z, 13Z, 20Z)-strobilinin and (7Z, 13Z, 20Z)-felixinin: new furanosesterterpene tetronic acids from marine sponges of the genus Ircinia. Chem. Pharm. Bull. 45, 181–184.). Besides, properties such as analgesic, antimicrobial, cytotoxic and anti-tumor of this furanosesterterpenes are well documented (Martínez et al., 1997Martínez, A., Duque, C., Sato, N., Fujimoto, Y., 1997. (8Z, 13Z, 20Z)-strobilinin and (7Z, 13Z, 20Z)-felixinin: new furanosesterterpene tetronic acids from marine sponges of the genus Ircinia. Chem. Pharm. Bull. 45, 181–184.).


The other two promising QSI sponges, S. tubulosa, formerly known as Pseudoaxinella tubulosa, have no reports about its chemistry, but the sponges of this genus are known as source of long chain fatty acids, steroidal, and peptide compounds, some of them with antibacterial activities (Kong et al., 1992Kong, F., Burgoyne, D.L., Andersen, J., Allen, T.M., 1992. Pseudoaxinellin, a cyclic heptapeptide isolated from the Papua New Guinea sponge Pseudoaxinella massa. Tetrahedron Lett. 33, 3269–3272.; Sjöstrand et al., 1981Sjöstrand, U., Kornprobst, J.M., Djerassi, C., 1981. Minor and trace sterols from marine invertebrates 29 (22E)-ergosta-5,22,25-trien-3β-ol and (22E,24R)-24,26-dimethylcholesta-5,22,25(27)-trien-3β-ol. Two new marine sterols fromthe sponge Pseudopxinella lunachata. Steroids 38, 355–364.). In our preliminary QSI bioassay-guided fractionation of this sponge, we found that organic fractions are responsible for the activity, and these fractions are rich in epidioxysterols and lipids, which could be the QS inhibitors in the crude extract (unpublished results) considering that, for some fatty acids, QSI activity is reported (Osorno et al., 2012Osorno, O., Castellanos, L., Ramos, F.A., Arévalo, C., 2012. Gas chromathography as a tool in quorum sensing studies. In: Salih, B., Çelikbıçak, Ö. (Org.). Gas Chromatography – Biochemicals, Narcotics and Essential Oils. Intech, Croatia, pp. 67–96.). The genus Neopetrosia is known as a source of quinolinic type alkaloids and other nitrogen compounds that exhibit cytotoxic, antimicrobial activity, among others (Nakao et al., 2004; De Almeida et al., 2008Nakao, Y., Shiroiwa, T., Murayama, S., Matsunaga, S., Goto, Y., Matsumoto, Y., 2004. Identification of Renieramycin A as an antileishmanial substance in a marine sponge Neopetrosia sp. Mar. Drugs 25, 55–62.). Particularly from N. carbonaria, previously known as Xestospongia carbonaria, it was possible to isolate acridine type polar alkaloids (Thale et al., 2002Thale, Z., Johnson, T., Tenney, K., Wenzel, P.J., Lobkovsky, E., Clardy, J., 2002. Structures and 24 cytotoxic properties of sponge-derived bisannulated acridines. J. Org. Chem. 67, 9384–9391.) with recognized anticancer activity. In a previous study, these alkaloids were present in the butanolic fraction which, in the present study, is also active disrupting QS biosensor systems. Consequently, it could be assumed that these alkaloids are responsible for the QSI activity in our extract, but it has not been demonstrated yet.

In conclusion, we were able to identify the extracts of the sponges N. carbonaria, S. tubulosa and I. felix as the most promising Quorum sensing inhibitors disrupting the expression of both biosensors' phenotypes without affecting their growing process. Different from these extracts, the extracts of the sponges A. citrina, A. tubulata, I. arenosa, and T. ophiraphidites showed antibacterial activity against E. coli and P. putida, and the extracts of the sponges N. caycedoi, C. tenuis, Ptilocaulis wallpersi, P. pellasarca and the algae L. catarinensis and Laurencia obtuse were highly antibacterial against C. violaceum. From the sponge I. felix, a furanosesterterpenes enriched-fraction showed a moderate QSI activity without affecting the biosensor's growth. In general, these furanosesterterpenes are structurally related with AHL and other QSI, which could explain the observed activity against C. violaceum, additionally possessing a similar mechanism of action. Notwithstanding, other studies are required to improve the real potential of these compounds as QSI exploitable by man.

Acknowledgments

This work was conducted as part of a joint project financially supported by CNPq/MCT/Brazil (grant number 490151/2007-8) and COLCIENCIAS/Colombia (grant number 358-2007). The work was also financed by other grants from Colciencias and "Fundación para la Promoción de la Investigación y la Tecnología del Banco de la República". The Colombian authors greatly acknowledge Prof. Dr. Kathrin Riedel (Department of Microbiology, Institute of Plant Biology, University of Zürich) and Prof. Dr. Sven Zea from the Universidad Nacional de Colombia for their interest and assistance in this work, and Prof. Dr. Paulo Horta, Dr. Cintia Llhulier and Prof. Dr. Eloir P. Schenkel for the collection and identification of the algae.

References

  • Anta, C., Gonzáles, N., Santafé, G., Rodríguez, J., Jiménez, C., 2002. New Xenia diterpenoids from the Indonesian soft coral Xenia sp. J. Nat. Prod. 65, 766–768.
  • Assmann, M., Lichte, E., Pawlik, J.R., Köck, M., 2000. Chemical defenses of the Caribbean sponges Agelas wiedenmayeri and Agelas conifera Mar. Ecol. Prog. Ser.22, 255–262.
  • Blunt, J.W., Copp, B.R., Munro, M.H.G., Northcote, P.T., Prinsep, M.R., 2010. Marine natural products. Nat. Prod. Rep. 27, 165–237.
  • Boyer, M., Wisniewski-Dyé, F., 2009. Cell–cell signalling in bacteria: not simply a matter of quorum. FEMS Microbiol. Ecol. 21, 1–19.
  • Braekman, J.C., Daloze, D., Stoller, C., Van Soest, R.W.M., 1992. Chemotaxonomy of Agelas (Porifera: Demospongiae). Biochem. Syst. Ecol. 20, 417–431.
  • Cafieri, F., Carnuccio, R., Fattorusso, E., Taglialatela-Scafati, O., Vallefuoco, T., 1997. Anti-histaminic activity of bromopyrrole alkaloids isolated from Caribbean Agelas sponges. Bioorg. Med. Chem. Lett. 9, 2283–2288.
  • Carletti, I., Banaigs, B., Amade, P., 2000. Matemone a new bioactive bromine-containing oxindole alkaloid from the Indian Ocean sponge Iotrochota purpurea J. Nat. Prod. 63, 981–983.
  • Cuadrado, C.T., (Master's thesis) 2010. Aislamiento de N-acilhomoserinlactonas de algunas bacterias procedentes del Mar Caribe Colombiano, como evidencia de la existencia de circuitos de quorum sensing. Universidad Nacional de Colombia, Bogotá, 270 pp.
  • De Almeida, L.P., Carroll, A.R., Towerzey, L., King, G., McArdle, B.M., Kern, G., 2008. Exiguaquinol: a novel pentacyclic hydroquinone from Neopetrosia exigua that inhibits Helicobacter pylori MurI. Org. Lett. 19, 2585–2588.
  • Dickschat, J.S., 2010. Quorum sensing and bacterial biofilms. Nat. Prod. Rep. 27, 343–369.
  • Dobretsov, S., Teplitski, M., Paul, V., 2009. Mini-review: quorum sensing in the marine environment and its relationship to biofouling. Biofouling 25, 413–427.
  • Dobretsov, S., Teplitski, M., Bayer, M., Gunasekera, S., Proksch, P., Paul, V., 2011. Inhibition of marine biofouling by bacterial quorum sensing inhibitors. Biofouling 27, 893–905.
  • Fan, G., Li, Z., Shen, S., Zeng, Y., Yang, Y., Xu, M., 2010. Baculiferins A-O, O-sulfated pyrrole alkaloids with anti-HIV-1 activity, from the Chinese marine sponge Iotrochota baculifera Bioorg. Med. Chem. 18, 5466–5474.
  • Greenberg, E.P., 2003. Bacterial communication and group behavior. J. Clin. Invest. 112, 1288–1290.
  • Gunasekera, P.S., Sennett, S.H., Kelly-Borgues, M., Bryant, R.W., 1994. Ophirapstanol trisulfate, a new biological active steroid sulfate from the deep water marine sponge Topsentia ophiraphidites J. Nat. Prod. 57, 1751–1754.
  • Harvell, D., Jordán-Dahlgren, E., Merkel, S., Rosenberg, E., Raymundo, L., Smith, G., Weil, E., 2007. Coral disease environmental drivers, and the balance between coral and microbial associates. Oceanography 20, 172–195.
  • Keifer, P.A., Schwartz, R.E., Koker, M.E.S., Hughes Jr., R.G., Rittschof, D., Rinehart, K.L., 1991. Bioactive bromopyrrole metabolites from the Caribbean sponge Agelas conifera J. Org. Chem. 56, 2965–2975.
  • Kim, J.S., Kim, Y.H., Seo, Y.W., Park, S., 2007. Quorum sensing inhibitors from the red alga Ahnfeltiopsis flabelliformis Biotechnol. Bioproc. Eng. 30, 308–311.
  • Kong, F., Burgoyne, D.L., Andersen, J., Allen, T.M., 1992. Pseudoaxinellin, a cyclic heptapeptide isolated from the Papua New Guinea sponge Pseudoaxinella massa Tetrahedron Lett. 33, 3269–3272.
  • Lhullie, C., Falkenberg, M., Ioannou, E., Quesada, A., Papazafiri, P., Antunes-Horta, P., Schenkel, E.P., Vagias, C., Roussis, V., 2010. Cytotoxic halogenated metabolites from the Brazilian red alga Laurencia catarinensis J. Nat. Prod. 73, 27–32.
  • Lowery, C.A., Dickerson, T.J., Janda, K.D., 2008. Interspecies and interkingdom communication mediated by bacterial quorum sensing. Chem. Soc. Rev. 37, 1337–1346.
  • Martin, C.A., Hoven, A.D., Cook, A.M., 2008. Therapeutic frontiers: preventing and treating infectious diseases by inhibiting bacterial quorum sensing. Eur. J. Clin. Microbiol. 27, 635–642.
  • Martinez, A., Duque, C., Hara, N., Fujimoto, Y., 1995. Variabilin 11-metiloctadecanoate a branched-chain fatty acid ester of furanosesterpene tetronic acid, from sponge Ircinia felix Nat. Prod. Lett. 6, 281–284.
  • Martínez, A., Duque, C., Sato, N., Fujimoto, Y., 1997. (8Z, 13Z, 20Z)-strobilinin and (7Z, 13Z, 20Z)-felixinin: new furanosesterterpene tetronic acids from marine sponges of the genus Ircinia Chem. Pharm. Bull. 45, 181–184.
  • Meseguer, D., Kiil, S., Dam-Johansen, K., 2004. Antifouling technology – past, present and future steps towards efficient and environmentally friendly antifouling coatings. Progr. Org. Coat. 50, 75–104.
  • Mora-Cristancho, J.A., Arevalo-Ferro, C., Ramos, F.A., Tello, E., Duque, C., Lhullier, C., Falkenberg, M., Schenkel, E.P., 2011. Antifouling activities against colonizer marine bacteria of extracts from marine invertebrates collected in the Colombian Caribbean Sea and on the Brazilian Coast (Santa Catarina). Z. Naturforsch. C 66, 515–526.
  • Nakao, Y., Shiroiwa, T., Murayama, S., Matsunaga, S., Goto, Y., Matsumoto, Y., 2004. Identification of Renieramycin A as an antileishmanial substance in a marine sponge Neopetrosia sp. Mar. Drugs 25, 55–62.
  • Ng, W.L., Bassler, B.L., 2009. Bacterial quorum-sensing network architectures. Annu. Rev. Genet. 17, 197–222.
  • Osorno, O., Castellanos, L., Ramos, F.A., Arévalo, C., 2012. Gas chromathography as a tool in quorum sensing studies. In: Salih, B., Çelikbıçak, Ö. (Org.). Gas Chromatography – Biochemicals, Narcotics and Essential Oils. Intech, Croatia, pp. 67–96.
  • Peters, L., König, G.M., Wright, A.D., Pukall, R., Stackebrandt, E., Eberl, L., 2003. Secondary metabolites of Flustra foliacea and their influence on bacteria. Appl. Environ. Microbiol. 69, 3469–3475.
  • Qian, P.Y., Lau, S.C.K., Dahms, H.U., Dobretsov, S., Harder, T., 2007. Marine biofilms as mediators of colonization by marine macroorganisms: implications for antifouling and aquaculture. Mar. Biotechnol. 12, 399–410.
  • Rasmussen, T.B., Givskov, M., 2006. Quorum sensing inhibitors as anti-pathogenic drugs. Int. J. Med. Microbiol. 296, 149–161.
  • Shen, X., Perry, T.L., Dunbar, C.D., Kelly-Borges, M., Hamann, M.T., 1998. Debromosceptrin, an alkaloid from the Caribbean sponge Agelas conifera J. Nat. Prod. 61, 1302–1303.
  • Sjöstrand, U., Kornprobst, J.M., Djerassi, C., 1981. Minor and trace sterols from marine invertebrates 29 (22E)-ergosta-5,22,25-trien-3β-ol and (22E,24R)-24,26-dimethylcholesta-5,22,25(27)-trien-3β-ol. Two new marine sterols fromthe sponge Pseudopxinella lunachata Steroids 38, 355–364.
  • Skindersoe, M., Ettinger-Epstein, P., Rasmussen, T., Bjarnsholt, T., De Nys, R., Givskov, M., 2008. Quorum sensing antagonism from marine organisms. Mar. Biotechnol. 20, 56–63.
  • Teplitski, M., Ritchie, K., 2009. How feasible is the biological control of coral diseases? Trends Ecol. Evol. 24, 378–385.
  • Thale, Z., Johnson, T., Tenney, K., Wenzel, P.J., Lobkovsky, E., Clardy, J., 2002. Structures and 24 cytotoxic properties of sponge-derived bisannulated acridines. J. Org. Chem. 67, 9384–9391.
  • Yang, S.W., Chan, T.M., Pomponi, S.A., Chen, G., Wright, A.E., Patel, M., 2003. A new bicyclic guanidine alkaloid, Sch 575948, from a marine Sponge, Ptilocaulis spiculifer J. Antibiot. 56, 970–972.

Publication Dates

  • Publication in this collection
    May-Jun 2015

History

  • Received
    14 Nov 2014
  • Accepted
    26 Mar 2015
Sociedade Brasileira de Farmacognosia Universidade Federal do Paraná, Laboratório de Farmacognosia, Rua Pref. Lothario Meissner, 632 - Jd. Botânico, 80210-170, Curitiba, PR, Brasil, Tel/FAX (41) 3360-4062 - Curitiba - PR - Brazil
E-mail: revista@sbfgnosia.org.br