Screening of extracts of algae from Baja California Sur, Mexico as reversers of the antibiotic resistance of some pathogenic bacteria

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1 European Review for Medical and Pharmacological Sciences Screening of extracts of algae from Baja California Sur, Mexico as reversers of the antibiotic resistance of some pathogenic bacteria 2010; 14: M. MUÑOZ-OCHOA, J.I. MURILLO-ÁLVAREZ, L.A. ZERMEÑO-CERVANTES, S. MARTÍNEZ-DIAZ, R. RODRÍGUEZ-RIOSMENA* Departamento de Desarrollo de Tecnologías, Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz (Mexico); *Departamento de Biología Marina, Universidad Autónoma de Baja California Sur, La Paz (Mexico) Abstract. Background and Objectives: Sixty ethanol extracts of marine flora of Baja California Sur (Mexico) were screened to evaluate the reversing effect of the bacterial resistance to antibiotics in combination with a sublethal concentration of ampicillin or erythromycin. Materials and Methods: The activity was assayed by using a modification of the classical agar-diffusion method against 3 resistant, pathogenic bacteria; Escherichia coli (ATCC BAA196), Staphylococcus aureus (ATCC BAA42), and Streptococcus pyogenes (ATCC BAA946). Results: From the 60 ethanolic extracts, 12 (20%) of them in combination with ampicillin were able to reverse the resistance of Staphylococcus aureus and 8 (13%) with erythromycin yielded the same reversal with Streptococcus pyogenes. An extract from Sargassum horridum was the only one that reversed the resistance to antibiotics against both Staphylococcus aureus and Streptococcus pyogenes. Conclusions: Our findings suggest that some algae may be source of compounds with the potential to reverse the antibiotic resistance of some bacteria. In addition, of the assayed extracts, 35 (57%) showed inhibitory activity against Staphylococcus aureus, 48 (78%) were active against Streptococcus pyogenes, but none was active against Escherichia coli. The most active extracts were from Laurencia spp., Gelidium robustum, Chnoospora implexa, Padina mexicana, Gracilaria subsecundata, and Dictyopteris undulata. Key Words: Escherichia coli, Streptococcus, Staphylococcus, Antimicrobial. Introduction Today, an increasing demand of new compounds to combat the bacterial resistance to antibiotics is globally recognized. Resistance is a phenomenon characterized by having serious health and socioeconomic implications. Because of the bacterial resistance, a number of infectious diseases are becoming difficult to treat, making it necessary to be more aggressive and using expensive treatments over a longer time 1. The problem is global, complex, and includes a large number of bacteria of pathological importance. The multicausality of the resistance makes it a difficult problem with which to deal 2. Highly populated areas, inadequate control of infections, and the transference of patients between hospitals are important factors causing the increasing of the resistance. The changes in the ecology of infections observed since the introduction of the antimicrobial agents have been well-documented 2. In response to those changes, bacteria have developed a number of adaptive strategies; enzyme inactivation, efflux pumps, target alteration, and permeability changes 3,4. The most successful mechanisms of resistance are the expression of enzymes able to degrade the β-lactam ring of penicillins and cephalosporins 5 and efflux pumps that are able to extrude structurally diverse compounds, including antibiotics, from the cell. To combat the rapid spread of bacteria expressing resistance, many Authors 5-8 agree for the need to discover new antibacterials and resistance-reversing agents. This latter involves the Corresponding Author: Mauricio Muñoz-Ochoa, MD; mmo6709@gmail.com 739

2 M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena discovery of compounds able to inhibit those mechanisms that confer resistance to antibiotics by the bacteria. With this in mind, in our research a library of extracts from marine macroalgae from Mexico were screened as reversers of the resistance to antibiotics of three pathogenic bacteria to select unexplored sources of resistancereversing compounds. This choice is justified because marine algae are important sources of metabolites, such as acetogenins, polyphenolics, aromatic compounds, terpenes, and other related structures 9. To the best of our knowledge this is the first time in which marine macroalgae collected from the coasts of Baja California Sur, Mexico have been assessed as reversers of the resistance to antibiotics. Our finding has allowed us to select a number of algae for further investigation in search for the active compounds. Materials and Methods The algae studied were gathered from several locations along the coast of Baja California Sur (Figure 1) between June 2004 and November The collection was done by free diving at 1- to 2-m depth. Algal samples were cleaned of epiphytes, rinsed with tap water to remove any associated debris, and the cleaned fresh materials were dried in the sun, ground, and stored at 20 C until extraction. Voucher specimens were preserved for taxonomic identification and future reference. The extracts were obtained by maceration. Briefly, 100 g of each algal sample was soaked with 250 ml of distilled ethanol at C. After 48 h, the mixture was filtered through paper. The residual algal tissue was extracted once again under same conditions. After filtration, both extracts were pooled and concentrated to dryness under vacuum at 40 C (Yamato RE500, San Francisco, CA, USA). The crude extracts were kept in dark and dry conditions at 20 C until biological testing. A modification of the classical agar disc-diffusion method 10 was used to test the effect of extracts on the reversion of the resistance of Escherichia coli (ATCC BAA-196, expressing extended spectrum β-lactamases), Staphylococcus aureus (ATCC BAA-42, β-lactamases), and Streptococcus pyogenes (ATCC BAA-946, efflux pump). Each strain was first cultured on Mueller- Hinton agar plates at 37 C. After 18 h, the cell biomass was harvested and suspended in a 0.85% sodium chloride solution. The cell density was adjusted by spectrophotometry (Merck SQ118, Darmstadt, Germany) at λ 585 nm until 1.0 AU (equivalent to cells ml -1 ). The suspensions of each strain were used to inoculate the surface of Mueller-Hinton agar plates with an added sublethal concentration of an antibiotic. Escherichia coli and Staphylococcus aureus were cultured on Mueller-Hinton agar with added ampicillin (12 mg L -1, equivalent to 75% of the minimum inhibitory concentration (MIC)). The medium for Streptococcus pyogenes was added erythromycin (1.5 mg L -1, equivalent to 25% of the MIC). The drug susceptibility pattern of each target strain was previously determined. The culturing conditions of strains under sublethal concentrations of antibiotic were extensively tested and optimized. The assays were done by putting, on the inoculated surface of the agar plate, extract-impregnated paper discs (6.5 diameter, Whathman No. 4). Each paper disc was loaded with 100 µl of a stock solution of each algal extract (20 mg ml -1 ). The impregnation of the disc was done under aseptic conditions. Solvent evaporation from the discs left 2.0 mg disc -1. Plates were incubated at 37 C. The inhibition zone (when observed) around discs was measured after 24 h, and expressed in mm. Every algal extract was tested in a medium added with antibiotic (as mentioned before) and its effect was contrasted by testing the same extract, at the same time, in Mueller-Hinton agar without an antibiotic. Those extracts active against bacteria cultured on a medium with added antibiotic, but inactive in an antibioticfree medium were considered as reversers of the resistance. Measures of the inhibition zones are presented as the mean values of two measurements and its SD. Ethanol was always used as a negative control. Statistical Analysis We have found a great difficult to perform a statistical analysis. However, we have taken into mind that: The substances produced by each alga do not necessarily correspond to the same chemical group as well as we cannot assume that they are in the same concentration, therefore a comparison between them we could give an erroneous interpretation of the activity of each of the species. 740

3 Reversion of the antibiotic resistance Figure 1. Map of Baja California peninsula (Mexico) showing the different collection locations. The effect of each alga on the three test bacteria cannot be compared because the mechanisms used by bacteria to resist antibiotics are different (β-lactamases and efflux pump as mentioned in the introduction). Thus although in some cases there is a tendency to increase the diameter of the halos when the algae extract is placed in the presence of the antibiotic, it cannot be directly interpreted as an effect of inhibition of antibiotic resistance, because the 741

4 M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena extract has antibacterial activity and therefore both mechanisms may be acting simultaneously. In future work, surely we must focus to describe the nature of the compounds responsible for the antibacterial activity and effect of each of the molecules in the presence of antibiotics. Therefore we consider the only valid criterion to define some attribute the extracts was attributable to the presence of antibiotic that inhibition is present whereas in the absence of antibiotic inhibition was not observed. With the foregoing, we dismiss the ability of the extracts per se to inhibit the process of bacterial resistance to antibiotics. In this case the values were zero for the diameter of halo in the absence of antibiotic and values of 8 mm or more for the diameter of halo in the presence of antibiotic. Results As seem in Table I, from all the 60 algal extracts assessed, 9 extracts, 15%, were able to reverse the resistance of Staphylococcus aureus to a sublethal concentration of ampicillin. The effect was denoted by the inhibition of growth in combination of the antibiotic and extracts from Padina crispata (sample ID: ), Hypnea valentiae (04-011), Sargassum horridum (04-003), Rosenvingea. intricata (04-015), Spyridia filamentosa (04-025), Liagora californica (06-008), Chondracanthus canaliculatus (07-010), Codium amplivesiculathum (04-004) and Codium cuneatum (06-011). The extracts from Sargassum horridum (04-003), Cystoseira osmundacea (06-023), Chondracanthus canaliculatus (06-026), Hypnea johnstonii (06-035), Pterosiphonia bipinnata (07-003), Spyridia filamentosa (07-006), Gracilaria marcialana (07-007), and Colpomenia sinuosa (07-008) showed the same effect on the resistance of Streptococcus pyogenes to erythromycin. From the 3 tested extracts of Sargassum horridum collected in different years, just the extract from the specimen was active. In addition, Sargassum horridum (04-003) was the one that showed a reverser effect on the antibiotic resistance of Staphylococcus aureus and Streptococcus pyogenes against ampicillin and erythromycin. Surprisingly, none of extracts tested was able to affect the growth of Escherichia coli. Discussion Objective of this study was to evaluate the ability of extracts of marine algal species collected from several locations from Baja California Sur (Mexico) to inhibit (or reverse) the mechanism of resistance to antibiotics of three pathogenic bacteria. In the literature there are numerous reports about a broad range of antimicrobial activities from algal extracts from around the world 11-20, including algae from Mexican waters A number of algal natural products with antimicrobial activity have been described in the past few decades 9. After an extensive bibliographic review through electronic data bases, reports of a reverser effect of the algal extracts on antibiotic-resistance of bacteria were not found. Probably our study is a pioneer in this area. As mentioned, just those extracts that were active against bacteria cultured in a medium with an added sublethal concentration of antibiotic (but necessarily an extract inactive by itself) were considered as inhibitors of the mechanisms of bacterial resistance. That interpretation was done under the assumption of that some compounds present in the extracts were able to inhibit the β- lactamase activity in Staphylococcus aureus andor the efflux pump in Streptococcus pyogenes allowing the added antibiotic (ampicillin or erythromycin) to cause cell growth inhibition, denoted by inhibition zones around the discs. In the search for inhibitors of the mechanisms of bacterial resistance, inhibitors have been found mainly in bacteria or by chemical synthesis. For example, the clavulanic acid produced by Streptomyces clavuligerus acts as an inhibitor of β-lactamases, which are a defensive response to the β- lactam antibiotics 25. Competitive inhibition is the mode of action of the clavulanic acid caused by a structure identical to the natural β-lactamase substrate. We believe that competitive inhibitors of β- lactamases can be found in macroalgae and that is our explanation for the observed effect of Sargassum horridum (04-003) on the resistance of Staphylococcus aureus against ampicillin. This idea is supported by the isolation of sargassumlactam from Sargassum kjellmanianum 26. Considering that similar compounds may be synthesized by other brown algae, the effect shown by Padina crispata (04-002) and Rosenvingea intricata (04-015) on the resistance of Staphylococcus aureus may be explained by the presence of structures similar to the sargassumlactam. 742

5 Reversion of the antibiotic resistance Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on Mueller Hinton agar plates add with a sublethal concentration of antibiotic. Diameter of the inhibition zone, in mm a S. aureus S. pyogenes E. coli MAWA CWA MAWE CWA MAWA CWA Family Sample ID, species name, and authority (collection place) Bangiaceae , Porphyra perforata Agardh, 1883 (2) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Chnoosporaceae , Chnoospora implexa Agardh, 1848 (8) 10.0 ± ± ± ± 0 0 ± 0 0 ± 0 Cladophoraceae , Cladophora sp. (7) 10.0 ± ± ± ± ± 0 0 ± 0 Codiaceae , Codium amplivesiculatum Setchell & Gardner, 1924 (8) 8.0 ± 0 0 ± ± ± 0 0 ± 0 0 ± 0 ( , Codium amplivesiculatum Setchell & Gardner, 1924 (6) 0 ± 0 0 ± ± ± 0 ± 0 0 ± , Codium cuneatum Setchell & Gardner, 1924 (8) 10.0 ± ± ± ± 0 0 ± 0 0 ± , Codium cuneatum Setchell & Gardner, 1924 (6) 9.5 ± ± ± ± ± 0 0 ± , Codium simulans Setchell & Gardner, 1924 (6) 9.5 ± ± ± ± 0 0 ± 0 0 ± 0 Corallinaceae , Amphiroa valonioides Yendo, 1902 (7) 0 ± 0 0 ± ± ± ± 0 0 ± , Corallina vancouveriensis Yendo, 1902 (4) 9 ± ±0 9.5 ± ± ± 0 0 ± , Corallina sp. (2) 15.5 ± ± ± ± ± 0 0 ± 0 Cystocloniaceae , Hypnea johnstonii Setchell & Gardner, 1924 (2) 0 ± ± ± 0 0 ± 0 0 ± 0 0 ± , Hypnea valentiae (Turner) Montagne, 1841 (8) 8.0 ± 0 0 ± ± ± ± 0 0 ± 0 Dictyotaceae , Dictyota flabellata Setchell & Gardner, 1924 (6) 10.0 ± ± ± ± ± 0 0 ± , Dictyopteris undulata Holmes, 1896 (2) 18.0 ± ± ± ± ± 0 0 ± , Dictyopteris delicatula Lamouroux, 1809 (2) 14.5 ± ± ± ± 0 0 ± 0 0 ± , Padina mexicana Thivy, 1945 (8) 9.5 ± ± ± ± 0 0 ± 0 0 ± , Padina mexicana Thivy, 1945 (8) 14.5 ± ± ± ± 0 0 ± 0 0 ± , Padina concrescens Thivy, 1945 (2) 10.5 ± ± ± ± ± 0 0 ± 0 (Continued) 743

6 M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on Mueller Hinton agar plates add with a sublethal concentration of antibiotic. Diameter of the inhibition zone, in mm a S. aureus S. pyogenes E. coli MAWA CWA MAWE CWA MAWA CWA Gelidiaceae , Gelidium robustum Hollenberg & Abbott, 1965 (3) 11.0 ± ± ± ± 0 0 ± 0 0 ± , Gelidium robustum Hollenberg & Abbott, 1965 (4) 14.0 ± ± ± ± 0 0 ± 0 0 ± , Gelidium robustum Hollenberg & Abbott, 1965 (2) 12.5 ± ± ± ± ± 0 0 ± 0 Gigartinaceae , Chondracanthus canaliculatus Harvey, 1993 (2) 9.5 ± ± ± ± 0 0 ± 0 0 ± , Chondracanthus canaliculatus Harvey, 1993 (1) 9.0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Gracilariaceae , Gracilaria marcialana Setchell & Gardner, 1924 (8) 0 ± 0 0 ± ± 0 0 ± 0 0 ± 0 0 ± , Gracilaria veleroae Dawson, 1944 (8) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± , Gracilaria vermiculophylla (Ohmi) Papenfuss, 1967 (5) 0 ± 0 0 ± ± ± 0 0 ± 0 0 ± , Gracilaria subsecundata Setchell & Gardner, 1924 (8) 13.5 ± ± ± ± ± 0 0 ± 0 Laminariaceae , Macrocystis pyrifera Agardh, 1820 (2) 9.0 ± ± ± ± ± 0 0 ± 0 Lessoniaceae , Eisenia arborea Areschoug, 1876 (2) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Liagoraceae , Ganonema farinosum Fan & Wang, 1974 (8) 12.5 ± ± ± ± 0 0 ± 0 0 ± , Liagora californica Zeh, 1912 (8) 9.0 ± 0 0 ± ± ± ± 0 0 ± , Liagora californica Zeh, 1912 (6) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Rhodymeniaceae , Rhodymenia californica Kylin, 1931 (7) 7.0 ± ± ± ± 0 0 ± 0 0 ± , Rhodymenia californica Kylin, 1931 (2) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 (Continued) 744

7 Reversion of the antibiotic resistance Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on Mueller Hinton agar plates add with a sublethal concentration of antibiotic. Diameter of the inhibition zone, in mm a S. aureus S. pyogenes E. coli MAWA CWA MAWE CWA MAWA CWA Rhodomelaceae , Acanthophora spicifera (M. Vahl) Børgesen, 1910 (8) 0 ± 0 0 ± ± ± ± 0 0 ± , Laurencia gardneri Hollenberg, 1943 (7) 0 ± 0 0 ± ± ± ± 0 0 ± , Laurencia johnstonii Setchell & Gardner, 1924 (8) 30.0 ± ± ± ± ± 0 0 ± , Laurencia johnstonii Setchell & Gardner, 1924 (8) 12.0 ± ± ± ± ± 0 0 ± , Laurencia pacifica Setchell & Gardner, 1924 (8) 19.5 ± ± ± ± ± 0 0 ± , Laurencia pacifica Setchell & Gardner, 1924 (6) 21.0 ± ± ± ± ± 0 0 ± , Laurencia sp. (8) 19.0 ± ± ± ± ± 0 0 ± , Neorhodomela larix (Turner) Masuda, 1982 (2) 12.0 ± ± ± ± 0 0 ± 0 0 ± , Pterosiphonia bipinnata Falkenberg, 1901 (7) 0 ± 0 0 ± ± ± 0 0 ± 0 0 ± 0 Sargassaceae , Cystoseira osmundacea (Turner) Agardh, 1820 (4) 0 ± 0 0 ± ± ± 0 0 ± 0 0 ± , Sargassum horridum Setchell & Gardner, 1924 (8) 9.0 ± 0 0 ± ± 0 0 ± 0 0 ± 0 0 ± , Sargassum horridum Setchell & Gardner, 1924 (8) 9.0 ± ± ± ± ± 0 0 ± , Sargassum horridum Setchell & Gardner, 1924 (8) 9.5 ± ± ± ± ± 0 0 ± 0 Scytosiphonaceae , Hydroclathrus clathratus (Agardh) Howe, 1920 (8) 10.5 ± ± ± ± 0 0 ± 0 0 ± , Hydroclathrus clathratus (Agardh) Howe, 1920 (8) 9.5 ± ± ± ± 0 0 ± 0 0 ± , Rosenvingea intricata (Agardh) Børgesen, 1914 (8) 11.5 ± ± ± ± 0 0 ± 0 0 ± , Rosenvingea intricata (Agardh) Børgesen, 1914 (8) 7.5 ± ± ± ± 0 0 ± 0 0 ± , Rosenvingea intricata (Agardh) Børgesen, 1914 (8) 9.0 ± ± ± ± ± 0 0 ± , Colpomenia tuberculata Saunders, 1898 (6) 10.5 ± ± ± ± ± 0 0 ± , Colpomenia tuberculata Saunders, 1898 (2) 13 ± ± 0 13 ± 0 10 ± 0 0 ± 0 0 ± , Colpomenia sinuosa Derbés & Solier, 1851 (8) 11.0 ± ± ± ± 0 0 ± 0 0 ± 0 Spyridiaceae , Spyridia filamentosa (Wulfen) Harvey, 1833 (5) 10.5 ± ± ± ± 0 0 ± 0 0 ± , Spyridia filamentosa (Wulfen) Harvey, 1833 (5) 11.0 ± ± ± ± 0 0 ± 0 0 ± 0 Ulvaceae , Ulva rigida Agardh, 1823 (8) 0 ± 0 0 ± 0 15 ± ± 0 0 ± 0 0 ± , Ulva dactylifera Setchell & Gardner, 1920 (7) 11.0 ± ± ± ± 0 0 ± 0 0 ± 0 a Mean values (n = 2) ± standard deviation. MAWA = cultured on medium added with 75% of the MIC of ampicillin; CWA = cultured without antibiotic; MAWE = cultured on medium added with 25% of the MIC of erythromycin. 745

8 M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena The synthetic inhibitors of a bacterial efflux pump have been investigated in the activity of the NorA pump expressed in some species of the genus Staphylococcus and Streptococcus 27,28. Among the compounds investigated as pump inhibitors are the indole alkaloid reserpine, and some tyrosine derivatives such as MC and INF240. Other compounds containing bromophenol as structural moieties have been investigated 29,28. The literature shows that compounds related to those inhibitors of the NorA pump are present in extracts from Hypnea johnstonii (06-035), Chondracanthus canaliculatus (06-026), Gracilaria marcialana (07-007), Pterosiphonia. bipinnata (07-003), Colpomenia osmundacea (06-023), Sargassum horridum (04-003), Colpomenia sinuosa (07-008), and Spyridia filamentosa (07-006) 9, and we believe that may explain the effect observed by the extracts on the resistance of Streptococcus pyogenes. In this study, a different response was measured by extracts from same species collected in different locations and-or year. Temporal and spatial variation of the biological responses of crude extracts of marine organisms as a consequence of biotic and nonbiotic conditions has been extensively documented One can note that 80% of the extracts were active against one, and 51% were active against two target organisms. Conclusions The results clearly suggest that macroalgae collected from Baja California Sur (Mexico) are an interesting resource for the search for compounds that may be developed as potential reversers of some mechanisms of bacterial resistance to antibiotics. The finding of new reversers of resistance may have important implications for public health. References 1) ANONYMOUS. The world health report Geneve: WHO Press; ) SMITH PA, ROMESBERG FE. Combating bacteria and drug resistance by inhibiting mechanisms of persistence and adaptation. Nat Chem Biol 2007; 3: ) DAVIES J. Inactivation of antibiotics and the dissemination of resistance genes. Science 1994; 264: ) TENOVER FC. Mechanisms of antimicrobial resistance in bacteria. Am J Med 2006; 119: S3-S10. 5) LIVERMORE DM. Evolution of β-lactamase inhibitors. Intens Care Med 1994; 20: S10-S13. 6) MAHAMOUD A, CHEVALIER J, ALBERT-FRANCO S, KERN WV, PAGÈS JM. Antibiotic efflux pumps in Gramnegative bacteria: the inhibitor response strategy. J Antimicrob Chemother 2007; 59: ) STAVRI M, PIDDOCK LJV, GIBBONS S. Bacterial efflux pump inhibitors from natural sources. J Antimicrob Chemother 2007; 59: ) DZIDIC S, SUSKOVIC J, BLAZENKA K. Antibiotic resistance mechanisms in bacteria: biochemical and genetic aspects. Food Technol Biotechnol 2008; 46: ) BLUNT JW, COPP BR, HU WP, MUNRO MHG, NORTH- COTE PT, PRINSEP MR. Marine natural products. Nat Prod Rep 2007; 24: ) BAUER AW, KIRBY WM, SHERRIS JC, TURK M. Antibiotic susceptibility testing by standardized single disk method. Am J Clin Pathol 1966; 45: ) NAQVI SWA, KAMAT SY, FERNANDES L, REDDY CVG. Screening of some marine plants from the Indian coast for biological activity. Bot Mar 1980; 24: ) CACCAMESE S, TOSCANO RM, FURNARI G, CORMACI M. Antimicrobial activities of red and brown algae from southern Italy coast. Bot Mar 1985; 28: ) HODGSON LM. Antimicrobial and antineoplastic activity in some south Florida seaweeds. Bot Mar 1984; 27: ) BALLANTINE DL, GERWICK WH, VELEZ SM, ALEXANDER E, GUEVARA P. Antibiotic activity of lipid-soluble extracts from Caribbean marine algae. Hydrobiologia 1987; 151/152: ) CAMPOS-TAKAKI GM, DIU MBS, KOENING ML, PEREIRA EC. Screening of marine algae from Brazilian northeastern coast for antimicrobial activity. Bot Mar 1988; 3: ) BALLESTEROS E, MARTIN D, URIZ MJ. Biological activity of extracts from some Mediterranean macrophytes. Bot Mar 1992; 35: ) KUMAR AK, RENGASAMY R. Evaluation of antibacterial potential of seaweeds occurring along the coast of Tamil Nadu, India against the plant pathogenic bacterium Xanthomonas oryzae pv. oryzae (Ishiyama) dye. Bot Mar 2000; 43: ) BHOSALE SH, NAGLE VL, JAGTAP TG. Antifouling potential of some marine organisms from India species of Bacillus and Pseudomonas. Mar Biotechnol 2002; 4:

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