Tropical Atlantic marine macroalgae with bioactivity against virulent and antibiotic resistant Vibrio

The antibacterial activity of ethanol, methanol, hexane and acetone-based extracts of the macroalgae Padina gymnospora (PG), Hypnea musciformes (HM), Ulva fasciata (UF) and Caulerpa prolifera (CP) was investigated. The disk diffusion method was used to evaluate the algae antimicrobial effect against standard strains of Vibrio parahaemolyticus, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella enterica and five virulent antibiotic-resistant strains of V. brasiliensis, V. xuii and V. navarrensis (isolated from the hemolymph of Litopenaeus vannamei). Ethanol extracts of PG and HM inhibited all Vibrio strains. E. coli and P. aeruginosa were only susceptible to ethanol extracts of PG. Among the methanol extracts, only UF was bioactive, inhibiting V. navarrensis. The observed inhibitory effect of ethanol extracts of PG, HM and UF against virulent antibiotic-resistant bacteria suggests these macroalgal species constitute a potential source of bioactive compounds.

Research on natural compounds as alternatives to synthetic antibiotics is garnering increasing attention in the scientific community (Al-Haj et al., 2010;Vieira et al., 2010).Investigations on bioactive compounds have mostly been focused on phanerogams (Nair & Chanda, 2007;Peixoto et al., 2011), but macroalgae have also been shown to contain compounds capable of inhibiting bacterial growth, including that of well-known human pathogens (Taskin et al., 2007;Kolanjinathan & Stella, 2009).
Macroalgae are multicellular, eukaryotic and autotrophic organisms lacking a specialized vascular system (Pádua et al., 2004).They have traditionally been classified according to their photosynthetic pigments, although much new taxonomic information has become available.Macroalgae may be found throughout marine and freshwater environments and on soils, rocks, snow and plant surfaces, given appropriate light and moisture conditions (Vidotti & Rollemberg, 2004).As demonstrated by Lima-Filho et al. (2002), a number of macroalgae endemic to northeastern Brazil contain bioactive compounds against a broad spectrum of gram-positive and gramnegative bacteria.
The objective of this study was to evaluate the bioactivity of extracts of the marine macroalgae species Padina gymnospora, Hypnea musciformes, Ulva fasciata and Caulerpa prolifera against standard strains of Vibrio parahaemolyticus, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella enterica, and against antibiotic-resistant vibrios carrying virulence factors.
All strains used in this study were supplied by the microbe bank of the Laboratory of Seafood and Environmental Microbiology (LABOMAR/UFC).The standard strains belonged to five species: V. parahaemolyticus ATCC 17802, E. coli ATCC 25922, Staphylococcus aureus ATCC 25923, P. aeruginosa ATCC 27853 and Salmonella IOC (Instituto Oswaldo Cruz).The virulent vibrio strains belonged to three species: Vibrio brasiliensis (n = 3), V. xuii (n = 1) and V. navarrensis (n = 1), originally isolated from the hemolymph of the Pacific white shrimp (Litopenaeus vannamei).
The algae powder (2 g) was submitted to extraction with ethanol, methanol, acetone and hexane at 1:100 (p/v) in a Soxhlet extractor at 78.4, 64.7, 56.0 and 69.0°C, respectively.After 16 h under reflux conditions, the extracts were evaporated to a final concentration of 100 mg mL -1 .The extracts were stored at 4°C.Sterilized 5 mm white disks (Laborclin) were soaked with 100 µL extract.Control disks were soaked with 100 µL of each solvent (ethanol, methanol, acetone and hexane).
Strains adjusted to 10 8 CFU mL -1 (as described above), were inoculated with swabs on Mueller-Hinton agar (CLSI, 2009).Subsequently, disks soaked in extract were applied in triplicate.A negative control disk (soaked in solvent only) and a positive control disk (soaked in 5 µg ciprofloxacin) were used for each strain.The plates were incubated at 35°C for 24 h.Inhibition halos ≥ 6 mm were considered evidence of antibacterial activity (Engel et al., 2006).
The results of the antibiogram test are shown in Table 2.Only ethanol and methanol extracts were bioactive.The greatest inhibition halos were observed for ethanol extracts of PG, ranging from 10.2 ± 1.6 (in E. coli culture) to 16.7 ± 2.1 mm (in V. brasiliensis culture).
The smallest inhibition halos were observed for ethanol extracts of UF, when compared to extracts of PG or HM.However, UF was the only algal species with antibacterial activity when methanol was used as solvent.In this case, bioactivity was observed only against V. navarrensis (average halo size: 11.3 ± 0.8 mm).
PG, HM and UF all displayed vibrocidal activity, especially the first two (Phaeophyta and Rhodophyceae, respectively), which inhibited the growth of all the vibrio species tested.No extract of CP displayed antibacterial activity.
S. aureus and S. enterica were resistant to all extracts.The standard strains of E. coli and P. aeruginosa were susceptible to the ethanol extract of PG only.
Vibrio strains presenting proteases, lipase, phospholipase and β-hemolytic activity may be considered potentially pathogenic (Zhang & Austin, 2005).In view of the presence in our vibrio strains of these markers of virulence along with resistance to one or more commonly used antibiotics, the ability of PG, HM and UF to inhibit these isolates is a very important finding (Table 1), indicating the presence of effective bioactive compounds in the algal species tested.Shanmughapriya et al. (2008) found macroalgae of the classes Phaeophyta, Rhodophyceae and Chlorophyceae to contain possibly lipolytic compounds inhibiting gram-positive and gram-negative bacteria, demonstrating the relevance of investigating the antibacterial activity of algal extracts against pathogens resistant to multiple antibiotics.In fact, according to Watson & Cruz-Rivera (2003), algae may contain a wide range of bactericidal agents, including amino acids, terpenoids, phlorotannins, acrylic acid, phenolic compounds, steroids, haloge-nated ketones, alkanes, cyclic polysulphides and fatty acids.
In our study, ethanol PG extracts presented the broadest spectrum of antibacterial activity, inhibiting all vibrio strains, E. coli and P. aeruginosa (Table 1).Vallinayagam et al. (2009) reported an even broader range of bioactivity for PG extracts prepared from algae collected in India: in addition to inhibiting Vibrio and P. aeruginosa, extracts were also effective against S. aureus and Salmonella.
Likewise, ethanol extracts of PG prepared by Manivannan et al. (2001) presented bioactivity against not only V. parahaemolyticus, E. coli and P. aeruginosa, matching our findings, but also against Salmonella.The authors observed significant inhibition halos when challenging cultures of V. cholerae, V. fluvialis and V. splendidus, confirming the vibriocidal potential of ethanol extracts of PG.This potential was evidenced in the present study by the inhibition of V. xuii, V. brasiliensis and V. navarrensis, in addition to V. parahaemolyticus.In this context, the antibacterial activity of red seaweed (Gracilaria fisheri) was reported by Kanjana et al. (2011), who verified that ethanol, methanol and chloroform extracts showed activity against a virulent strain of V. harveyi.
Extracts prepared with HM and UF were only bioactive against vibrio strains.However, in a study on the bioactive properties of these algal species, Selvin & Lipton (2004) observed a much broader antibacterial spectrum for both UF extracts (inhibiting all the bacterial species tested, including V. fischeri, V. alginolyticus, V. harveyi, Micrococcus luteus, Bacillus cereus, B. subtilis, E. coli, Aeromonas hydrophila, P. aeruginosa and Aeromonas sp.) and HM extracts (inhibiting V. fischeri, M. luteus, B. cereus, B. subtilis, A. hydrophila and P. aeruginosa).The antibacterial potential of UF was observed by Selvin et al. (2011).These authors suggested that the macroalgae U. fasciata may be a source for developing a medicated feed for shrimp disease caused by Vibrio and Aeromonas.
In our study, none of the 10 strains tested were susceptible to CP extracts.Similar findings were reported by Freile-Pelegrín & Morales ( 2004) who observed no inhibition halos when challenging strains of E. coli, P. aeruginosa, S. aureus and Salmonella with ethanolic CP extracts.Only three of the eight bacterial species tested (Bacillus subtilis, Strepto-coccus faecalis and Micrococcus luteus) were susceptible to their extracts.Plaza et al. (2010) reported significant variations in antibacterial potential between extracts of the macroalga Himanthalia elongata prepared with different solvents.A similar variation was observed in the present study, as none of the four algal species tested (PG, HM, UF and CP) were effective against any of the ten bacterial species when the extracts were prepared with acetone and hexane.This is additionally supported by the absence of antibacterial effects of hexane-based UF and CP extracts upon strains of E. coli, S. aureus, P. aeruginosa and Salmonella reported by Lima-Filho et al. (2002).
When extracts were prepared with methanol, UF was the only algal species displaying bioactivity, and only one bacterial species was susceptible (V.navarrensis).In contrast, Chiheb et al. (2009) reported methanol UF extracts to be effective against both S. aureus and E. coli.However, the bioactive properties of macroalgae are known to vary according to geographic location and season; thus, extracts prepared with the same solvent and the same algal species may differ significantly in antibacterial potential, depending on sampling location and time.
In a study covering 80 species of marine macroalgae, Padmarkumar & Ayyakkannu (1997) demonstrated that while extracts of chlorophycean species remained bioactive all year round, extracts of phaeophycean species lacked antibacterial activity when prepared with algae collected in certain periods.
The bioactivity of extracts of rhodophycean species varied from season to season, but was never completely absent.
In view of the observed inhibitory effect of ethanol extracts of P. gymnospora, H. musciformes and U. fasciata against virulent antibiotic-resistant microorganisms, it may be concluded that these macroalgae represent a potential source of bioactive compounds.

Table 1 .
Virulence factors and antibacterial resistance profile of Vibrio strains isolated from shrimp (Litopenaeus vannamei) hemolymph.