Screening of Antibacterial and Antifungal Activities of Six Marine Macroalgae from Coasts of Yucatán Peninsula

Abstract

Ethyl acetate partition of methanol extracts of Avrainvillea nigricans. Decaisne (Udoteaceae), Codium decorticatum. (Woodward) Howe (Codiaceae), Halymenia floresia. (Clemente) C. Agardh (Grateloupiaceae), Laurencia obtusa. (Hudson) Lamouroux (Rhodomelaceae), Sargassum filipendula. C. Agardh (Sargassaceae), and Sargassum hystrix. J. Agardh, marine macroalgae from Yucatán peninsula coasts, were screened using the well-diffusion technique against three Gram-positive bacteria, four Gram-negative bacteria, two yeasts, and two molds. The extracts inhibited the growth of Staphyloccocus aureus. at 6.25–1.56 mg/mL and Bacillus subtilis. at 6.25–0.78 mg/mL. Trichophyton mentagrophytes. growth was inhibited by extracts of Laurencia obtusa., Sargassum filipendula., and Sargassum hystrix. at 6.25–3.13 mg/mL. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) were obtained by the microplate method.

Keywords:

• Antibacterial
• antifungal
• marine algae
• MIC
• Yucatán peninsula

Introduction

Infectious diseases are one of the main causes of high morbidity and mortality in human beings around the world, mainly in developing countries (Waldvogel, 2004). In Mexico, infections caused by bacteria and fungi have been an important public health problem for many years (WHO, 2001). These diseases have increased in recent years and have been magnified by the increase of HIV infections and the emergence of multidrug-resistant strains (Martinez-Donate et al., 2004).

The importance of terrestrial biodiversity as a resource to obtain new bioactive molecules is well-known. Due to their easy accessibility, terrestrial plants have served as the major source of medicinally useful products. The marine environment covers a wide thermal range, pressure range, and nutrient range, and it has extensive photic and nonphotic zones. This extensive variability has facilitated an extensive biodiversity that far exceeds that of the terrestrial environment. Despite this fact, research into the use of marine natural products as pharmaceutical agents is still in its infancy in many countries around the world, mainly in developing countries (Kumar Jha & Zi-rong, 2004).

Mexico has been considered one of three areas in the world with the highest biologic terrestrial and cultural diversity (Bye et al., 1995). The chemical diversity of the Mexican medicinal flora has long been studied, and several bioactive compounds have been isolated; however, the chemical potential of Mexican marine resources has not been intensively investigated.

The Mexican territory with 2,946,825 km² of maritime extension and 11,122 km of continental littorals extends from the Pacific Ocean to the Caribbean Sea and the Gulf of Mexico, including large peninsulas, Baja California (northwest) and Yucatán (southeast), with rich marine flora and fauna.

The Gulf of Mexico and the Caribbean Sea are two outstanding marine ecosystems joining in the Yucatán channel. This particular geographical situation promotes the existence of a high diversity and abundance of different marine algae species, which represent a potential source of bioactive compounds and food. However, phycological research in Mexico and Yucatán peninsula has focused mainly on taxonomic, ecologic, and distribution studies, and the evaluation of algae as a source of bioactive compounds with potential activity against pathogenic microorganism is very incipient (Freile, 2001). Thus, it is necessary to increase the number of studies on the chemistry of algae from Mexican coasts.

Based on these facts, we have initiated studies on the chemistry and biological activity of algae species from Mexico. In this paper, we wish to report our results on the antimicrobial and antifungal activity of the extracts of macroalgae species from coasts of the Yucatán peninsula. We started the studies with specimens of the Chlorophyta, Rhodophyta, and Phaeophyta divisions. The six investigated species were selected because they are the most abundant in the marine ecosystems of this area, and four of them are exclusively distributed in Mexico, in the Yucatán peninsula (Dreckmann, 1998; Dreckmann, 2000).

Materials and Methods

Plant material

The tested algae were Avrainvillea nigricans. Decaisne (Udoteaceae) (I. Sánchez-Molina 855), Codium decorticatum. (Woodward) Howe (Codiaceae) (I. Sánchez-Molina 859), Halymenia floresia. (Clemente) C. Agardh (Grateloupiaceae) (I. Sánchez-Molina 860), Laurencia obtusa. (Hudson) Lamouroux (Rhodomelaceae) (I. Sánchez-Molina 856), Sargassum filipendula. C. Agardh (Sargassaceae) (I. Sánchez-Molina 862), and Sargassum hystrix. J. Agardh (I. Sánchez-Molina 864).

Collection of marine algae

Six different algae species were collected, by scuba diving, at Puerto Chelem (21°15′ 46.11″ N, 89°44′25.2″ W), Santa Clara (21°03′ 00″ N, 88°57′50″ W), Isla Cerritos (21°34′ 00″ N, 88°17′00″ W), and Tulum (20°12′41.5″ N, 87°21′03.3″ W), in the states of Yucatán and Quintana Roo, during July–November 2002. The material was cleaned and kept at 4°C during transportation to the laboratory. One specimen of each species was fixed in methanol for identification, carried out by Isabel Sánchez according to Taylor (1972) and Wynne (1986). Vouchers were deposited at the herbarium “Alfredo Barrera Marin” of the Universidad Autónoma de Yucatán (UADY), Mérida Yucatán, Mexico.

Preparation of extracts

Samples of 200 g of each algae species were blended in 200 mL of methanol, the volume completed to 1 L with 800 mL of methanol and extracted at room temperature for 3 days. The methanol extracts were filtered and distilled until dryness at 40°C under vacuum to yield thick syrups that were resuspended in 1000 mL of distilled water and partitioned with 500 mL of ethyl acetate (3×). The ethyl acetate fractions were concentrated to give the organic extracts.

Microorganisms

Microorganisms used were Staphylococcus aureus. (4012), Bacillus subtilis. (465), Streptococcus agalactiae. (4768), Escherichia coli. (128), Pseudomonas aeruginosa. (260), Klebsiella pneumoniae. (4209), Shigella flexneri. (9748), Candida albicans. (752), Saccharomyces cerevisae. (287), Aspergillus niger. (16888), and Trichophyton mentagrophytes. (4807). All of them were purchased from the American Type Culture Collection.

Antimicrobial tests

Organic extracts were screened, using the agar-well diffusion method (Rios et al., 1988). A 5% (w/v) test solution of each extract was prepared in dimethylsulfoxide. Amikacin (31.25 µg/µL), nystatin (5.00 IU/µL), and itraconazole (25.00 µg/µL) were used as positive controls for bacteria, yeasts, and molds, respectively. Dimethylsulfoxide was used as negative control. Mueller-Hinton agar was used as a culture media for bacteria and yeasts and Sabouraud dextrose agar was used for filamentous fungi. Each extract solution (100 µL) and controls were dropped in a 6-mm-diameter well. Plates were incubated for 24 h at 37°C for bacteria and yeasts and for 3 days at room temperature for filamentous fungi; all cultures were kept under aerobic conditions. The diameter of the inhibition zone around each well was measured and recorded. Antimicrobial activity was expressed as the ratio of the average of inhibition zones produced by the extract under test and the average of inhibition zones caused by the positive controls. Each test was performed in triplicate.

The minimum inhibitory concentration (MIC) was determined using the microplate method (Eloff, 1998). DMSO test solution (100 µL) of each selected extract (12.5 mg/mL) was serially diluted 50% with Mueller-Hinton broth, and 100 µL of a culture of the microorganisms was added to 12 wells of a 96-well microplate. The microplates were incubated overnight at 37°C for bacteria and 3 days at room temperature for molds. To indicate bacterial growth, 40 µL of a 0.02% aqueous solution of p.-iodonitrotetrazolium violet were added to the microplates and incubated at 37°C for 30 min. Sabouraud dextrose broth was used as mycological media and Mueller-Hinton broth was used for antibacterial activities.

To determine the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC), broth was taken from the microplate wells and cultured as described above for bacteria and fungi. When the microorganism did not grow, the sample was considered bactericidal.

Results and Discussion

The results of the antimicrobial activity of the tested algae extracts are displayed in Table 1. It can be observed that all extracts were active against Gram-positive bacteria and one filamentous fungus species. Although the extracts were nonpolar, none of them showed activity against Gram-negative bacteria. However, the results followed the common pattern in antibacterial screening of extracts from terrestrial plants (Rabe & Van Staden, 1997; Mothana & Lidenquist, 2005) and extracts from marine organisms, mainly algae, according to several reports in the literature (Encarnación et al., 2000; Lima, 2002; Magallanes et al., 2003). The observed behavior is due to the more complex cell wall of Gram-negative bacteria compared with the less resistant Gram-positive bacteria (Koneman et al., 1998).

Table 1. Inhibition ratio, MIC, MBC, and MFC of organic extracts of six marine macroalgae from Yucatán peninsula

	Microorganisms used. Ratio (MIC, MBC, or MFC) mg/ml
Macroalgae	S. aureus.	B. subtilis.	T. mentagrophytes.
A. nigricans.	0.38 (6.25, >12.5)	0.36 (6.25, >12.5)	—
C. decorticatum.	0.50 (1.56, 12.5)	0.38 (0.78, 3.13)	—
H. floresia.	—	0.31 (12.5, >12.5)	—
L. obtusa.	0.48 (3.13, >12.5)	0.36 (12.5, >12.5)	0.73 (3.13, >12.5)
S. hystrix.	0.52 (6.25, >12.5)	0.36 (1.56, >12.5)	0.77 (6.25, >12.5)
S. filipéndula.	0.50 (1.56, >12.5)	0.38 (3.13, >12.5)	0.68 (6.25, >12.5)

MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration.

None of the six studied species had been tested against filamentous fungi; however, antimicrobial activity of A. nigricans., C. decorticatum., H. floresia., and L. obtusa. extracts had been studied before against Gram-positive bacteria and the yeast Candida albicans. (Olesen et al., 1964; Jones & Seaton, 1994). In addition, H. floresia. has also shown antiviral activity against A-PR8 influenza virus (Berti et al., 1962).

Our results indicated a moderate antimicrobial activity of all tested species against two Gram-positive bacteria. However, no activity was observed against S. agalactiae. L. obtusa. had been reported before as active against S. aureus. and C. albicans., but not against B. subtilis. and T. mentagrophytes. H. floresia. showed selective activity against B. subtilis., but MIC and MBC values were very high.

Besides antimicrobial activity, L. obtusa., S. hystrix., and S. filipendula. also showed moderate activity (based on MIC and MFC) against T. mentagrophytes., a keratinophilic filamentous fungus and the causal agent of dermatophytosis infections of hair, skin, and nails (Sabota et al. 1996; Evans, 1998; Aly et al., 2000; Roldan et al., 2000; Aman et al., 2001). Its ability to invade keratinized tissues and the possession of several enzymes, such as acid proteinases, elastases, keratinases, and other proteinases, are the major virulence factors of these fungi (Weitzman & Summerbell, 1995). In addition, Trichophyton. species may cause invasive infections in immune-compromised hosts (Squeo et al., 1998). In spite of the moderate activity shown by the tested algae, this is an important fact because of the lack of effective antifungal drugs, its toxicity, and the pathogenicity of this species.

Conclusions

Ethyl acetate partition of methanol extracts of all of the antimicrobial screened species exhibited a moderate activity against Bacillus subtilis. and Staphylococcus aureus.. For the first time, six marine macroalgae from coasts of the Yucatán peninsula were screened against Trichophyton mentagrophytes., and three species, Laurencia obtusa., Sargassum hystrix., and Sargassum filipendula., have documented activity against that mold. No activity against Gram-negative bacteria, Streptococcus agalactiae. (Gram-positive), yeasts, and Aspergillus niger. (mould) was recorded for any extract of the investigated algae. The correlation observed for inhibition zones and MICs confirm that the antimicrobial activity quantification is necessary.

The results encourage us to continue investigating marine macroalgae species from Yucatán peninsula coasts to isolate the bioactive constituents and to determine their antifungal activity.

Acknowledgments

The current study was supported by the Programa de Impulso y Orientación a la Investigación of the Universidad Autónoma de Yucatán (PRIORI), grant FQUI-02-002. The authors are grateful to Dr. Leovigildo Quijano for critical review of the paper and to M.Sc. Marina Vera-Kú for help in document translation.