SHORT COMMUNICATION/COURTE COMMUNICATION

Antifungal and Antibacterial Activities of FourMalaysian Sponge Species (Petrosiidae)Activite antifongique et antibacterienne de quatre eponges (Petrosiidae)isolees en Malaisie

H. Qaralleh a,*, S. Idid a, S. Saad b, D. Susanti a, M. Taher c, K. Khleifat d

aDepartment of Biomedical Science, Faculty of Science, International Islamic University Malaysia, Jalan Istana,25200 Kuantan, Pahang, Malaysiab Institute of Oceanography and Maritime Studies, Faculty of Science, International Islamic University Malaysia,Jalan Istana, 25200 Kuantan, Pahang, MalaysiacDepartment of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia,Jalan Istana, 25200 Kuantan, Pahang, MalaysiadDepartment of Biology, Mutah University, Karak, Mutah, 61710 Jordan

Received 10 July 2010; accepted 9 October 2010Available online 18 November 2010

KEYWORDSAntibacterial activity;Antifungal activity;Marine sponges;Neopetrosia exigua

Summary Aqueous and organic extracts from four marine sponges collected from the coast-line of Malaysia were screened against six microorganisms, including the bacteria Staphylococcusaureus ATCC25923, Bacillus cereus ATCC11778, Pseudomonas aeruginosa ATCC27853, Escher-ichia coli ATCC35218, the yeasts Candida albicans ATCC10231 and Cryptococcus neoformansATCC90112. All the sponge species in this study showed antibacterial activity against at least onebacterial strain and only one sponge species was significantly active against C. albicans. P.aeruginosa was considered resistant to all tested samples, since no inhibition zone was observedwhile the Gram-positive B. cereus was shown to be the most sensitive microorganism followed byC. albicans and S. aureus. The highest activity was obtained for the aqueous extract ofNeopetrosia exigua against the Gram-positive bacteria B. cereus (inhibition zone 25 mm andMIC 0.07 mg/mL) and S. aureus (17.5 mm and 0.12 mg/mL) and against C. albicans (21 mm and0.32 mg/mL). This extract is currently undergoing further analysis to identify the activecompounds.# 2010 Elsevier Masson SAS. All rights reserved.

MOTS CLÉSActivité antifongique ;

Résumé Des extraits aqueux et organiques de quatre éponges marines récoltées sur la côtemalaysienne ont été testés contre six microorganismes (Staphylococcus aureus ATCC25923,Bacillus cereus ATCC11778, Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC35218,Candida albicans ATCC10231 et Cryptococcus neoformans ATCC90112). Toutes les éponges

Journal de Mycologie Médicale (2010) 20, 315—320

* Corresponding author.E-mail address: haithym2006@yahoo.com (H. Qaralleh).

1156-5233/$ — see front matter # 2010 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.mycmed.2010.10.002

Activitéantibactérienne ;Eponges marines ;Neopetrosia

étudiées ont montré une activité antibactérienne contre au moins une espèce bactérienne etseulement une éponge a été significativement active contre C. albicans. P. aeruginosa a étéconsidéré comme résistant à tous les échantillons testés car il n’a pas été observé de zoned’inhibition. B. cereus a été le plus sensible suivi par C. albicans et S. aureus. La plus forteactivité a été observée avec les extraits aqueux de Neopetrosia exigua contre B. cereus(diamètre d’inhibition = 25 mm and CMI = 0.07 mg/mL), S. aureus (17.5 mm and 0.12 mg/mL)et C. albicans (21 mm and 0.32 mg/mL). Cet extrait est actuellement en cours d’analyse pouridentifier ses principes actifs.# 2010 Elsevier Masson SAS. Tous droits réservés.

316 H. Qaralleh et al.

Introduction

The development of safe and effective antimicrobial drugshas revolutionized medicine in the past 70 years [9]. How-ever, the widespread use of antibiotic has promoted theemergence of antibiotic-resistant pathogens [22]. The devel-opment of drug resistance in human pathogens against com-monly used antibiotics has necessitated a search for newantimicrobial substances from other sources including nat-ural sources from any terrestrial or marine source. Marineorganisms are known to produce different compounds toprotect themselves against the variety of their own patho-gens and therefore can be considered as a potential source ofmany classes of antimicrobial substances [2]. More than15,000 natural products, used for different purpose, havebeen mainly isolated from marine sponges, jellyfish, seaanemones, corals, bryozoans, molluscs, echinoderms, tuni-cates and crustaceans [2,21,30].

Among the marine organisms, sponges, which are themost primitive invertebrates, are considered as the majorrich phyla with novel bioactive compounds [2]. Mayer and hiscolleagues [16,17] reported that more than 90 compoundswere isolated from sponges in the period from 2003 to 2006,representing 36% of the total isolated compounds reported inthese studies. The compounds exhibit a wide range of activ-ities including antibacterial, antifungal [8], antiviral [29],and antitumor [19]. The isolated compounds also show a widevariation in chemical structure such as terpens, peptides,alkaloids and fatty acids [16].

In this report, we describe the screening of aqueousand organic extracts of four species of sponges collectedfrom the coastline of Malaysia for antibacterial and anti-fungal activities against six human pathogenic microorgan-isms.

Table 1 Sponge species investigated in this study and yield of eEponges testees et rendement d’extraction.

Voucher specimennumber

Sponge species Depth(m)

Pc

1L82008 Neopetrosia exigua 1—2 L7S82008 Xestospongia testudinaria 5—10 L8S82008 Xestospongia testudinaria 5—10 S9S82008 Xestospongia spp 5—10 S

Yield (%) = (weight of crude/weight of dried sponge tissue) � 100%.

Materials and methods

Sampling and identification

Four species of sponges were collected using SCUBA divingfrom Langkawi (6812047.0100N 99844039.3200E) and South ChinaSea, Snake Island (4803016.7800 N 103824029.3300E) in 2008 atdepths of 1—10m. Each sample was cut into small pieces andthen immediately frozen and maintained at �20 8C. prior toextraction. Information on the organism, the place of col-lection, date of collection and depth were recorded. TheVoucher of the specimen was deposited in the department ofbiomedical science, IIUM, Malaysia, to facilitate the subse-quent taxonomic identification.

Identification of sponges sample was carried out by Mr.Lim Swee-Cheng (Tropical marine science institute, Nationaluniversity of Singapore, Singapore) based on skeletal slidesand dissociated spicule mounts. The species investigated inthis study are listed in Table 1.

Extraction

The frozen samples were freeze dried, macerated with dryice (CO2) by blender and soaked overnight in distilled water.The supernatants were then removed, centrifuged at10,000 rpm, filtered and collected. The collected waterextracts were freeze-dried (aqueous extract). The insolublesolid materials were then successively extracted with metha-nol—dichloromethane (1:1 v/v), followed by methanol(100%) for 24 h each. The organic extracts were combinedand the solvent removed by rotary evaporation at no morethan 40 8C to avoid degradation of compounds. All crudeextracts were kept in a deep freezer until used [5,26].

xtraction.

lace ofollection

Date ofcollection

Aqueousextractionyield (%)

Organicextractionyield (%)

angkawi 24/8/2008 6.14 0.52angkawi 24/8/2008 5.14 0.21outh China sea 5/9/2008 5.48 0.16outh China sea 5/9/2008 7.44 0.24

Antifungal and Antibacterial Activities of Four Malaysian Sponge Species (Petrosiidae) 317

Samples preparation

A sample of 100 mg from each aqueous extract was dissolvedin 1 mL deionized water while the organic extraction wasprepared by Methanol-deionized water (4:1). The extractwas then clarified by filtration through sterile syringe filterwith 0.2—0.45 mm pore. Finally the filtered extract wasstored as aliquots until it was used.

Microbial strains

Six reference strains of human pathogens were used in thisstudy including two Gram-positive (Staphylococcus aureusATCC25923, Bacillus cereus ATCC11778), two Gram-negative(Pseudomonas aeruginosa ATCC27853, Escherichia coliATCC35218) and two fungal strains (Candida albicansATCC10231 and Cryptococcus neoformans ATCC90112).

Antimicrobial assay

Disc diffusion methodThe agar disc diffusion method was employed for the deter-mination of antimicrobial activities of the extracts. Briefly,inoculums containing 107 CFU/mL was spread on Mueller-Hinton agar plates for bacteria and 104 CFU/mL was spreadon potato dextrose agar for fungus strains. Using sterileforceps, the sterile filter papers (6 mm diameter) containingthe crude extracts (2.5 mg), standard antibiotics (30 mg ofchloramphenicol or 100 mg of amphotericin B) or negativecontrol (Deionized water or methanol: water 4:1) were laiddown on the surface of inoculated agar plate. The plateswere incubated at 37 8C. for 24 h for the bacteria and at roomtemperature (18—20 8C.) for 24—48 h for yeasts strains. Eachsample was tested in triplicate and the zone of inhibition wasmeasured as millimeter diameter.

Microdilution methodMinimum inhibitory concentration (MIC) was performed aspreviously described [13] with some modification. MIC was

Table 2 Antimicrobial activity of different marine sponge extraActivite antimicrobienne des different extraits d’eponge.

Species Extract Zone of inhibition (mm

S. aureus B. cereus

N. exigua Aqueous 17.5 25Organic 15 14.5

X. testudinaria (7S82008) Aqueous 0 0Organic 11.5 12

X. testudinaria (8S82008) Aqueous 0 0Organic 11 10.5

Xestospongia spp Aqueous 0 0Organic 9.5 9.5

Chloramphenicola (30 mg) 24.5 26Amphotericin Ba (100 mg) — —Negative controlb 0 0

0: no inhibition zone; —: not determined.a Reference antibiotic.b Deionized water or methanol-deionized water (4:1).

measured by determining the smallest amount of extract orstandard antibiotic needed to inhibit the growth of a testmicroorganism. This was done using 96-well plates andperformed on Versa MaxTM Tunable microplate reader. Theassay plates were filled with Mueller-Hinton broth medium(MHB) containing different concentrations of Neopetrosiaexigua extracts, Chloramphenicol or solvent control and thetest microorganism (107CFU/mL). After 24 h incubation per-iods at 37 8C, the turbidity in each well was measured at600 nm.

MIC for fungal strains was performed using 96-well plate.Each well contained potato dextrose broth, different con-centration of N. exigua extracts, Amphotericin B or solventcontrol and the test yeast strains (104 CFU/mL). Incubationwas performed at room temperature (18—20 8C.) for 48 h.The yeast growth was measured at 494 nm using Versa MaxTM

Tunable microplate reader.

Results

Table 1 shows the results of extracts yield of tested sponges.In general, the yields of aqueous crude extracts were higherthan organic crude extracts. The ranges of percentage dryweights isolated from aqueous extracts were between 5.14and 7.4% and between 0.16 and 0.52% using organic solvents.

The antibacterial and antifungal activity of four spongesextracts are summarized in Table 2. Generally, all of thesponges extracts tested showed antimicrobial activityagainst at least one of the tested strains.

Particularly, the results showed that the organic extractsof all species, except N. exigua, showed higher antimicrobialactivities than water extracts. As shown in Table 2, inaqueous extracts antibacterial activity was found in onlyone species namely N. exigua and the inhibition zones were25 mm against B. cereus and 17.5 mm against S. aureuswhich were larger than those from the organic extracts(15 and 14.5 mm, respectively). All aqueous extracts forthe three species of Xestospongia have no antimicrobialactivity, while the organic extracts are active against some

cts.

)

E. coli P. aeruginosa C. albicans C. neoformans

8 0 21 13.59 0 16.5 80 0 0 09 0 0 00 0 0 00 0 0 07.5 0 0 07.5 0 0 037 33 — —— — 20 190 0 0 0

Table 3 MIC of N. exigua extracts and standard antibiotics.CMI des extraits de N. exigua et des temoins antibiotiques.

Microorganism N. exigua (mg/mL) Chloramphenicol (mg/mL) Amphotericin B (mg/mL)

Aqueous Organic

S. aureus 0.12 0.37 0.02 —B. cereus 0.07 0.12 0.02 —C. albicans 0.37 1.10 — 0.40C. neoformans 3.30 — — 0.30

—: not determined.

318 H. Qaralleh et al.

tested bacteria. The three extracts showed similar levels ofactivity and the inhibition zone values ranged from 7.5 to12 mm. Furthermore, in vitro antifungal activity was foundin only one sponge species. N. exigua generally exhibitedhigher activity against C. albicans than C. neoformans. It isclear that the tested species showed higher activity againstGram-positive bacteria than the yeasts species.

MIC of Chloramphenicol, Amphotericin B and N. exiguaextracts which showed maximum antimicrobial activity isdepicted in Table 3. The results of inhibition zones werereflected in lower MICs. The inhibition of microbial growth atconcentrations as low as 0.07 mg/mL indicated the potentantimicrobial activity of N. exigua extracts.

On the other hand, P. aeruginosa was considered resistantto all tested samples, since no inhibition zone was observedwhile the Gram-positive B. cereus was shown to be the mostsensitive microorganism followed by C. albicans and S. aur-eus.

Discussion

Marine sponges are a highly biodiverse group and the phylo-genetic age of these animals is more than 600 million years[20]. These organisms were successful during evolution andable to avoid extinction. The secret behind this might be thefact that sponges, as sessile filter feeders, do not suffer fromnutrient shortage and have strong defense system to defeatforeign invaders. Sponges have developed an amazinglyefficient immune system, which is reminiscent of that foundin invertebrates [24]. In addition, sponges seem to havestrategies to produce secondary metabolites to protectthemselves against foreign organisms [1,25].

On the other hand, infectious diseases represent a seriouspublic health problem and they remain the major cause ofdeath throughout the world [34]. Currently, there is a pro-blem of microbial drug resistance and an increase of oppor-tunistic infections due to a growth in immunocompromisedpopulation such as organ transplant recipients and HIVpatients. These problems coupled with the toxicity effectof continued use of several antifungal drugs [11] highlightsthe need to search for new drugs to treat opportunisticfungal infections [8].

The wide range of variation in antimicrobial activityshown by the sponge species might be reflecting the differ-ences in the chemical concentration and composition amongthe species. In this report, an efficient extraction procedurewas performed to extract components of the whole sampleusing a combination of water and organic solvents [12], since

water is able to extract the hydrophilic compounds whileorganic solvents are generally able to extract multivariabletypes of hydrophobic compounds; polar and non-polar. In thepresent study, although the majority of the active compo-nents were extracted by water, indicating that most of thecompounds present in sponge species are with hydrophilicproperties (polar compounds), aqueous extract of all testedspecies displayed no antimicrobial activity except N. exiguawhich showed strong antimicrobial activity in both aqueousand organic extracts. Touati and his colleagues [33] whoexamined seven sponges and found that aqueous extractsfrom the seven sponges showed very weak antibacterialactivity compared to the ethyl acetate extracts. El-Amraouiand his co-authors [7] found that the ethanol fraction ofhydroalcoholic extracts from Cliona and Haplosclerida genushave no antimicrobial activity, while the dichloromethanefractions are active against all bacteria tested.

N. exigua gave the highest activities detected againstboth Gram-positive bacteria and yeasts, proposing it as avaluable source of novel substances for future drug discov-ery. When the MIC values of N. exigua extracts were com-pared with chloramphenicol and amphotericin B, it wasfound all tested strains are more susceptible to standardantibiotics than to the extracts. The only exception was theMIC value of aqueous extract against C. albicans (0.37 mg/mL) which was very close to the MIC value of amphotericin B(0.40 mg/mL). Rifai et al. found that the activity of theHippospongia communis extracts were less active thanamphotericin B. Another study of Rifai et al. [27,28] showedthe MIC of (�)-untenospongin B, which isolated from H.communis, has the same activity as amphotericin B in inhi-biting the growth of C. albicans.

These may lead us to conclude that the presence of theimpurities in the extracts might have an influence on theantimicrobial activity and the pure active metabolite couldbe stronger. In the case of the aqueous N. exigua extractagainst C. albicans, this could probably be due to the pre-sence of high concentrations of active antifungal compoundsin N. exigua. A synergistic effect arising from several com-pounds could be also account for the high activities that areobserved.

Many compounds have been isolated from N. exigua withdifferent biological activities. Exiguaquinol, hydroquinonethat inhibited Helicobacter pylori glutamate racemase, hasbeen isolated from a species collected from Australia [14].Renieramycin J, tetrahydroisoquinoline alkaloid as a potentcytotoxin, has been isolated from this species collected fromJapan [23], alkaloid araguspongine M, has been isolated fromthis species collected from Japan [15], neopetrosiamdes

Antifungal and Antibacterial Activities of Four Malaysian Sponge Species (Petrosiidae) 319

A and B, tricyclic peptides that inhibit amoeboid invasion ofhuman tumor cells, has been isolated from this speciescollected from Guinea [6] and Njaoamines G and H, poly-cyclic alkaloids as a potent brine shrimp toxins, were isolatedfrom a species collected from Tanzania [31].

The organic extracts from Xestospongia species in thisstudy showed moderate activity against Gram-positivebacteria. The sponges from the genus Xestospongia areamong the most widely studied in terms of their naturalproducts [10]. A number of different compounds have beenisolated and characterized, some of them being antimi-crobial such as bromopolyacetylenes which has been iso-lated from X. testudinaria [3]. Calcul et al. [4] isolatedeight compounds from an unidentified species from thegenus Xestospongia and they found that aaptamine 1 andisoaaptamine 2 exhibited antifungal activity towardsC. tropicalis.

In this present investigation, Gram-positive bacteria aremore sensitive than Gram-negative bacteria and yeasts. Thisis in agreement with many antimicrobial screening studies.McCaffrey and Endean [18] reported that Gram-positivebacteria were particularly sensitive to sponge extracts whileGram-negative bacteria were not sensitive. Tadesse et al.[32] showed that 29% of the total number of extracts testedshowed strong activity against Gram-positive bacteria,whereas 13% of the extracts tested were active againstGram-negative and 5% were active against fungi.

Conclusion

The species of sponge collected from Malaysia have beenshown to possess antimicrobial activity. Among the speciesinvestigated, N. exigua was the most promising speciestested, suggesting the possibility of finding potent antibac-terial and antifungal agents from aqueous and organic N.exigua extracts. These extracts are being submitted tobioassay-guided fractionations in order to isolate the com-pounds responsible for the activity.

Conflict of interest statement

There is no conflict of interest.

Acknowledgments

This work was supported by a grant from Research Manage-ment Center, International Islamic University Malaysia,IIUM (EDW B 0903-235 and EDW A 09-390). The authorswould like to thank the staff of INOCEM (Institute ofOceanography and Maritime Studies, IIUM) for their hospi-tality and kind assistance in field collection. Acknowledge-ments to Mr. Lim Swee Cheng (Tropical Marine scienceinstitute, National university of Singapore, Singapore) forassistance during sponge identification.

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