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International Biodeterioration & Biodegradation 98 (2015) 53e58

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Inhibition of bacterial fouling by soft coral natural products

Sergey Dobretsov a, *, Aisha S.M. Al-Wahaibi a, b, Daowan Lai c, d, Jamal Al-Sabahi e,Michel Claereboudt a, Peter Proksch c, Bassam Soussi f, g

a Department of Marine Science and Fisheries, College of Agricultural and Marine Sciences, Sultan Qaboos University, Omanb Center of Excellence in Marine Biotechnology, Sultan Qaboos University, Omanc Institut für Pharmazeutische Biologie und Biotechnologie, Düsseldorf, Germanyd MOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University,Beijing 100193, PR Chinae College of Agricultural and Marine Sciences, Sultan Qaboos University, Omanf UNESCO Chair of Marine Biotechnology, Sultan Qaboos University, Omang Institute for Clinical Sciences, Sahlgren's Academy University of Gothenburg, Sweden

a r t i c l e i n f o

Article history:Received 2 July 2014Received in revised form31 October 2014Accepted 31 October 2014Available online

Keywords:Cnidarian secondary metabolitesOman SeaAntimicrobial activityMarine bacteriaBiofoulingFatty acid esterCladiella

* Corresponding author. Tel.: þ968 2414 3657.E-mail address: sergey@squ.edu.om (S. Dobretsov

http://dx.doi.org/10.1016/j.ibiod.2014.10.0190964-8305/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Six common soft coral species (Sarcophyton sp., Sinularia sp.1 and sp.2, Cladiella sp., Scleronephthya sp.and Dendronephthya sp.) from Bandar Al-Khayran (Sultanate of Oman) had significantly lower bacterialdensity in comparisonwith surfaces of empty shells. Methanol: chloroform (1:1) extracts of these specieswere tested against Gram positive (Micrococcus luteus, Staphlococcus aureus, Bacillus subtilis, Bacillus sp.)and Gram negative (Salmonella enterica, Pseudomonas aeruginosa, Escherichia coli, Cytophaga sp., Pseu-domonas sp., Shewanella sp.) marine biofouling and pathogenic bacterial strains. All tested extracts hadsome activity against human pathogens and the highest antimicrobial activity was observed for extractsof Sinularia sp.1 and Cladiella sp. (inhibited 50% and 60% of the strains, respectively). Only ethyl acetateextracts of Cladiella sp. inhibited growth of biofouling bacteria. The active fraction was purified andidentified as a mixture of hexadecyl palmitate and hexadecyl stearate. Pure hexadecyl palmitateinhibited growth (Bacillus sp. and Psychrobacter sp.) and attachment (Bacillus sp., Cytophaga sp., Pseu-domonas sp., Psychrobacter sp., Shewanella sp.) of the marine biofouling bacteria. The results of this studysuggest that soft corals have developed mechanisms to combat microbial infections and inhibit bacterialfouling.

© 2014 Elsevier Ltd. All rights reserved.

Introduction

Any natural or artificial substrata submerged into the marineenvironment are quickly colonized bymicro- andmacro-organismsin a process known as “biofouling” (Clare, 1996). Biofouling causesserious problems for marine industries and navies around theworld (Yebra et al., 2004). Usually biofouling is prevented bybiocidal antifouling and fouling release coatings (Rittschof, 2000,2001). Modern antifouling biocides (Sea-Nine, Irgarol, iso-thiazolone and copper) are harmful to marine organisms (Karlsonet al., 2010). These biocides are not effective against some marinemicrobes and their biofilms (Molino et al., 2009; Dobretsov andThomason, 2011), which increase drag, fuel consumption and

).

affect the performance of antifouling coatings (Yebra et al., 2006).Moreover, micro-fouling on the surface of reverse osmosis mem-branes of desalination plants cannot be prevented by toxic biocidesor chlorination. Thus, there is a need for the development of“environmentally friendly” non-toxic antifouling protection effec-tive against marine microbes.

Marine organisms, especially sessile ones, are faced with theproblem of biofouling. They evolved chemical and physical de-fences to combat marine biofouling (Wahl, 1989; Rittschof, 2000,2001). Thus, natural products from marine organisms can bepotentially used for antifouling defence (Clare, 1996). Soft bodiedsessile organisms, such as soft corals (Alcyonacea), are of particularinterest as they have higher chemical diversity of natural productscompared to other marine organisms (Gerhart et al., 1990; Paul andRitson-Williams, 2008). Soft corals have been shown as an affluentresource of terpenoids, eicosanoids, steroids and cembranoids thatare responsible for a range of biological activities that include

S. Dobretsov et al. / International Biodeterioration & Biodegradation 98 (2015) 53e5854

anticancer (Shen et al., 2008; Lin et al., 2010; Xio et al., 2013),antimicrobial (Gerhart et al., 1990; Aceret et al., 1998; Vanisree andSubbaraju, 2002; Kumar and Lakshmi, 2006; Hunt et al., 2012),antiviral (Cheng et al., 2010; Mayer et al., 2013), and anti-inflammatory activities (Cheng et al., 2009; Tai et al., 2013).

Most studies on soft corals metabolites are based on isolation ofnovel compounds (see reviews: McClintock and Baker, 2001; Paulet al., 2011) and only limited studies have demonstrated that ex-tracts of soft corals and their symbionts are able to inhibit growth ofenvironmental microbes (Slattery et al., 1997; Aceret et al., 1998;Kelman et al., 1998; Bhosale et al., 2002; Harder et al., 2003; Huntet al., 2012). Only few anti-microfouling compounds from softcorals have been isolated and characterized so far (see reviews:Gerhart et al., 1990; Fusetani, 2003; Qian et al., 2010). Awaterbornecompound homarine produced by Antarctic soft corals Alcyoniumpaessleri and Gersemia antarctica inhibited growth of biofoulingbacteria (Slattery et al., 2001). The soft coral Juncella juncea pro-duced juncellin 1 and 2 which inhibited growth of bacteria asso-ciated with the barnacle Balanus amphitrite (Avelin Mary et al.,1993). Desoxyhavannahine isolated from the soft coral Xenia mac-rospiculata inhibited growth of environmental bacteria with MIC1.25 mg ml�1 (Kelman et al., 1998).

This study was designed to investigate the antimicrobial activityof soft corals in Oman waters as a possible source of bacterialfouling inhibitors. The main aims of this study were: (1) to inves-tigate the presence of microorganisms on the surface of variousoctocorallia from Bandar al-Khayran, Oman; (2) to quantify theantimicrobial activity of extracts of octocorals against bacterialpathogens and fouling bacteria; and (3) to identify the substancesresponsible for the observed activity.

Material and methods

Soft corals collection

Six abundant soft coral (Octocorallia: Alcyonacea) species werecollected by SCUBA divers from 10 to 15 m at Bandar Al-Khayranarea (Muscat, Sultanate of Oman) (N 23.5227�, E 58.7475�). Thesoft corals were immediately placed into separate plastic bags withseawater and kept on ice until they were processed in the labora-tory. A voucher sample for each soft coral was taken and kept inethanol for taxonomic identification. Another sample was used forscanning electron microscopy and preparation of soft coral extract(see below). The soft coral specimens were identified by Mr. KavehSamimi-Namin (Nationaal Natuurhistorisch Museum, Leiden,Netherlands).

Scanning electron microscopy

Small pieces of each soft coral (n ¼ 5) and empty shells (n ¼ 5)from Bandar Al-Khayran were fixed in 5% buffered formalin anddehydrated in an increasing ethanol series, dried by the criticalpoint procedure, and sputtered with gold (for details see Dobretsovand Qian, 2002). The specimens were examined by a JEOL 6300F(70 eV) scanning electron microscope (SEM) at magnifications of1000x and 5000x. Bacteria were counted in 10 selected fields ofview (8000 mm2) per replicate.

Preparation of soft coral extracts

The wet weight of the coral and its volume were measured.Then, the soft coral whole tissues were chopped into small piecesand soaked in 1:1 methanol: chloroform solutions at þ25 �C. Afterone week, extracts were filtered through the filter (Whatman No1)and the filtrates were collected. The same extraction and filtration

steps were repeated three times and the resulting crude extracts foreach species were combined. These extracts were dried underreduced pressure by a rotary evaporator (Büchi, Switzerland) andweighed on an analytical balance to the nearest 0.001 g. Driedextracts were re-dissolved in 1:1 methanol: chloroform and kept inthe fridge at þ4 �C until used for separation and bioassays.

Antimicrobial bioassays

Two types of bioassays were performed. First, we tested theability of the extracts to inhibit growth of pathogenic andbiofouling bacteria. Second, we investigated inhibition of attach-ment of biofouling bacteria by purified compounds. All extractswere tested at the tissue level concentrations.

A disc diffusion assay was performed on several Gram positive(Micrococcus luteus, Staphlococcus aureus, Bacillus subtilis, Bacillussp.) and Gram negative (Salmonella enterica, Pseudomonas aerugi-nosa, Escherichia coli, Cytophaga sp., Pseudomonas sp., Shewanellasp.) marine biofouling and pathogenic bacterial strains according toDobretsov and Qian (2002). Pathogenic bacteria were obtainedfrom the culture collection at Sultan Qaboos University hospital.Biofouling bacteria (Bacillus sp., Cytophaga sp., Pseudomonas sp.,Shewanella sp.) were isolated from biofilms covering stones in thearea of the investigation. Before the bioassay, each bacterium wascultivated in marine broth (Oxoid, USA) for 48 h at þ25� C (forbiofouling bacteria) or atþ37� C (for pathogens). Sterile paper discs(diameter ¼ 5.5 mm and surface area 1 cm2) made of WhatmanNo1 paper with extracts, fractions or pure compounds were used inthe bioassay. Two ml of extracts or pure compounds were applied todisks and solvent were evaporated prior the bioassay. The standardantibiotic streptomycin (SigmaeAldrich) was used as a positivecontrol at a concentration of 1 g L�1. 1:1 methanol: chloroformwasused as a negative control at a concentration of 10 ml disk�1. After48 h, the diameter of the inhibition zone around the paper diskswas measured with a ruler to the nearest 0.5 mm.

Purified compounds, hexadecanoic and octadecanoic acid (SC6-FA), hexadecanol (SC6Al) and hexadecyl palmitate (SigmaeAldrich)(see below)were used in the disk diffusion experiments (see above)and in the bacterial attachment bioassays. The experiments wereperformed with biofouling bacteria Bacillus sp., Psychrobacter sp.,Cytophaga sp., Pseudomonas sp., Psychrobacter sp. and Shewanellasp. Bacterial attachment assays were performed in 96-well plates.Firstly, bacteriawere cultured inmarine broth until the exponentialphase of growth. Series of dilutions of the compounds were madeand the concentrations ranging from 3 � 10�5 to 10 mg ml�1 weretested. In each well 100 ml of bacterial culture was inoculated at anoptical density of 0.01 at 600 nm (OD600). After 2 h of incubation at25 �C, the wells were emptied and washed with distilled water.Bound cells were stained with 0.2% (wt/vol) crystal violet solutionin ethanol at room temperature for 10 min. The wells were thenwashed with water three times, and dried at room temperature.The dye was solubilized with 95% ethanol. Alteration between thecontrol and the treatment colour indicated differences in attach-ment of tested bacteria. Minimum inhibitory concentrations (MIC)were determined. The experiments were repeated 3 times.

Fractionation of soft coral extracts

Crude soft coral extracts were partitioned by liquideliquidextraction using a separating funnel (Ebada et al., 2008). Differenceswere based on the properties of the soft corals extracts and theirfraction solubility. Crude extract of Sarcophyton sp. was subsequentlypartitioned using hexanes, chloroform, butanol and water. Extract ofSinularia sp.1 was subsequently partitioned between ethyl acetate,butanol, and water. Extract of Sinularia sp.2 was subsequently

Soft coralscontrol SC1 SC2 SC3 SC4 SC5 SC6

01slleclairetcabfo

ytisneD

3 m

m-2

0

5

10

15

20

25

30

35

**

** *

*

Fig. 1. Densities (cells mm�2) of bacteria on shells (control) and on the surfaces ofinvestigated soft corals. Bacteria were counted by a scanning electronic microscope(SEM) at 5,000x magnification. Data plotted are means (n ¼ 5) ± SE. Densities statis-tically different from the control ones according to the Dunnett test (ANOVA: P < 0.05)are indicated by asterisks. Soft corals: SC1 e Sarcophyton sp., SC2 e Sinularia sp.1, SC3 e

Sinularia sp.2, SC4 e Scleronephthya sp., SC5 e Dendronepthya sp., SC6 e Cladiella sp.

S. Dobretsov et al. / International Biodeterioration & Biodegradation 98 (2015) 53e58 55

partitioned using hexanes, butanol and water. Extracts of Scle-ronephthya sp. and Dendronepthya sp. were separated using chloro-form and water. Extract of Cladiella sp. was subsequently partitionedbetween ethyl acetate and water. Here and later unless specified allanalytical grade solvents were from Fisher Scientific (USA). Individ-ual fractions were collected and evaporated till dry using the rotaryevaporator (Büchi, Switzerland). Their bioactivity was tested usingthe disk diffusion test (see above).

Isolation and identification of antibacterial compound

In order to isolate compound that inhibits bacterial fouling, ethylacetate fraction of the soft coral Cladiella sp. (SC6EtOAc) was purifiedby normal phase column chromatography (Ebada et al., 2008). Thecolumn was packed with silica gel 60 (SigmaeAldrich) dissolved inhexanes. The column was eluted gradually with 100% hexanes thenwith 95:5, 90:10, 80:20, 70:30, and 60:40 hexanes: chloroform (vol:vol) mixtures. Finally, the columnwaswashedwith 100% chloroformand with 15: 85 methanol: chloroform. Corresponding fractionswere collected. Thin layer chromatography (TLC) was employedthroughout the purification process to assist the process of separa-tion. Fractions having similar compounds were then combined. Theresulting fractions were evaporated by the rotary evaporator andtheir bioactivity was tested using the disk diffusion assay. The activefraction was purified using normal phase column chromatographyand the column and conditions were similar to the first purificationstep. All fractions were collected, evaporated and their bioactivitywas tested using the disk diffusion test. Purity of the bioactive frac-tion was determined by thin layer chromatography (TLC) andconfirmed by gas chromatography mass spectroscopy (GCeMS) and1H and 13C nuclear magnetic resonance (NMR). In order to identifythe mixture of fatty acid esters we hydrolyzed it. First, about 5 mg ofthe compound were dissolved in 1 ml of ethanol. To this solution2ml of 10% KOH solution in 90% aqueous ethanol was added. Second,this mixture was kept for 6.5 h in a water bath at 70� C. The solventwas evaporated completely and the residuewas partitioned betweenethyl acetate: hexanes (5:95) and distilled water. Finally, the upperlayer containing the alcohol was collected and evaporated (SC6-AL).The lower layer contained the potassium salt of the acid. It wasacidified (pH ¼ 3.5) with HCl. This solution was extracted with ethylacetate in order to get the free fatty acid (SC6-FA). The structures ofthe fatty acids and the alcohol were elucidated by GCeMS. Beforethis the fatty acids were derivatized which involved methylation bytrimethylsulfonium hydroxide (TMSH) (SigmaeAldrich). For this1 mg of SC6-FA was dissolved in 200 ml of dichloromethane. Then,15 ml of TMSHwas added to 50 ml of this solution (SC6-FA-TMSH). Asthe control, 1 mg of palmitic acid (C16H32O2, SigmaeAldrich) wasdissolved in 200 ml of dichloromethane and this solution was mixedwith TMSH as described previously. Derivatized fatty acids weresequentially injected into GCeMS. The GC conditions were asfollowing. The split ratio was 1:50. The column used was an OPTIMAHP-5 column (crosslinked 5% PH ME siloxane) 25 m in length,0.25 mm in diameter, film 0.25 mm (MachereyeNagel). The carriergas was nitrogen (generator: UHPNO751, Domnick Hunter) with aflow rate of 1.3mlmin�1. The temperature of the injector was 300 �C.The temperature program during each runwas as follows: starting at180 �C with a slope of 10 �C/min until 270 �C, and then for 15 min at270 �C. The final structure of the compound was elucidated byanalysis ofmolecular weight of alcohol and fatty acids fragments andresults of NMR.

Statistical analysis

The effect of soft coral extracts on growth of bacteria wasinvestigated using non-parametrical statistical tests:

PERMANOVAþ (Anderson, 2001). PERMANOVA (PermutationANOVA) analyzes univariate or multivariate data in response tofactors, groups or treatments in an experimental design analysis. Itis a form of analysis of variance in which statistical significance istested against a large number of artificial data sets obtained byrandom permutations of the original data. Its advantage is theabsence of normality and homoscedasticity assumption of the dataset. All calculations were performed with the help of the statisticalpackage PRIMER-E PERMANOVAþ (Plymouth Marine Laboratory,UK). The densities of bacteria and diatoms on the surface of coralsand shells were analysed by ANOVA followed multiple pair-wisecomparison versus a control (Dunnett's test). In all cases thethreshold level for significance was 5%.

Results

Coral species and bacterial densities

Six species of soft corals (Octocorallia: Alcyonacea; Sarcophytonsp., Sinularia sp.1 and Sinularia sp.2, Cladiella sp., Scleronephthya sp.and Dendronephthya sp.) all belonging to the families Alcyoniidaeor Nephtheidae were collected from Bandar al-Khayran. The colourof Sinularia sp.1 was lighter than the one of Sinularia sp.2.

Scanning electron microphotographs showed the presence ofonly few attached bacteria on the soft coral surfaces. Surfaces ofshells (control) were heavily colonized by bacteria and diatoms. Thehighest bacterial abundance was observed on the control surfaces,while the lowest abundance was found on Cladiella sp. surface(Fig. 1). Other soft corals had intermediate densities of bacteria ontheir surfaces. Densities of bacteria on the soft coral surfaces weresignificantly lower (ANOVA, Dunnett, p < 0.05) in comparison withthe control (Fig. 1).

Antimicrobial activity

Only crude extracts of the soft coral Cladiella sp. inhibitedgrowth of the marine biofouling strain Bacillus sp. (Table 1). Ex-tracts of five out of six octocoral species inhibited growth of thepathogen M. luteus. The largest inhibition zones were observed for

Table 1The antibacterial effect of the crude soft corals extracts on marine biofouling and pathogenic bacteria. All extracts were tested at the tissue level concentrations. 1:1 methanol:chloroform was used as a negative control at a concentration of 10 ml disk�1. Streptomycin was used at a concentration of 1 mg ml�1 as a positive control. Data are the meandiameters (n ¼ 3) ± SD of inhibition zones.

Tested strains Soft corals & controls

Sarcophyton sp. Sinularia sp.1 Sinularia sp.2 Scleronephthya sp. Dendronephthya sp. Cladiella sp. Control Streptomycin

Biofouling bacteriaBacillus sp. 0 0 0 0 0 7.2 ± 1.5 0 0Cytophaga sp. 0 0 0 0 0 0 0 0Pseudomonas sp. 0 0 0 0 0 0 0 0Shewanella sp. 0 0 0 0 0 0 0 0

PathogensMicrococcus luteus 9.4 ± 3.3 6.9 ± 0.9 9.9 ± 2.7 0 6.2 ± 1.2 6.6 ± 1.2 0 32.1 ± 0.6Staphylococcus aureus 12.9 ± 0.3 9.9 ± 0.3 6.9 ± 2.1 0 0 12.6 ± 2.4 0 13.5 ± 1.2Bacillus subtilis 10.2 ± 2.4 15.0 ± 2.1 0 0 0 9.0 ± 1.2 0 27.3 ± 2.1Salmonella enterica 0 6.3 ± 1.8 15.3 ± 2.7 16.5 ± 4.8 6.6 ± 1.5 12.0 ± 3.6 0 12.3 ± 1.2Pseudomonas aeruginosa 0 0 0 0 0 9.6 ± 3.6 0 18.0 ± 2.4Escherichia coli 0 11.1 ± 3.6 0 27.6 ± 2.1 0 0 0 0

S. Dobretsov et al. / International Biodeterioration & Biodegradation 98 (2015) 53e5856

the antibiotic control and extracts of Sinularia sp. 2. Only extracts ofScleronephthya sp. and Dendronephthya sp. did not inhibit growth ofStaphylococcus aureus (Table 1). Half of the soft coral extractsinhibited growth of B. subtilis with extracts of Sinularia sp.1 beingthe most effective. Extracts of five out of six coral species inhibitedgrowth of the pathogen S. enterica and extracts of Sinularia sp.2performed better than the antibiotic control. Only extracts of Cla-diella sp. inhibited growth of the strain Pseudolateromonas aerugi-nosa. Growth of the antibiotic resistant E. coli was inhibited byextracts of Sinularia sp.1 and Scleronephthya sp. (Table 1). Usually,coral extracts affected Gram positive but not Gram negative bac-teria. Overall, extracts of Cladiella sp. and Sinularia sp.1 were themost active ones against growth of tested bacteria. These specieshad low density of bacteria on their surfaces in comparison withother corals (Fig. 1).

Statistical analysis revealed that extracts from different softcorals had significantly different activity (PERMANOVA, p < 0.001)against bacterial strains (Table 2). Choice of bacterial strainssignificantly (PERMANOVA, p < 0.001) affected the performance ofsoft coral extracts. There was also a significant interaction betweenspecies of tested bacteria and activity of soft coral extracts (Table 2).

Antibacterial compounds

Only the ethyl acetate fraction (SC6EtOAc) but not water fraction(SC6W) from Cladiella sp. showed antibacterial activity (Table 3).Purification of active fraction resulted in the isolation of a mixtureof long chain carboxylic acid esters (Fig. 2, Appendix 1 and 2). Afterhydrolysis of this mixture 1-hexadecanol (SC6-AL) (Appendix 3), aswell as hexadecanoic and octadecanoic acids (SC6-FA) were ob-tained (Appendix 4). The relative amount of hexadecanoic acid washigher than that of octadecanoic acid. Therefore, the antibacterial“compound” (HPHP) comprises a mixture of hexadecyl palmitate(C32H64O2) and hexadecyl stearate (C34H68O2) (Fig. 2A and B). Inthis mixture, hexadecyl palmitate is the dominating constituent.

Table 2Results of PERMANOVA þ analysis of the effect of soft coral extracts on bacterial growth. dvalues, F critical: the critical Pseudo-F value at p ¼ 0.001. Probability values for tested fa

Source Df SS MS

Coral species 5 441.21 88.242Bacterial species 9 609.21 121.84Coral species � Bacterial

species45 1237.8 49.513

Residue 120 356.25 9.8958Total 179 2644.5

Experiments with mixtures of compounds and pure hexadecylpalmitate showed inhibition of growth of the marine biofoulingbacteria Bacillus sp. and Psychrobacter sp. (Table 3). Hexadecylpalmitate (1 mg ml�1) inhibited growth of Bacillus sp. with themean inhibition zone (±SD) 8.0 ± 1.0 mm. The minimum inhibitoryconcentration (MIC) of hexadecyl palmitate (HP) was 1.8 mg ml�1

(Table 3). Hexadecyl palmitate was less effective in inhibition ofPsychrobacter sp. The MIC was 0.1 mg ml�1 and 1 mg ml�1 hex-adecyl palmitate produced the mean inhibition zone 7.1 ± 0.6 mm.Hexadecanoic and octadecanoic acids (SC6-FA) as well as 1-hexadecanol (SC6-AL) did not affect growth of biofouling bacteria(Table 3). Only hexadecyl palmitate and its mixture with hexadecylstearate inhibited attachment of all tested biofouling bacteria(Table 3). The MIC ranged from 0.9 to 23.7 mg ml�1. The highestinhibitory activity was observed for Bacillus sp., while the lowestactivity was found for Shewanella sp. (Table 3).

Discussion

In this study, six species of the soft corals were collected fromBandar Al-Khayran area. These corals were identified as Sarcoph-yton sp., Sinularia sp.1 and sp.2, Scleronephthya sp., Dendronephthyasp., and Cladiella sp. Only Sinularia spp. were reported previouslyfrom Bandar Al- Khayran area (UNEP/IUCN, 1988) and Den-dronephthya sp. were found west of Musandam in the Arabian Gulf(Sheppard et al., 1992). A recent study carried out in Iran demon-strated that soft corals belonging to genera Sarcophyton, Den-dronephthya and Sinularia dominate some coral communities in theGulf (Samimi-Namin and van Ofwegen, 2009).

All crude extracts of the investigated soft corals demonstratedsome antimicrobial activity against pathogens. Extracts of Sinulariasp.1 and Cladiella sp. were among the most active ones in terms ofthe number of inhibited strains. It is interesting to note that Sinu-laria sp.1 and Cladiella sp. had the lowest bacterial abundanceamong the investigated species and extracts of these corals

f: degrees of freedom, SS: sum of squares, MS: mean square, Pseudo-F: the Pseudo-Fctors and their combinations were always significant, p < 0.001.

Pseudo-F F-critical Unique permutations

8.917 4.416 99812.312 3.379 9985.004 2.060 999

Table 3Inhibition of growth and attachment of marine bacteria (Bacillus sp., Cytophaga sp., Pseudomonas sp., Psychrobacter sp., and Shewanella sp.) by compounds and extracts isolatedfrom Cladiella sp. Tested fractions: ethyl acetate (SC6EtOAc) and water (SC6W) fractions. Tested compounds: mixture of hexadecanoic and octadecanoic acids (SC6-FA), 1-hexadecanol (SC6Al), mixture of hexadecyl palmitate and hexadecyl stearate (HPHP), hexadecyl palmitate (HP). Data are the mean MIC (mg ml�1) of 3 repeats ± SD.

Bacterial strains Growth Attachment

SC6EtOAc SC6W SC6-FA SC6-Al HPHP HP SC6EtOAc SC6W SC6-FA SC6-Al HPHP HP

Bacillus sp. 5.5 ± 0.7 0 0 0 3.6 ± 0.4 1.8 ± 0.2 0 0 0 0 1.8 ± 0.3 0.9 ± 0.1Psychrobacter sp. 478 ± 24 0 0 0 190 ± 15 100 ± 55 0 0 0 0 5.3 ± 1.0 3.1 ± 1.0Cytophaga sp. 0 0 0 0 0 0 0 0 0 0 20.7 ± 3.0 10.4 ± 1.5Pseudomonas sp. 0 0 0 0 0 0 0 0 0 0 10.7 ± 2.5 5.2 ± 1.3Shewanella sp. 0 0 0 0 0 0 0 0 0 0 23.7 ± 2.5 11.3 ± 1.5

S. Dobretsov et al. / International Biodeterioration & Biodegradation 98 (2015) 53e58 57

inhibited both Gram-positive and Gram-negative bacterial strains(Table 1). Antimicrobial activity against bacterial pathogens hasbeen reported earlier for octocoral extracts (Slattery et al., 1997;Aceret et al., 1998; Kelman et al., 1998; Vanisree and Subbaraju,2002; Kumar and Lakshmi, 2006; Hunt et al., 2012). For example,Ata et al. (2003) reported that the compounds cladiella peroxide,(6E)-2a,9a-epoxyeunicella-6,11(12)-dien-3b-ol, and polyanthellinA isolated from Cladiella sp. showed antibacterial activity againstStreptococcus pyogenes, E. coli and Pseudomonas aeruginosa. Ourfindings suggest that extracts of soft corals contain some antibac-terial compounds.

Only the extract of Cladiella sp. inhibited growth of thebiofouling bacterium Bacillus sp. The antifouling compound fromthis soft coral was purified and identified as a mixture of hexadecylpalmitate (C32H64O2) and hexadecyl stearate (C34H68O2) (Fig. 2).Pure hexadecyl palmitate inhibited growth of the biofouling bac-teria Bacillus sp. and Psychrobacter sp. and attachment of thebiofouling bacteria Bacillus sp., Cytophaga sp., Pseudomonas sp.,Psychrobacter sp. and Shewanella sp. The MIC concentrations ofhexadecyl palmitate (0.9e100 mg ml�1) were similar to onesobserved in the bioassay with Staphylococcus epidermis and stan-dard antibiotics (Cerca et al., 2005). The estimated natural con-centrations of hexadecyl palmitate in the soft coral Cladiella sp.were 1000-fold higher than the observed MIC. This may suggestthat the soft coral Cladiella sp. produces these compounds in orderto prevent bacterial fouling on its surface.

In comparison with other known inhibitors of bacterial foulingfrom soft corals, hexadecyl palmitate was 694-fold more activethan desoxyhavannahine isolated from X. macrospiculata (Kelmanet al., 1998). Similarly, hexadecyl palmitate was 256-fold moreeffective in inhibition of bacterial attachment in comparison ofethanolic extracts of the asteroid Goniaster tesselatus (Bryan et al.,1996). Antibacterial properties of pure hexadecyl stearate werenot tested in this study. Antimicrobial activities of mixtures of longchain carboxylic acid esters and structural similarities of thiscompound with hexadecyl palmitate allowed us to propose that

A:

B:

Fig. 2. The structure of carboxylic esters A: hexadecyl palmitate (C32H64O2) and B:hexadecyl stearate (C34H68O2).

hexadecyl stearate would have activity against the biofouling bac-teria as well.

To our knowledge this is the first report that long chain car-boxylic acid esters inhibit bacterial fouling (Blunt and Munro,2007). At the same time, antimicrobial activity of fatty acids hasbeen previously reported (Fusetani, 2003; Flemming et al., 2008;Qian et al., 2010). A study by Plouguern�e et al. (2010) demon-strated that twelve non-polar fractions from chloroform extracts ofthe invasive brown alga Sargassum muticum had anti-microfoulingactivity. Antimicrobial compounds were identified as mixtures ofsaturated and unsaturated hydrocarbons, fatty acids (arachidonic,palmitic, linoleic and palmitoleic acids), as well as galactoglycer-olipids. In opposite, our data suggested that neither palmitic andoctadecanoic acids nor 1-hexadecanol inhibited growth andattachment of marine fouling bacteria. Themechanisms of action oflong chain carboxylic acid esters need to be further investigated infuture studies.

Conclusions

Our results suggested that soft corals from Oman waters havepromising antibacterial activities. For the first time it was demon-strated that long chain carboxylic esters from Cladiella sp. inhibitgrowth and attachment of biofouling bacteria. In the industry theuse of natural products as antifoulants is blocked by the cost andlack of sufficient supply (Clare, 1996; Rittschof, 2000). In opposite,long chain carboxylic esters can be easily synthesized (Metzger andBornsheuer, 2006) which can help to use them in the industry.

Acknowledgements

This project was supported by HM Fund for Strategic Research(SR/AGR/FOOD/05/01) and the Sahlgen's Academy, University ofGothenburg to BS, as well as the Sultan Qaboos University internalgrants IG/AGR/FISH/09/03 and IG/AGR/FISH/12/01 to SD. SDacknowledged the Alexander von Humboldt Foundation for spon-soring his research stay in Germany. The authors thank Mr. KavehSamimi-Namin (Nationaal Natuurhistorisch Museum, Leiden,Netherlands) for the help in identification of the soft corals. Adviceof Dr. Sarath P. Gunasekera (Fort Pierce SmithsonianMarine Station,USA) in identification of compounds is acknowledged.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ibiod.2014.10.019.

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