In this study, the extract was tested for in vitro activity against oxytetracycline-resistant V. harveyi. Toxicity of the methanolic extract was evaluated by Brain Shrimp Lethality test. Geodia sp. was characterized by three spicula types (oxeas, trianes and oxyaster euaster), encrusting growth formation, hispid surface features and skeletal structure of paratangential ectosome. The methanolic extract of Geodia sp. exhibited anti-oxytetracycline-resistant V. harveyi activity with MIC of 31.25 μg mL^-1. The extract also was able to inhibit the growth of oxytetracycline-resistant V. harveyi in broth culture at concentrations of 1 and 2xMICs and able to kill almost V. harveyi cells at 4xMIC. Interestingly, the extract did not show any toxic effect in Artemia salina up to 125 μg mL^-1. It is the first report for the antibacterial activity of methanolic extract of Geodia sp. against oxytetracycline-resistant V. harveyi, a pathogenic bacterium in marine aquaculture. This results suggest that Geodia sp. might be used as a source of alternative compound to control marine bacterial pathogen especially oxytetracycline-resistant V. harveyi.

INTRODUCTION

Vibrio harveyi is a significant pathogenic bacterium in marine aquaculture (Austin and Zhang, 2006) and recognized as the main causative agent of luminous vibriosis, which often results in mass mortality in cultured marine animals. The bacterium infects almost all cultured marine animals such as crustacean, mollusk and fish. Crustacean, including shrimp, crab, lobsters and Artemia are very susceptible to this opportunistic pathogenic bacterium (Jivaranichpaisal et al., 1994; Karuna Sagar et al., 1994; Liu et al., 1996; Robertson et al., 1998; Diggles et al., 2000; Soto-Rodriguez et al., 2003; Bourne et al., 2007). V. harveyi is also well known as a bacterial pathogen in almost all cultured marine fish species (Kraxberger-Beatty et al., 1990; Saeed, 1995; Hispano et al., 1997; Company et al., 1999; Zhang and Austin, 2000; Tendencia, 2002; Pujalte et al., 2003a, b; Zorrilla et al., 2003; Liu et al., 2003; Sivaram et al., 2004; Gauger et al., 2006; Oh et al., 2006). This bacterium is also determined as a causative agent for the disease in seahorse Hippocampus sp. (Alcaide et al., 2001; Tendencia, 2004), sea cucumber Holothuria scabra (Becket et al., 2004), abalone Holiotis discus hannai (Sawabe et al., 2007) and stony corals (Luna et al., 2007).

Oxytetracycline that is effective against a broad range of both gram positive and negative bacteria is usually used as feed additive to control a natural infection in aquaculture (Saeed, 1995). The use of this antibiotic causes the development of resistance in Vibrio spesies including V. harveyi. The high incidence of resistance to oxytetracycline has been found in Vibrio in larvae and post-larvae of Macrobrachium rosenbergii (Hameed et al., 2003) and fish intestine (Nonaka et al., 2000). Vibrio sp. isolated from diseased fish are also reported as the bacteria harboring oxytetracycline resistance gene determinant, tet 34 which show high resistant to the antibiotic with MICs 125-500 μg mL^-1 (Nonaka et al., 2002; Kim et al., 2003). Vibrio sp. have been determined as the main reservoir of another oxytetracycline resistance gene, tet(M) (Nonaka et al., 2007).

In particular, V. harveyi has been found to be able to develop the resistance to oxytetracycline with the increase in the MIC up to 250 times (Nakayama et al., 2006). This bacterium has been reported to be multi-antibiotic resistant to almost all available antibiotics (Tjahjadi et al., 1994; Ottaviani et al., 2001; Nakayama et al., 2006). The emergences of antibiotic-resistant bacteria in aquaculture suggest that the development of alternative counter measures to control aquatic bacterial diseases is urgent.

We conducted screening to explore the antibacterial activity from sponge to develop alternative practices for diseases control in aquaculture and found that Geodia sp. has potential antibacterial activity. Geodia sp. have been reported as producers of several bioactive substances (Tinto et al., 1998; Sjögren, 2006; Rangel et al., 2006; Uy et al., 2002, 2003; Ohta et al., 2006; Rangel et al., 2005), but the in vitro activity of Geodia sp. extract against bacterial disease in aquaculture is little known. This study aimed to evaluate in vitro antibacterial activity of Geodia sp. extract against the most important pathogenic bacterium in marine aquaculture, oxytetracycline resistant-V. harveyi, as well as its toxicity.

MATERIALS AND METHODS

V. harveyi strain and medium: V. harveyi was kindly given by Brachishwater Aquaculture Development Center, Jepara, Central Java, Indonesia. The bacterium was cultured in Zobell medium (pH 7.5) {polypepton (Nihon Seiyaku, Japan), 5 g L^-1; yeast extract (Oxoid), 1 g L^-1 dissolved in filtered 75% of 30 ppt seawater}.

Resistance test of V. harveyi to oxytetracycline: Resistance of V. harveyi to oxytetracycline was examined by Minimum Inhibitory Concentration (MIC) test using agar dilution method (Clinical Laboratory Standards Institute, 2006). Zobell agar medium was supplemented with various concentrations of oxytetracycline (Zalmweg Raamsdonksveer, Nentherland) and used for culture of V. harveyi.

Sample of Geodia sp.: Sample of Geodia sp. was collected from intertidal zone of Wediombo coast, Gunungkidul, Yogyakarta, Indonesia in January-September 2005 and May 2006. Identification of the sponge based on spicula by bleaching digestion, skeletal structure by simple clearing method, surface structure and growth formation (Hooper, 2000).

Extraction of Geodia sp.: Geodia sp. sample was washed by freshwater and sliced. Then the sample was extracted with methanol (MeOH) at the ratio sponge and methanol of 1:4 (w/v). The extraction was done by mean of a homogenizer for 15 min and then the sample was centrifuged at 4,500 g for 20 min to obtain the supernatant as MeOH extract. The extraction was carried out twice in the same volume of MeOH. The extract was concentrated by a rotary evaporator at 40°C.

Anti-oxytetracycline resistant V. harveyi activity test and determination of the Minimum Inhibitory Concentration (MIC): Anti-V. harveyi activity of the MeOH extract was evaluated by paper disk diffusion method using double layer agar of Zobell medium as previously described by Horikawa et al. (1999), Miller et al. (2003) and Isnansetyo and Kamei (2005) after 20-fold concentrated. Sterile paper disks (ø 8 mm, Advantec, Tokyo) were impregnated with 50 μL of the MeOH extract and dried at 30°C. ZoBell medium with 0.7% agar kept in a water bath at 48°C was inoculated with an overnight culture of V. harveyi to give an initial bacterial density of 10⁶ cells mL^-1 and overlaid onto Zobell agar medium plate. Before inoculation, the bacterial density in the inoculum was estimated by a spectrophotometer (UV-VIS spectrophotometer, UV-1650PC, Shimadzu) at 625 nm with McFarland standard. The MeOH extract-impregnated paper disks were placed on the plates and incubated at 30°C for 24 h. The Minimum Inhibitory Concentration (MIC) of the MeOH extract was determined by the same method used for the anti-V. harveyi activity test with serial dilution of the MeOH extract concentrations.

Bactericidal assay: The time-kill experiment was conducted by the method described by Aeschlimann and Rybak (1998) and Entenza et al. (1998). The experiment was conducted in 25 mL-Erlenmeyer flasks containing 15 mL fresh Zobell medium inoculated with an overnight V. harveyi to give an initial bacterial density of 10⁶ cells mL^-1. The inoculation was carried out immediately just after addition of the MeOH extract at the final concentrations of 1, 2 and 4xMIC in duplicates. The flasks were further incubated at 30°C agitating with shaker. The bacterial cells density at various incubation times was estimated by a spectrophotometer (UV-VIS spectrophotometer, UV-1650PC, Shimadzu) at 625 nm. Viable bacterial cells were estimated by plating on TCBS agar medium (Oxoid).

Brine shrimp lethality test (BST): Brine Shrimp Lethality Test (BST) was used to evaluate toxicity of MeOH extract of Geodia sp. based on the procedure previously described by Bailey et al. (2005) and Libralato et al. (2007). A conical container was used to hatch the brine shrimp cysts (A Quality Cysts, Great Salt Lake, Inve) in 30 ppt filtered seawater with enough aeration. Test was performed in a multiwell test plate (6x4 wells) with 30 ppt of filtered seawater. Ten Artemia nauplii instar II were exposed with the MeOH extract at the concentrations of 0 (control treatment), 1, 2, 4 and 8xMIC in duplicates. After 24 h exposure, the mortality of Artemia was observed.

RESULTS AND DISCUSSION

The resistance test of V. harveyi to oxytetracycline showed that the bacterium used in this study has high resistance to the antibiotic with MIC 140 μg mL^-1. The resistance of marine Vibrio to oxytetracycline have been reported by Nonaka et al. (2002) and Kim et al. (2003) with MIC 125-500 μg mL^-1. Nakayama et al. (2006) reported that the MIC of oxytetracycline against oxytetracycline-resistant V. harveyi is 250 times higher than the MIC of the same antibiotic against the sensitive one.

Geodia sp. is a sponge characterized by oxeas of monaxonid megascleres and trianes of tetraxonid megascleres of spicula, while its microsclere was oxyaster euaster, with encrusting growth forms, hispid surface features and paratangential ectosome of skeletal structure (Hooper, 2000). The sponge sample was found at intertidal zone.

The extract of Geodia sp. exhibited high anti-oxytetracycline-resistant V. harveyi activity indicated by wide and clear inhibition zone at various concentrations (Fig. 1a, b). Furthermore, MIC of the methanolic extract of Geodia sp. was 31.25 μg mL^-1 indicated that the extract was very potent against oxytetracycline-resistant V. harveyi, the causative agent of luminescence disease. Geodia sp. have been reported as the source of several bioactive substances including an anti cancer, Geodiamolide H (Tinto et al., 1998), exiguamide that inhibits the cell fate specification of sea urchin embryogenesis (Uy et al., 2002, 2003), macrocyclic polyketide lactam tetramic acid, an anti-nematode substance (Yan, 2004), barettin with antifouling activity (Sjögren et al. 2006), exiguolide that inhibits the fertilization of sea urchin gametes (Ohta et al., 2006), crude extract of G. corticostylifera with antibacterial, antifungal, cytotoxic, haemolytic and neurotoxic activities and the cyclic peptide geodiamolides A, B, H and I from this sponge with anti-cancer activity (Rangel et al., 2006). The mouse acute toxicity, neurotoxic and haemolytic activities were also reported from the extract of Geodia sp. (Rangel et al., 2005). The geodiamolide H, a peptide from G. corticostylifera inhibits migration and invasion of Hs578T cells derived from breast cancer through modifications in actin cytoskeleton (Freitas et al., 2008). Encarnacion et al. (2000) described antibacterial activity of ethanol extract from Geodia sp. against a gram positive bacterium, Mycobacterium avium, but did not prove against other bacterial strains including Vibrio.

Fig. 1:	(a, b) Anti-oxytetracycline-resistant V. harveyi activity of the MeOH extract of Geodia sp. A: 4,000 μg disk-1; B: 2,000 μg disk-1; C: 1,000 μg disk-1; D: 500 μg disk-1; E: 250 μg disk-1; F: 125 μg disk-1; G: 62.5 μg disk-1; H: 31.25 μg disk-1; I: 15.63 μg disk-1; J: 7.81 μg disk-1; K: 3.91 μg disk-1; M: Negative control (methanol); C: Oxytetracycline at 10 μg disk-1

The anti-oxytetracycline-resistant V. harveyi activity of Geodia sp. extract has not been reported yet so far. The result of this study suggests that Geodia sp. may have the important ecological role in controlling the outbreak of luminescence disease caused by V. harveyi in marine aquaculture. This finding also shows that Geodia sp. is the potential source of antibacterial substance for the alternative counter measure against marine bacterial diseases, especially V. harveyi.

The use of antibiotic in veterinary and aquaculture contributes the increase in the resistant of pathogenic bacteria to commercial available antibiotics (Alderman and Hasting, 1998; Teuber, 2001). The resistance of V. harveyi to antibiotics might be caused by the use of various antibiotics in Indonesian aquaculture especially in 1980’s. V. harveyi has been reported to be multi-antibiotic resistant to ampicillin, amoxicillin, carbenicillin, cephalothin, colistin sulphate, kanamycin, lincomycin, neomycin, novobiocin, nitrofurantoin, penicillin, polymyxin, rifampicin, streptomycin, sulphamethoxazole, tetracycline and trimethoprim (Tjahjadi et al., 1994; Ottaviani et al., 2001). The resistance development of Vibrio is encoded by R-plasmid which is transferable to others bacterial cells (Aoki, 1992). The oxytetracycline resistance determinants, tet 34 (Nonaka et al., 2002; Kim et al., 2003) and tet M (Nonaka et al., 2007) in Vibrio have been determined. Two genetic determinants of tetracycline resistance in V. harveyi have been also found by Teo et al. (2002). The resistance determinants are easily transferred to other bacterial cells including bacterial pathogen in animal and human, which cause serious impacts in global environment and human health. In addition, another mechanism of resistance to β-lactam antibiotics has been found in V. harveyi harboring β-lactamase genes, blaVHW-1 and blaVHH-1 (Teo et al., 2000).

Bactericidal activity of the methanolic extract of Geodia sp. was tested by time course study. The optical density of V. harveyi treated with the extract at the concentrations of 1, 2 and 4xMICs decreased slightly. In contrast, the optical density of the bacterium in the control treatment increased constantly after 3 h incubation (Fig. 2). This result also showed that the extract has stable activity from the early incubation to 24 h incubation period. The absorbance of bacterial suspension did not increase in the broth medium added with the extract at 1xMIC (31.25 μg mL^-1) for 24 h incubation suggesting the MIC obtained from paper disk diffusion on double layer agar had the same inhibitory effect to V. harveyi in broth culture.

Fig. 2:	Bactericidal activity of the methanolic extract of Geodia sp. against oxytetracycline-resistant V. harveyi

Although, there was no difference in the optical density of V. harveyi treated with the extract at 1, 2 and 4xMICs, the viable bacterial density of V. harveyi after 24 h incubation in each treatment estimated by pour plate method was different. The viable bacterial density in control treatment, at the concentrations of 1, 2 and 4xMICs were 2.39x10¹⁰, 2.01x10⁸, 2.01x10⁵ and 9.2x10³ cells mL^-1, respectively. This result shows that the methanolic extract of Geodia sp. was able to kill 99.1 and 79.9% bacterial cells of oxytetracycline-resistant V. harveyi at the concentration of 4 and 2xMICs, revealing that the extract was bactericidal at 4xMIC, but bacteriostatic at 2xMIC.

In this study, the viable bacterial density of V. harveyi could not be estimated merely by a spectrophotometer as there was no difference in the optical density, but considerably different in viable cells densities in each treatment. This time course study also indicated that the extract did not lyse the cells of V. harveyi as the antibacterial mode of action since the absorbance of the bacterium did not decrease as the decrease of the viable bacterium density. This finding suggests that the mechanism of antibacterial activity of the mathanolic extract of Geodia sp. is not lysis the bacterial cells of V. harveyi. This mechanism is likely resembled to methdilazine (Chattopadhyay et al., 1998), MC21 (Isnansetyo and Kamei, 2003) and microcin E492 (MccE492) (Destoumieux-Garzón et al., 2003) which cause bacterial cell death without cellular lysis. The further investigation on the antibacterial mechanism should be conducted after purifying the antibacterial substance from the extract.

Brain shrimp lethality test showed that the methanolic extract of Geodia sp. had low toxicity with no mortality at 0, 31.25 μg mL^-1 (1xMIC), 62.5 μg mL^-1 (2xMIC) and 125 μg mL^-1 (4xMIC) and caused 20% mortality at 250 μg mL^-1 (8xMIC). This result revealed that the extract might be applicable to control V. harveyi by immersion because the extract did not cause any mortality to Artemia nauplii at concentration up to 4xMIC. Comparing to oxytetracycline, the toxicity of the extract is higher because the LC_50-24 and LC_50-48 of oxytetracycline to A. parthenogenetica are 871 and 806 μg mL^-1, respectively (Ferreira et al., 2007). The toxic effect of the methanol extract of Geodia sp. might be not only caused by principle constituent of antibacterial substances in the extract but also by others substances in the extract. Therefore, purification and chemical elucidation as well as evaluation of in vivo activity are necessary for further study. Although, the methanolic extract of Geodia sp. showed non toxic to Artemia at 4xMIC, the toxicity of the extract to cultured aquatic organisms should be determined before application.

The results of this study can be summarized that the MeOH extract of Geodia sp. exhibited potent antibacterial activity against oxytetracycline-resistant V. harveyi with MIC 31.25 μg mL^-1. The extract showed bacteriostatic activity at low concentration and bacterisidal activity at the concentration of 4xMIC. This extract might be applied to control the disease caused by the bacterium as the extract showed no toxicity up to 125 μg mL^-1 (4xMIC) based on brain shrimp lethality test.

ACKNOWLEDGMENTS

This study was financially supported in part by Competitive Grant XIII from Directorate General of Higher Education, Department of National Education, the Republic of Indonesia. We thank to the Laboratory of Fish and Environment Health, Brackishwater Aquaculture Development Center, Jepara, Indonesia for the gift of V. harveyi isolate.