Introduction
Syzygium aromaticum flower bud, commonly known as clove, is well known in food preparation, as an anticarcinogenic agent (
Zheng et al., 1992), and as a traditional remedy for asthma (
Kim et al., 1998), disorder of digestive system (
Baytop, 1999), dental disorders, respiratory disorders, headaches and sore throat in Asian countries (
Domaracky et al., 2007). Besides the reported antimicrobial, antifungal and antiviral properties, the essential oil of
S. aromaticum shows anti-inflammatory, cytotoxic and anesthetic activities (
Chaieb et al., 2007).
Cai and Wu (1996) reported that
S. aromaticum possessed antimicrobial activity against oral bacteria commonly associated with dental caries and periodontal disease. Therefore,
S. aromaticum is used as an ingredient in toothpaste and mouth fresheners in India (
Banerjee et al. 2006).
Nowadays, antibiotics residues are reported to be ineffective in controlling diseases in aquaculture due to the misuse or overuse of antibiotics by fish farmers. Furthermore, antibiotics residues are found as a threat to human health (
Alderman and Hastings, 1998) and the environment (
Cabello, 2006). Therefore, scientists have come out with an idea of applying natural antimicrobial agents for aquaculture use, especially for fish bacterial disease treatment. Many studies have reported on the antimicrobial property of
S. aromaticum against various types of bacteria. For instance,
Fu et al. (2007) reported that the essential oil of
S. aromaticum showed inhibitory activity against
Staphylococcus aureus and
Escherichia coli. Another study by
Betoni et al. (2006) showed that the methanol extract of
S. aromaticum possessed antimicrobial activity against clinical strains of
Staphylococcus aureus. Furthermore,
Lopez et al. (2005) claimed that the essential oil of
S. aromaticum possessed inhibitory activity against four Gram-positive bacteria (
Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, and
Listeria monocytogenes) and four Gram-negative bacteria (
Escherichia coli, Yersinia enterocolitica, Salmonella choleraesuis and
Pseudomonas aeruginosa). Another study of
Yano et al. (2006) claimed that the aqueous extract of
S. aromaticum, at a concentration of 0.04 mg·mL
-1, was able to control
Vibrio parahaemolyticus, a foodborne pathogen. However, until the present, no study has been conducted to reveal the antimicrobial property of the essential oil of the
S. aromaticum flower bud against fish bacterial diseases in aquaculture. Therefore, our study was carried out to reveal the potential of
S. aromaticum essential oil as an antimicrobial agent as an alternative to commercial antibiotics in aquaculture use.
Materials and methods
Essential oil preparation
Syzygium aromaticum flower bud (Clove) was purchased from a market in Terengganu, Malaysia. The sample was subjected to steam distillation for 6 h using a piece of steam apparatus to produce the essential oil in a yield of 3.0% (v/w) based on the dry weight of the sample. The essential oil was stored in the dark at 4°C until use.
Bacterial isolates
A total of 36 bacterial strains isolated from 10 types of aquatic animals (Penaeus vannamei, Penaeus monodon, Scylla sp., Rana catesbeiana, Macrobrachium rosenbergii, Trachinotus blochii, Clarias gariepinus, Tilapia sp., Monopterus albus and Trichogaster pectoralis) were applied in the present study. There were Vibrio spp. (n=6), Edwardsiella spp. (n=21), Aeromonas spp. (n=2), Escherichia coli (n=2), Flavobacterium spp. (n=1), Salmonella spp. (n=2), Streptococcus spp. (n=1) and Pseudomonas spp. (n=1). Escherichia coli (ATCC 25922), Citrobacter freundii (ATCC 8090), Aeromonas hydrophila (ATCC 49140), Pseudomonas aeruginosa (ATCC 35032), Streptococcus agalactiae (ATCC13813), Edwardsiella tarda (ATCC 15947) and Yersinia enterocolitica (ATCC 23715) used as bacterial reference strains.
Minimum inhibitory concentration (MIC) test
The values of the minimum inhibitory concentration (MIC) of the essential oil of
S. aromaticum as well as the positive controls, namely kanamycin and eugenol (Analar, UK), against bacterial isolates were determined through a two-fold broth micro dilution method (
Daud et al., 2005). The essential oil was diluted using 0.01% methanol. The bacterial isolates were cultured in tryptic soy broth for 24 h at room temperature and the concentration of these cultures was adjusted to 10
9 CFU·mL
-1 by using saline (0.85% of NaCl), and monitored with Biophotometer (Eppendorf, Germany). The bacterial suspensions were then inoculated into a microtiter plate that contained a serial dilution of the isolated compound and the microplate was incubated at room temperature for 24 h. The MIC values were defined as the lowest concentration of the isolated compound in the wells of the microtiter plate that showed no visible turbidity after the 24 h incubation.
Gas chromatography mass spectrometry (GC–MS)
The chromatographic procedure was carried out using a Shimadzu QP2010-GC-MS with autosampler. The sample was diluted 25 times with acetone, with 1 μL injected into the column. A fused silica capillary column HP5-MS (30 m×0.32 mm, film thickness 0.25 μm) was used. Helium was used as the carrier gas, and a split ratio of 1/100 was applied. The oven temperature used was maintained at 60ºC for 8 min. The temperature was then gradually raised at a rate of 3°C per min to 180°C per min and maintained at 180°C for 5 min. The temperature at the injection port was 250°C. The components of the test solution were identified by comparing the spectra with those of known compounds stored in the internal library.
Results and discussion
In the present study, the essential oil of Syzygium aromaticum was able to inhibit the growth of 36 bacterial isolates from ten types of freshwater and marine aquatic animals as well as seven ATCC reference bacterial strains. The minimum inhibitory concentration (MIC) values of the essential oil of S. aromaticum against the tested bacterial strains ranged from 0.015 µg·mL-1 to 0.062 µg·mL-1 (Table 1). Meanwhile, the MIC values of kanamycin and eugenol against the tested bacterial isolates ranged from 15 µg·mL-1 to 125 µg·mL-1 and 15625 µg·mL-1 to 250000 µg·mL-1, respectively. A total of nine chemical compounds were detected in the essential oil, with eugenol (49.0%) and caryophyllene (7.5%) being the major compounds.
Our study is the first report on the potential use of the essential oil of
S. aromaticum as an alternative antimicrobial agent to commercial antibiotic for aquaculture use. The essential oil successfully inhibited the growth of all bacterial isolates from various types of aquatic animals including seven ATCC reference bacterial strains, with an MIC value as low as 0.015 μg·mL
-1. On the other hand,
Fu et al. (2007) reported on the MIC value of essential oil of
S. aromaticum against human pathogens;
Staphylococcus epidermidis,
Escherichia coli and
Candida albicans ranged from 0.62 mg·mL
-1 to 5 mg·mL
-1. Other studies showed that the MIC value of the essential oil of
S. aromaticum against human pathogens, four Gram positive bacteria (
Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, and
Listeria monocytogenes) and three Gram-negative bacteria (
Escherichia coli, Yersinia enterocolitica and
Salmonella choleraesuis) ranged from 17.5 mg·mL
-1 to 131 mg·mL
-1, however, the essential oil failed to show inhibitory activity against
Pseudomonas aeruginosa (
Lopez et al., 2005). In the study of
Moreira et al. (2005), the food grade essential oil of
S. aromaticum (Nelson and Russell, England) purchased commercially was able to inhibit the growth of
E. coli (ATCC 25158) at a concentration of 2.5 mg·mL
-1. Here, we may conclude that bacterial isolates from aquatic animals were more susceptible to the essential oil of
S. aromaticum compared with bacterial isolates from other hosts.
Many reports have claimed that eugenol is the major compound in the essential oil of
S. aromaticum. For instance,
Bauer et al. (2001) reported that the essential oil of
S. aromaticum consisted of 75% to 85% eugenol.
Farag et al. (1989) also mentioned that approximately 85% eugenol was found in the essential oil of
S. aromaticum. Another study by
Kong et al. (2004) claimed that the essential oil of
S. aromaticum possessed 68% eugenol. However, the essential oil of
S. aromaticum obtained in the present study only constituted 49.0% eugenol (Table 2). This may due to differences in methods for essential oil extraction, which resulted in a discrepancy of the percentage of eugenol in the essential oil. In the present study, a piece of self-designed steam apparatus was applied in obtaining the essential oil, whereas other studies used the Clevenger apparatus commonly used for essential oil extraction. Eugenol is reported to play an important role in inhibiting the growth of bacteria.
Thoroski et al. (1989) claimed that eugenol could inhibit the production of amylase and proteases in the cell of
Bacillus cereus. Another study of
Wendakoon and Sakaguchi (1993) reported that eugenol could inhibit the growth of
Enterobacter aerogenes by preventing enzyme action in the bacterial cells. Although the essential oil obtained in the present study constituted of a low percentage of eugenol compared with that of other studies, the MIC values of the essential oil in the present study against bacterial isolates from aquatic animals were lower than those in other studies. This may due to the combination of other chemicals with eugenol that are present in the essential oil, which synergically increased the potency of the antimicrobial property of the essential oil. This is supported by the finding of the present study that the MIC values of eugenol alone against the present bacterial isolates ranged from 15625 µg·mL
-1 to 250000 µg·mL
-1 and
Kim et al. (1995) reported that the MIC value of eugenol against foodborne pathogens,
E. coli and
Salmonella typhimurium, was 1000 µg·mL
-1 and 500 µg·mL
-1, respectively, which were much higher than the MIC values of the essential oil obtained in the present study. However, further studies should be carried out before we come to a final conclusion.
In short, the result of our present study showed that the potential of the essential oil of S. aromaticum as an antimicrobial agent for aquaculture use is promising. However, further studies should be carried out to evaluate the efficacy of this essential oil in controlling bacterial disease infections in fish as well as production costs before the essential oil is introduced to fish farmers.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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