Antibacterial Potential of Marine Ascidian Phallusia arabica Against Isolated Urinary Tract Infections Bacterial Pathogens
S. Mohamed Hussain
In-vitro antibacterial activity of marine ascidian Phallusia arabica was investigated against urinary tract infection bacterial pathogens using disc diffusion method. The crude ethyl acetate extract was more active exhibiting a broad-spectrum antibacterial activity than that of the crude methanol extract against each of the microbes tested. Maximum inhibition zone (12 mm) was observed against Proteus mirabilis in crude ethyl acetate extract (1 mg mL-1 concentration) and the minimum inhibition zone (1.5 mm) was observed against Pseudomonas aeruginosa in methanol extract (1 mg mL-1 concentration). The range of MICs and MBCs were high in ethyl acetate extract and it was low in the methanol extract. This result suggest that P. arabica can be used as effective inhibitor of urinary tract infection pathogens making them applicable to medical devices and antimicrobial control systems.
Received: November 01, 2010;
Accepted: March 15, 2011;
Published: March 25, 2011
Patients with non-infectious disease who have stay in hospital have high risk
to acquire nosocomial infection. It has been reported that 10% hospital patients
acquire this infection while staying in hospital (Asefzadeh,
2005). The common pathogenic bacteria which include Escherichia coli,
Klebsilla pneumoniae, Haemophilus influenza, Streptococcus
pneumoniae and Proteus vulgaris, are the major causative agents of
nosocomial infections. Generally, nosocomial infections develop in urinary tract
(Saonuam et al., 2008). Ocean has potent bioactive
compounds isolated from marine organisms which are currently used as food. This
mechanism has proved to be timely alternative natural medicine to human beings.
The bioactive compounds from ascidians was recently initiated, it is significant
that the first marine natural product entering in to human clinical trials,
didemnin B, is an ascidian metabolite. Tunicates have been reported to be rich
sources of biologically active compounds and ranked third for their overall
activities, next to sponges and bryozoans (Davis and Bremner,
1999). Actinobacteria, the filamentous bacteria have provided many important
bioactive compounds of high commercial value and are being routinely screened
for new bioactive substances (Siva Kumar et al.,
2011). The first systematic search for antibiotics resulted in the discovery
of Actinomycetin from Actinomycete bacteria (Sultan et
al., 2002). A large proportion of natural compounds have been extracted
from marine invertebrates, especially sponges, ascidians, bryozoans and molluscs
and some of them are currently in clinical trials (Proksch
et al., 2002). The bioactive substance which possesses potent anticancer
activity Ecteinascidin-743 was isolated from Caribbean Sea squirt Ecteinascidia
turbinate (Russo et al., 2008). Such potential
ascidians need to be explored for the pharmaceutical purpose. The study was
carried out to establish the occurrence of antibacterial activity of ascidians
extracts, collected from Southeast coast of Tamil Nadu, India
MATERIALS AND METHODS
Collection and preparation of samples: The ascidian Phallusia arabica (Chordata: Ascidiacea: Pleurogona: Pyuridae) were collected during the low tide of the intertidal area at Thoothukudi coast, Tamil Nadu, Southeast coast of India, between 20 and 23 August 2010. The collected samples were rinsed with sterile seawater to remove associated debris and salt. The samples were weighed (10 g) and preserved separately in methanol and ethyl acetate (1:2) and brought to the laboratory. Samples were then soaked in the above mentioned solvents for 48 h, the extracts were then obtained from the soaked samples by grinding, using pestle and mortar and filtering through Whatman No. 1 filter paper, the filtrate was centrifuged at 3000 rpm. The solvent was evaporated under reduced pressure using desiccator and the residue was weigh and dissolved in dimethyl formamide for using them for the antibacterial activity. All the urinary tract infection bacterial pathogen strains were obtained from Raja Muthiah Medical College, Annamalai University.
Antibacterial succeptiblility assay: Ascidian crude extract was tested
for inhibition of bacterial growth against urinary tract infections bacterial
pathogens. The extract of P. arabica was tested for antibacterial activity
at two different concentrations by disc diffusion method (Avelin
et al., 1991).
Minimum Inhibitory Concentration (MIC): Minimum inhibitory concentration
was determined by the following procedure (Collins et al.,
Minimum Bactericidal Concentration (MBC): Minimum bactericidal concentration
was experimented after the MIC in freshly prepared agar plates, followed by
standard method of Alade and Irobi (1993).
RESULTS AND DISCUSSION
In vitro antibacterial screenings of marine ascidians against selected
clinical isolates were performed and the inhibition zones of the extract against
the specific test organisms were given in Table 1. Maximum
inhibition zone (12 mm) was observed against P. mirabilis in crude ethyl
acetate extract (1 mg mL-1 concentration) of P. arabica and
the minimum inhibition zone (1.5 mm) was observed against P. aeruginosa
in methanol extract (1 mg mL-1 concentration). This view is consistent
with the findings of Anand and Edward (2002) reported
that ethyl acetate crude extract of ascidian exhibited strong antibacterial
activity than methanol extract and Petroleum ether of all the strain tested.
The methanol crude extract of P. Arabica, the range of inhibition of
the clinical isolates of the bacteria varied from 1.5-6 mm was observed in both
(500 μg mL-1 and 1 mg mL-1) concentration. This result
is lesser than the findings of Hussain and Ananthan (2009),
who reported that the crude methanol extract of Didemnum psammathodes,
the range of inhibition of the bacteria varied from 2-15 mm with an average
of 4.3 mm. On the other hand, Ramasamy and Murugan (2003)
have reported that the crude methanol extract of Didemnum psammathodes,
the range of inhibition of the bacteria varied from 6 and 10 mm with an average
of 7.1 mm. The Ethyl acetate extract of T. clinids, the range varied
between 1 and 8.5 mm with an average of 3.18 mm.
The crude ethyl acetate extract of P. arabica showed the maximum antibacterial activity in (1 mg mL-1 concentration) against P. mirabilis, followed by E. coli, P. vulgaris, Klebsiella sp and P. aeruginosa and the minimum activity was noticed in all 500 μg mL-1 concentration of the same extract. The crude methanol extract of P. arabica showed the maximum antibacterial activity in (1 mg mL-1 concentration) against P. mirabilis, followed by P. vulgaris, E. coli, Klebsiella sp. and P. aeurginosa. There is no activity against all 500 μg mL-1 concentration methanol extract (Table 1).
The minimum inhibitory concentrations (MICs) and minimum bactericidal concentration
(MBCs) of the crude extracts were shown in Table 2. The range
of MIC varied between 0.25-1.85 (500 μg mL-1 and 1 mg mL-1
concentrations) against all the bacterial strains used in this study and MBC
ranges between 0.20-2.10 (500 μg mL-1 and 1 mg mL-1
concentrations) against all the bacterial strains. Ascidians are already reported
for rich nitrogenous source with a wide range of biological activities (Biard
et al., 1994). The range of MICs is highest in ethyl acetate extract
1.85 (1 mg mL-1 concentration) and methanol extract 1.05 (1 mg mL-1
concentration). Range of MBCs was high in ethyl acetate extract 2.10 (1 mg mL-1
concentration) and methanol extract 1.15 (1 mg mL-1 concentration).
The range of MICs was low in the ethyl acetate extract 0.25 (500 μg mL-1
concentration) and methanol 0.25 (1 mg mL-1 concentration). The range
of MBCs was low in ethyl acetate extract 0.20 (500 μg mL-1 concentration)
and methanol 0.60 (1 mg mL-1 concentration). On the other hand, Natarajan
et al., (2010) reported that the Minimum Inhibitory Concentrations
(MICs) and Minimum Bactericidal Concentration (MBC) of the methanolic extract
of Polyclinum madrasensis, MIC varied between 0.70-0.95 mg mL-1
and MBC ranges between 0.85-1.10 mg mL-1 against all the bacterial
|| Antibacterial activity of Phallusia arabica
|-: No activity was observed
|| MIC and MBC of different concentration crude extracts of
|-: No activity was observed
Shobu and Pawlik (2007) reported that crude organic
extracts of whole tunic and internal tissues of P. nigra contained vanadium
metabolites (225 and 750 ppm dry mass, respectively) and were palatable to blue
head wrasse, Thalassoma bifasciatum. Crude extracts also exhibited no
antimicrobial effects against a panel of four marine bacteria (Vibrio parahaemolyticus,
Vibrio harveyi, Leucothrix mucor and Deleya marina). Jaffarali
et al. (2008) reported that the test of P. nigra exhibited
a bactericidal effect, with the zone of inhibition ranging from 2.3 to 12.3
mm against all pathogens tested, as referred to the mantle body. This result
more or less consistent to our results. Dowzicky and Park
(2008) reported that UTI bacterial pathogens have exhibited decreased susceptibility
rates to tigecycline over the years. Marine genus synthesizes active constituents
which are used in traditional and complementary medicine (Anbu-Jeba-Sunilson
et al., 2009). Antibacterial compounds from natural resources would
be the alternative to overcome the resistance problem. Since, the present study
indicated that the crude extract of P. arabica showed antibacterial activity
which could be used in pharmacological research.
Present study deduce that the continuing and overwhelming contribution of ascidians metabolites to the development of new pharmaceuticals are clearly evident and need to be explored. After taking in to consideration the immense side effects of synthetic drugs, great attention has to be paid for the discovery of novel drugs from marine natural products.
Authors thank Prof. Dr. T. Balasubramanian, Dean, CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University and the Ministry of Earth Sciences (OASTC) New Delhi for financial assistance. We acknowledge Dr. H. Abdul Jaffar Ali, Assistant Professor, Department of Biotechnology, Islamiah College, Vaniyambadi for his help and advice.
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