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INTRODUCTION

Background of the Study

Tilapia (Oreochromis spp.) is a very popular aquaculture species in the Philippines at

present. It has been reared by hatcheries and grow-out operators because of its high market

demand in the country (Yambot, 1998). Tilapia offers economical and social benefits for

rural communities. It also plays vital role in terms of worldwide employment (Badillo,

2010). However, in the early 1990s, severe disease outbreak threatened the growing tilapia

industry in the country attributed to Aeromonas hydrophila (Yambot and Inglis, 1994).

Aeromonas are water-borne pathogens that are common in almost all aquatic

environments including fresh, brackish and marine waters (Khan et al., 2008). Aeromonas

species are the cause of diseases in cultured and feral fishes in Europe (Cipriano et al., 2001).

“Motile Aeromonas Septicemia” (MAS), “Hemorrhagic Septicemia”, “Ulcer Disease”, or

“Red-Sore Disease” are some of the common diseases caused by A. hydrophila (White and

Swann, 1995). According to Khan et al. (2008), Aeromonas has been found to produce

exotoxin such as aerolysin-like hemolysin (ALH), Aeromonas Serine Protease (ASP) and

Aeromonas Metalloprotease (AMP) to survive undesirable condition. The study also showed

that ALH’s primary action is to rupture its cell victims; however, until today its mechanism

has been a mystery, while ASP has been found to promote plasma coagulation by means of

activating prothrombin (Khan et al., 2008). ASP is common and deadly consequence to

sepsis (blood poisoning) victims while the mode-of-action of AMP has not yet been

determined by the experts (Khan et al., 2008).

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In making curative remedies against bacterial infections, antimicrobial agents are

highly needed but the problem of increasing resistance against these agents posed challenges

to the scientific world (Kaskhedikar and Chhabra, 2009). In addition, commercial antibiotics

for large-scale treatment are unaffordable for most of the farmers (Siri et al., 2008).

Therefore, there is a need to develop alternative antibiotics from medicinal plants for the

treatment of infectious diseases (Agarwal et al., 1996).

According to Stuart (1993), in the Philippines, thousands of plants are known to have

medicinal value and the use of different plant parts to cure specific ailments since ancient

times and the antimicrobial medicinal effects of plant materials typically result from the

combinations of secondary products present in the plant that include alkaloids, steroids,

tannins, phenol compounds, flavonoids and resins fatty acids gums.

Significance of the Study

Nowadays, multiple drug resistance has developed due to the indiscriminate use of

commercial antimicrobial drugs commonly used in the treatment of infectious disease. In

addition to this problem, commercial antibiotics are costly and sometimes associated with

adverse effects on the host (Ravikumar et al., 2010). This situation necessitates searching for

new antimicrobial substances. Therefore, there is a need to develop alternative antimicrobial

drugs from medicinal plants for the treatment of infectious diseases.

The set of medicinal plants that will be used in this study are found effective against

bacterial pathogens in humans and animals (Stuart, 2011). Harnessing the potentials of these

plant materials against fish bacterial pathogens will be a great help to the aquaculture

industry.

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Objectives of the Study

The main objective of the study is to screen and evaluate the antibacterial potential of

the different plant extracts against A. hydrophila. Specifically, the study aims to compare the

antibacterial activity of the extracts and commercial antibiotics by measuring the zone of

inhibition.

Scope and Limitation

Leaves will only be used in this experiment. The screening screen of the antibacterial

potential of the different plant extracts against A. hydrophila will only be done in vitro.

Time and Place of the Study

The study will be conducted at the Fish Pathology Laboratory of the College of

Fisheries−Freshwater Aquaculture Center, Central Luzon State University (CF-FAC, CLSU),

Science City of Muñoz, Nueva Ecija from August to October, 2012.

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REVIEW OF RELATED LITERATURE

Medicinal Plants with Antibacterial Potential

Hundreds of medicinal plants are used in making powerful drugs nowadays (Stuart,

2011). Their potentials as a drug are mainly because of their secondary metabolites

constituents such as tannins, alkaloids and flavonoids (Ravikumar et al., 2010).

Presented below are lists of common medicinal plants found in the country with their

antimicrobial active components (Stuart, 2011).

Garlic (Allium sativum) is found to contain saponin, tannins, sulfurous compounds,

prostaglandins, alkaloids, volatile oils and allicin which is responsible for its pungent odor.

The most important chemical constituents are the cysteine sulfoxides (alliin) and the non-

volatile glutamylcysteine peptides which make up more than 82% of the sulfur content of

garlic. It also contains allyl disulphide which is responsible against helminths.

Duhat (Syszygium jambolanum) or Jambolan is claimed to contain alkaloid,

jambosine, glycoside and jambolin which halts the diastatic conversion of starch into sugar.

The leaf juice is effective in the treatment of dysentery. Jambolan leaves may be helpful as

poultices on skin diseases. The leaves, stems, flower buds, opened blossoms and barks have

some antibiotic activity.

Makabuhay (Menispermum crispum) is a popular insecticide for rice crop. Its stems

and leaves can be utilized against rheumatism, hay fever, allergic rhinitis and many more. It

has also antibacterial property because it contains diterpenes which was recorded to be

effective against P. aeruginosa and Bacillus subtilis.

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Noni (Morinda citrifolia) is found to increase immunity and also proven to have

antibacterial activity against Shigella and Salmonella. It is a very popular fruit juice due to

heavy marketing of some company. It was proven to contain antioxidant and anticarcinogen

properties. The fruit contains phytochemicals, lignans, polysaccharides, flavonoids, iridoids,

nonisides, scopoletin, catechin, epicatechin and damnacanthal.

Jathropa (Jathropa curcas) is a well-known piscicide and insecticide. The leaves of

Jathropa were found to contain phlobatannins and tannins that have antimicrobial activity

against Salmonella typhi, S. aureus and P. aeruginosa.

Acacia (Samanea saman) bark and seed contain saponin-like alkaloid and

pithecolobin. Aside from its antibacterial activity, it is also used against stomach ache. The

bark, stems, leaves and seeds are found to contain alkaloids. It can also be used as

antipyretic and antidermatoses.

Sampaguita (Jasminum sambac) is usually used against infection. Active

constituents are alkaloids, glycoside, flavonoids, terpines, tannin, resin and salicylic acid.

Sampaguita has antibacterial effect against Salmonella typhi and S. aureus.

Alugbati (Basella rubra) is an edible vine. Studies revealed that the aqueous,

ethanolic and petroleum ether extracts of leaves exhibited antimicrobial activity against some

bacteria. The ethanolic extract showed maximum effect against Escherichia coli.

Continuous study is needed to determine the active ingredient that causes its antimicrobial

activity.

Atsuete (Bixa orellana) seeds are used as antidote for cassava and Jathropa

poisoning. Poultice of leaves are diuretic and used for treatment of gonorrhea. Atsuete

extract showed significant effect against Bacillus cereus. The seeds contain carotenoids

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pigments such as bixin, norbixin, ß-carotene, cryptoxanthin, lutein, zeaxanthin and methyl

bixin.

Guava (Psidium guajava) is a famous edible backyard fruit. It is known for its

astringent, antispasmodic, antihelminthic and antiseptic properties. Leaves are commonly

used against toothache and wounds. Study showed that guava sprout is active against

diarrhea caused by E. coli- or S. aureus-produced toxin. The leaves extract showed

significant effect against Shigella spp., Vibrio spp., S. aureus, E. coli, P. aeruginosa and B.

subtilis.

Jackfruit (Arthocarpus heterophyllus) is a famous edible fruit because of its

delicious taste. Root is considered antiasthmatic and the bark is considered sedative. Many

medicinal effects have been noted about this plant. It has also antibacterial activity against B.

subtilis, B. cereus, S. aureus and E. coli. Active ingredient against bacteria has not been

found yet.

Pomelo (Citrus grandis) is a locally common citrus fruit. It contains phytochemicals

such as naringin, hesperidine, diosmin and naringenin. The leaves and flowers can be used

for nervous infections, coughs and ulcer. Naringenin, one of its active ingredients was found

to be effective against P. aeruginosa.

Indian Mango (Mangifera indica) is an effective agent against S. aureus, E. coli, P.

aeruginosa and this study provides a basis for its medical use in Uganda. Active components

are saponins, steroids and triterpenoids, alkaloids, coumarins, anthracenocides, flavonones,

tannins and reducing sugars.

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Zone of Inhibition

Zone of inhibition is the area without bacterial growth surrounding an antimicrobial-

impregnated disk an antimicrobial sensitivity test. It is widely used as the index of the

effectiveness of antimicrobial extract against bacteria.

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MATERIALS AND METHOD

Collection of Samples

All of the leaves of the medicinal plants that will be used in this study will be

collected at the CLSU compound. The selected medicinal plants will be composed of

Acacia, Alugbati, Atsuete, Duhat, Jackfruit, Jathropa, Guava, Indian Mango, Pomelo, Noni,

Makabuhay and Sampaguita. Leaves of the above plant samples will be subjected to

ethanolic extraction.

Ethanolic Extraction

Leaves of the plant samples will be sun-dried for 1 hour and then air dried for 7 hours

to remove the moisture content. The dried leaves will be powdered using a blender and later

stored in plastic containers until ready for use. Twenty-five grams of the powdered leaves

will be soaked in 100 ml ethanol for 48 hours at room temperature to give a concentration of

833 mg/ml. The resulting mixture will be filtered using single layer sterile filter paper and

the filtrate obtained will be concentrated on a water bath. The resulting residue will be

diluted in distilled water to give the desired doses of 25, 50, 100, 500 and 800 mg/ml

(Adesokan et al., 2012).

Bioassay

Plastic containers provided with aerator will be filled with 3.5 liters of tap water.

Five concentrations of each extract will be tested: Treatment 1 = 25 mg/ml, Treatment 2 = 50

mg/ml, Treatment 3 = 100 mg/ml, Treatment 4 = 500 mg/ml and Treatment 5 = 800 mg/l.

Every treatment will be replicated thrice. Each container will be stocked with 35 pre-

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conditioned tilapia fingerlings (size 24) (APHA, AWWA and WPCF, 1965). Mortality will

be recorded in 48 hours duration. Water quality parameters such as temperature, pH,

dissolved oxygen and hardness will be gathered daily. The concentration of each ethanolic

extract having the highest recorded survival will be used for the eradicant test described

below.

Determination of Minimum Inhibitory Concentration (MIC)

In a sterilized agar plate, 1 ml of the ethanolic extract (25, 50, 100, 500 and 800

mg/ml) and 9 ml of the Mueller Hinton Agar will be pipetted and will be mixed thoroughly.

The agar will be allowed to solidify at room temperature. Four to five isolated colonies will

be sub-cultured to a tube with 3 ml Mueller Hinton Broth. The broth will be incubated at 30

˚C for 18-24 hours until it achieves or exceeds the turbidity of 0.5 McFarland standards. The

standardized inoculum will be diluted at a dilution ratio of 1:10 in sterile saline solution to

obtain the desired concentration of 106 CFU/ml. From the standardized inoculum, 0.1 ml

will be streaked to the surface of the prepared agar. The agar plates will be incubated at 30

˚C for 18-24 hours. The MIC will be taken as the lowest concentration that completely

inhibits the growth of the organism as detected by the naked eye (Ruangpan and Tendencia,

2004).

Preparation of Aeromonas hydrophila

Pure culture of A. hydrophila will be obtained from the University of the Philippines,

Los Baños−National Institute of Molecular Biology and Biotechnology (UPLB-BIOTECH).

From the pure bacterial culture (which should not be more than 48 hours), four or five

colonies will be transferred to 5 ml Trypticase soy broth. The broth will be incubated at 30

10

˚C or at an optimum growth temperature until achieves or exceeds the turbidity of 0.5

McFarland standards. The turbidity of the test bacterial suspension with that of 0.5

McFarland will be compared against a white background with contrasting black line under

adequate light (Ruangpan and Tendencia, 2004).

Preparation of Eradicant Test Against the Bacteria

Whattman filter paper discs measuring 6 mm will be made using a paper puncher and

will be sterilized in an autoclave at 15 psi for 30 minutes. The sterilized discs will be soaked

into the ethanolic extracts (based on bioassay and MIC results) for 1 hour and will be air-

dried for 10 minutes in the inoculating chamber. The different ethanolic extracts will serve

as the treatments. Meanwhile, two commercially prepared antibiotic discs (Tetracycline and

Vancomycin) of the same diameter will be used as positive control and distilled water as the

negative control.

Sterile cotton swab will be dipped into the standardized bacterial suspension. The

swab containing the inoculum will be streaked in the prepared Mueller Hinton agar plates.

Using sterile forceps, the discs will be placed on the surface of the inoculated plate. The

discs will be positioned such that the minimum center distance is 24 mm and no closer than

10 to 15 mm from the edge of the petri dish. In inverted position, the plates will be incubated

at room temperature and will be observed after 12, 18, 24 and 36 hours after incubation.

Using ruler, the diameter of the zone of inhibition will be measured in millimeter. For each

control and treatment, six replicates will be used (Ruangpan and Tendencia, 2004).

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Statistical Analysis

Significant difference in the diameter of the zone of inhibition every time the plates

are observed will be analyzed using One-Way Analysis of Variance (ANOVA) under the

Statistical Package of Predictive Analytics Software (PASW) Statistics Version 18.

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LITERATURE CITED

Adesokan, A.A., M.A. Akanji and M.T.Yakubu. 2007. Antibacterial potentials of aqueous extract of Enantia chloranthas stem bark. African Journal of Biotechnology, 6(22):2502-2505.

Agarwal, P., V. Rai and R.B. Singh. 1996. Randomized, placebo-controlled, single-blind trial of holy basil leaves in patients with noninsulin-dependent diabetes mellitus. International Journal of Clinical Pharmacology and Therapeutics, 34:406-409.

American Public Health Association (APHA), American Water Work Association (AWWA) and Water Pollution Control Federation (WPCF). 1965. Standard methods for the examination of water and wastewater. Twelfth Edition. American Public Health Association, Inc. 1740 Broadway, New York, N.Y. 545-562 p.

Badillo, L.J. 2006. Age growth-models for Oreochromis aureus (Perciformes, Cichlidae) of the Infiernillo Reservoir, Mexico and reproductive behaviour. Rev. Biol. Trop. (Int. J. Trop. Biol., 54(2):577-588.

Cipriano, R.C. 2001. Aeromonas hydrophila and Motile Aeromonad Septicemias of fish. Fish Disease Leaflets, 68. (retrieved on September, 2011 at http://www.extension.org/mediawiki/files/1/1e/Aeromonas_hydrophila.pdf).

Kaskhedikar, M. and D. Chhabra. 2009. Multiple drug resistance in Aeromonas hydrophila isolates of fish. Vet. World, 3(2):76-77.

Khan, R., E., Takahashi, H. Nakura, M. Ansaruzzaman, S. Banik, T. Ramamurthy and K. Okamoto. 2008. Toxin production by Aeromonas sobria in natural environments: river water vs. seawater. Acta Med. Okayama, 62(6):363-371.

Ravikumar, S., G.P. Selvan and N.A.A. Gracetin. 2010. Antimicrobial activity of medicinal plants along Kanyakumari Coast, Tamil Nadu, India. Af. J. B. and App. Sci. 2 (5-6): 153-157. IDOSI Publications.

Ruangpan, L.A. and E.A. Tendencia. 2004. Laboratory manual of standardized method for antimicrobial sensitivity tests for bacteria isolated from aquatic animals and environment. Southeast Asian Fisheries Development Center-Aquaculture Department, Tigbauan 5021, Iloilo, Philippines. pp. 32-43

Siri, S., P. Wadbua, W. Wongphathanakzul, N. Kitancharoen and P. Chantaranotai. 2008. Antibacterial and phytochemical studies of 20 Thai medicinal plants against catfish-infectious bacteria, Aeromonas caviae. KKU Sci. J., 36 (Supplement):1-10.

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Stuart, G. 2011. Philippine medicinal plants. (Retrieved on December 9, 2012 from www.stuartxchange.org).

White, M.R., and L. Swann. 1995. Diagnosis and treatment of “Aeromonas hydrophila” infection of fish. Taken from” A guide to approved chemicals in fish production and fishery resource management”, 1989.

Yambot, A.V. 1998. Isolation of Aeromonas hydrophila from Oreochromis niloticus during fish disease outbreaks in the Philippines. Asian Fisheries Science, 10:347-354.

Yambot, A.V. and V. Inglis. 1994. Aeromonas hydrophila isolated from Nile tilapia (Oreochromis niloticus) with “eye disease”. In: M.K. Vidyadaran, M.T. Aziz and H. Sharif (eds.). International Congress of Quality Veterinary Services for 21st Century Kuala Lumpur. p. 87-88.