1. Introduction

Plants contain many biologically active compounds which have potential for development as medicinal agents. Herbal medicines already form the basis of therapeutic use in the developing countries, but of recent, there has been an increase in the use of herbal medicines in the developed world too (De and Ifeoma, 2002; El-Mahmood et al., 2010).

Plants provide an alternative strategy in search for new drugs. There is a rich abundance of plants reputed in traditional medicine to possess protective and therapeutic properties (Kayode and Kayode, 2011). It is likely that plants will continue to be a valuable source of new molecules which may, after possible chemical manipulation, provide new and improved drugs (Shah et al., 2006). Bacterial resistance to antibiotics represents a serious problem for clinicians and the pharmaceutical industry and great efforts are being made to reverse this trend, and one of them is the widespread screening of medicinal plants from the traditional system of medicine hoping to get some newer, safer, and more effective agents that can be used to fight infectious diseases (Natarajan et al., 2003).

Azadirachta indica is one of such medicinal plants belonging to the Meliaceae family and is indigenous to southern Asia (Akula et al., 2003). Azadirachta indica, commonly known as neem, has attracted worldwide prominence in recent years, owing to its wide range of medicinal properties. Neem has been extensively used in Ayurveda, Unani and Homoeopathic medicine and has become a cynosure of modern medicine. Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex. More than 140 compounds have been isolated from different parts of neem. All parts of the neem tree- leaves, flowers, seeds, fruits, roots and bark have been used traditionally for the treatment of inflammation, infections, fever, skin diseases and dental disorders. The medicinal utilities have been described especially for neem leaf. Neem leaf and its constituents have been demonstrated to exhibit immunomodulatory, anti-inflammatory, antihyperglycaemic, antiulcer, antimalarial, antifungal, antibacterial, antiviral, antioxidant, antimutagenic and anticarcinogenic properties (Talwar et al., 1997; Biswas et al., 2002; Subapriya and Nagini, 2005).

The objectives of this study therefore are to determine the phytochemical components of the leaf extracts of A. indica, to determine the minimum inhibitory concentration (MIC) of the extract on Pseudomonas aeruginosa, Klebsiella ozanae, Staphylococcus aureus and Escherichia coli and determine the minimum bactericidal concentration (MBC) of the extract on Pseudomonas aeruginosa, Klebsiella ozanae, Staphylococcus aureus and Escherichia coli.

2. Materials and Methods

Collection and preparation of samples

The bark of A. indica was collected from different locations in Minna metropolis. It was air-dried for six weeks in microbiology laboratory of Federal University of Technology, Minna. The dried materials were pulverised in mortar and packaged in bottles for analysis.

Collection of specimen

Pure cultures of Pseudomonas aeruginosa, Klebsiella ozanae, Staphylococcus aureus and Escherichia coli were obtained from General Hospital Minna. Niger State and were subcultured in agar slants.

Extraction of materials

Ethanol and water were used as solvents for the extraction of the plant materials. One hundred and fifty gram (150g) of pulverised sample was suspended in 750ml of 75% ethanol for 120hours. The extracts were decanted, filtered and evaporated in vacuole at 45^oC.

Phytochemical Screening of Extracts of A. indica

The phytochemical components of extracts of A. indica was determined using methods described by Odebiyi and Sofowora (1978) and Trease and Evans (1989). The phytochemical components analysed for were alkaloids, tannins, phenolics, glycosides, saponins, flavonoids, steroids, phlobatanins and triterpenes.

Antimicrobial susceptibility test

Susceptibility test of the test organisms to extracts of A. indica at concentrations of 500mg/ml, 50mg/ml and 5mg/ml was carried out using agar cup plate technique as described by Silver et al. (1997). Nutrient agar was sterilized using autoclave at 121^oC for 15 minutes. It was then poured on to plates and allowed to solidify. Standardized inoculum of each test organisms was spread on to agar plates so as to achieve a confluent growth. The impregnated discs with different concentrations of the extract were placed on the surface of the medium at three points equidistant from one another. The plates were then incubated at 37^oC for 24 hours.

Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) of the test organisms was determined using the tube dilution technique. Nine millilitre (9ml) of the nutrient broth was pippeted into various test tubes containing concentrations of 500mg/ml, 50mg/ml and 5mg/ml of the extract. The overnight culture of the test organisms diluted at 10⁶cfu/ml was added to the test tubes and then incubated at 37^oC for 24 hours. The least concentration of the extract that did not indicate any visible growth of the incubated organisms in broth culture was taken as the minimum inhibitory concentration (MIC) (Hugo and Russel, 1983).

3. Result

Phytochemical screening of the extracts

Table 1 shows the phytochemical screening of the extract of the A. indica. The phytochemical components of A. indica showed the presence of saponin and phlobatanin and the absence of alkaloids, tannins, phenolics, glycosides, flavonoids and triterpenes.

Antimicrobial activities of the extracts

Table 2 shows the zones of inhibitions (mm) of plant extracts at different concentrations (mg/ml). P. aeruginosa showed the highest susceptibility of 14±2mm at 500mg/ml, followed by S. aureus (12±2mm), K. ozanae 10±2mm and E. coli 8±2mm. At concentration of 50mg/ml, P. aeruginosa had the highest zone of inhibition (10±2mm), followed by S. aureus (8±2mm). The least was E. coli with zone of inhibition of 5±1mm. At concentration of 5.0mg/ml, both P. aeruginosa and K. ozanae had the same zone of inhibition of 8±2mm followed by S. aureus (5±1mm) and the least zone of inhibition of 4±1mm was recorded in E. coli.

Table 1. Phytochemical screening of the extracts Components A. indica Alkaloid - Tannins - Phenolics - Glycoside - Saponin + Flavonoid - Phlobtanin + Triterpenes -

Table 2. Antimicrobial activities of extract of A. indica Organisms Concentration of extract of A. Indica 500mg/ml 50mg/ml 5.0mg/ml P.aeruginosa 14±2mm 10±2mm 8±2mm K. ozanae 10±2mm 7.7±0.6mm 8±2mm S.aureus 12±2mm 8±2mm 5±1mm E. coli 8±2mm 5±1mm 4±1mm

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of extract

Table 3 shows the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the test organisms on the extract of A. indica. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 5mg/ml and 50mg/ml respectively for P. aeruginosa, Kl. ozanae, S. aureus and E. coli.

Table 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of extract Organisms MIC (mg/ml) MBC (mg/ml) P.aeruginosa 5 50 K.ozanae 5 50 S. aureus 5 50 E. coli 5 50

Susceptibility testing using standard antibiotics (Positive control)

Table 4 shows the susceptibility test results of some selected standard antibiotics. P. aeruginosa and S. aureus were susceptible to Amoxacillin (AMX) but K. Ozanae and S. aureus were resistant. All the test organisms were susceptible to Rifampin (RF) and Gentamycin (GEN), K. ozanae, S.aureus and E.coli were susceptible to Augmentin (AU) but P. aeruginosa was resistant. P. aeruginosa, K. ozanae and E.coli were susceptible to Streptomycin (S) but S.aureus was resistant. None of the test organisms were susceptible to Ciptrofloxacin (CPX) and Ceporex (CEP).

Table 4. Susceptibility testing using standard antibiotics (Positive con

Susceptibility testing using standard antibiotics (negative control)

Table 5 shows the susceptibility of the test organisms to some commonly used antibiotics K.ozanae and S. aureus were susceptible to Gentamycin P. aeruginosa were resistant. S.aureus was susceptible to Ciprofloxacin (CPX) but S. aureus, K ozanae and E.coli were resistant. P.aeruginosa, S. aureus and E.coli are susceptible to Ampicillin (AMP) but K. ozanae was resistant. Only P. aeruginosa was susceptible to AU- Augmentin (AU) and Streptomycin (S).

Table 5. Susceptibility testing using standard antibiotics (negative control)

4. Discussion

The photochemical screening of A. indica extract indicated the presence of saponin and phlobatanins (Table 1), the presence of these compounds may be responsible for the antibacterial activities of the extracts of A. indica on the test organisms. The phytochemical components of the A. indica have been established in previous studies and these include tannins, saponins, alkaloids, carbohydrates, phenols, flavonoids, anthraquinones, cardiac glycosides, sterols and resins (De and Ifeoma, 2002; Natarajan et al., 2003; Biswas et al., 2002, El-Mahmood et al,. 2010). Several studies have linked presence of these bioactive compounds in plant materials to antimicrobial activity.

The presence of these secondary metabolites in plants, produce some biological activity in man and animals and it is responsible for their use as herbs. These compounds also serve to protect the plant against infection by microorganisms, predation by insects and herbivores, while some give plants their odors and or flavors and some still are responsible for their pigments (El-Mahmood et al,. 2008). In some cases, the activity has been associated with specific compounds or classes of compounds. These active constituents can be used to search for bioactive lead compounds that could be used in the partial synthesis of more useful drugs (Ogbonnia et al., 2008; El-Mahmood et al., 2010).

The antibacterial effects of A. indica on the test organisms revealed that P. aeruginosa showed the highest zones of inhibition (mm) followed by S. aureus while E. coli had the least zone of inhibition (mm) at various extract concentrations of 500mg/ml, 50mg/ml and 5mg/ml (Table 2). Compared to commonly applied antibiotic (Tables 4 and Table 5), bark extracts of A. indica showed a higher value of zones of inhibition on the tested organisms. In a similar study hexane and aqueous extract of Azadirachta indica, inhibited Escherichia coli, P. aeruginosa, S. pyogenes and S.aureus (El-Mahmood et al.,2010).

The test organisms had the same minimum inhibitory concentration (MIC) value of 5mg/ml and minimum bactericidal concentration (MBC) value of 50mg/ml. This indicated that the bark of A. indica has similar potency on P. aeruginosa, Kl. ozanae, S. aureus and E. coli. This is similar to the findings of the National Library of Medicine at the National Institutes of Health (www.pubmed.com) who reported that in test tubes A. indica has been shown to have significant effects on both gram-positive and gram-negative organisms and other bacteria that cause a wide array of human and animal diseases.

5. Conclusions

The results of this study suggest that the bark of A. indica can be used as an antibacterial agent against infections caused by P. aeruginosa, K. ozanae, S. aureus and E. coli.