Full Project – ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT AND FRACTIONS OF MORINGA OLEIFERA LAM. ROOT BARK ON CLINICAL ISOLATES OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

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ABSTRACT

Development of antimicrobial resistance by bacteria is now a world wide health issue, as infection is one of the leading causes of death in the world today. This fact is also as a result of the emergence of multiple antibiotic resistant bacteria known as methicillin resistant Staphylococcus aureus (MRSA) with potential of cross resistance to other antibiotics of

choice like vancomycin. MRSA is often referred to as a potential killer and one of the tree top superbugs in hospitals multidrug resistant organisms (MDRO). The aim of this study was to evaluate the phytochemical components and antimicrobial activity of methanol extract and fractions of Moringa oleifera root bark as possible remedy for MRSA infections.

Staphylococcus aureus isolates from 3 different hospitals in South-east geopolitical region of Nigeria were confirmed by coagulase/staphylase test using Oxoid® reagents kits (DR0595A). The characterised S. aureus isolates were further identified as Methicillin resistant staphylococcus aureus by disc diffusion method as recommended by the Clinical Laboratory Standards Institute (CLSI), using standard antibiotic discs containing oxacillin (5 μg/ml), vancomycin (30 μg/ml), cephalexin (30 μg/ml), levofloxacin (5 μg/ml), ciprofloxacin (5 μg/ml), tetracycline (30 μg/ml), cotrimoxazole (25 μg/ml), gentamicin (30 μg/ml), clindamycin (2 μg/ml) and rifampicin (5 μg/ml). Methicillin resistant staphylococcus aureus

confirmation was done using Oxoid® DR0900 penicillin binding protein (pbp2ˈ) latex agglutination test kits. Pulverised Moringa oleifera root bark was defatted with n-hexane to yield hexane fraction (HEF). The dried marc was extracted with methanol using Soxhlet extractor to obtain crude methanol extract (ME). Methanol extract was adsorbed on Silical gel (60-200 mesh) and eluted in succession to obtain dichloromethane fraction (DMF), ethyl acetate fraction (EAF) and methanol fraction (MEF). Qualitative phytochemical analyses of the extracts were carried out using standard procedures. The antimicrobial activities of ME, HEF, DMF, EAF and MEF were evaluated on the MRSA, the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were recorded and compared with the standard disc antimicrobial test results. The extract fractions were analysed using gas chromatographic-mass spectrometry (GC-MS) for their bioactive compounds. The preliminary acute toxicity and sub-acute toxicity of ME and HEF were evaluated. Statistical analysis was done with ANOVA followed by Duncan post Hoc test using SPSS v 17 software. Characterised clinical isolates yielded 58 S. aureus strains. Antibiotic susceptibility tests indicated varied percentages of MRSA that were resistant to various antibiotics thus: oxacillin (62.1 ± 3.2%), vancomycin (60.4 ± 3.8%), cephalexin (55.2 ± 1.2%), levofloxacin (56.9 ±

2.2%), ciprofloxacin (56.9 ± 0.9%), tetracycline (65.5 ± 2.3%), cotrimoxazole (68.9 ± 0.8%), gentamicin (67.2 ± 1.3%), clindamycin (62.1 ± 3.3%) and rifampicin (62.1 ± 4.1%). Latex agglutination test confirmed 39 strains of the clinical isolates to be MRSA. The S. aureus isolates resistant to all the antibiotics including vancomycin at 30 μg/ml were sensitive to the

extract and all its fractions: ME: MIC (3.0 ± 0.1 to 5.0 ± 0.5 mg/ml) and MBC (3.0 ± 0.1 to 6.0 ± 0.5 mg/ml); EAF: MIC (5.0 ± 1.1 to 8.0 ± 0.5 mg/ml) and MBC (5.0 ± 0.5 to 8.0 ± 0.5 mg/ml); DMF MIC (8.0 ± 1.1 to 10 ± 0.5 mg/ml) and MBC (8.0 ± 0.5 to 10 ± 0.5 mg/ml); HEF: MIC (7.0 ± 0.5 to 8 ± 1.1 mg/ml) and MBC (7.0 ± 0.5 to 9 ± 0.5 mg/ml), MEF: MIC (9.0 ± 1.1 to 10.0 ± 0.5 mg/ml) and MBC (9.0 ± 0.5 to 10.0 ± 0.5 mg/ml).

Phytochemical analysis of the extracts showed the presence of alkaloids, glycosides, steroids, terpenoids, flavonoids, saponins, tannins, resins, reducing sugars, proteins, fats and oil and carbohydrates. GC-MS analysis revealed over 100 distinct compounds, some of which are stigmasterol (C29H48O), eugenol (C10H12O2), oxime (C3H7NO ) and ergosta-4, 22-dien-3-one (C28H44O). The oral acute toxicity test showed the LD50 of ME as 3663.96 mg/kg and HEF as 1934.15mg/kg, with no significant change (P > 0.05) in the hematological, serum biochemical parameters and weight of the rats.

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