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.