Susceptibility Profile of MethicillinResistant Staphylococcus aureus (MRSA) Isolates to Antibiotics and Methanolic Extracts of Parkia biglobosa

A. F. Obajuluwa, J. A. Onaolapo, A. R. Oyi1 and B. O. Olayinka

Purpose of the Study

To study the susceptibility profile of methicillin-resistant Staphylococcus aureus (MRSA) isolates from orthopaedic patients to antibiotics and methanolic extracts of Parkia biglobosa

Staphyloccocus
Gram-positive cocci

Facultative aerobes
Produce acid from glucose both aerobically and anaerobically

Resistant to drying
Tolerate high salt concentrations Forms cell clusters Normal microbial flora of upper respiratory tract Noses, Mouth and Skin Common parasites of humans and animals Cause serious infections

2 Major Species of Staphylococcus Staphylococcus Staphylococcus

epidermidis
Nonpigmented, nonpathogenic organism Found on skin or mucous membranes

aureus
Yellow-pigmented organisms Associated with pathological conditions like pimples, meningitis, arthritis, etc.

Staphylococcus aureus

Staphylococcus aureus
Able to resist the effects of an antimicrobial agent (methicillin) to which it is normally susceptible Methicillin-resistant Staphylococcus aureus was first discovered in 1961, they were isolates of S. aureus which have acquired genes encoding antibiotic resistance to all penicillins including methicillin and other narrow spectrum beta-lactamase resistant penicillin antibiotics. Methicillin
semi-synthetic penicillin Acid-stable Action: inhibit cell wall synthesis by blocking transpeptidation (cross-linking of two glycan-linked peptide chains)
Transpeptidase enzymes bind to penicillin -> penicillin-binding proteins: cannot catalyze transpeptidase reaction but cell wall synthesis continues -> formation of bacterial cell wall that is not cross-link and cannot maintain its strength

Staphylococcus aureus
Widespread hospital pathogen Pneumonia, Blood infections, Food poisoning, Carditis, Meningitis, Arthritis, Acne, Boils, Pimples

Infections occur when resistance of host is low because of hormonal changes, illnesses, wounds or treatment with drugs that compromise immunity
Most individuals are carriers, and infections can be transmitted by simple contact: prevention is hard Resistant to common antibiotics like penicillin-> treatment is very difficult Problem: Search for alternative anti-staphylococcal agents

Susceptibility Profile of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates to Antibiotics and Methanolic Extracts of Parkia biglobosa
Staphylococcal isolates from wound, skin and bed swab samples from orthopedic patients in Nigeria
Delay in the union of fractured bones

Parkia biglobosa: the African locust bean tree

Parkia biglobosa
Occur in agroecological zones from tropical rainforest where rain is high to arid zone where rain is low Bark used as mouthwash, vapour inhalant for toothache, ear complaints Extract used for leprosy, bronchitis, pneumonia, sores, etc. Leaves and roots used in lotion for sore eyes, hemorrhoids, toothache Seed taken for tension and pulp for fevers

Methodology
Suspected staphylococcal isolates: wound, skin and bed swabs Detection of Methicillin Resistance: isolates were spotinoculated onto MHA agar with 6g/mL methicillin: isolates with growth are methicillin resistant Antibiotic Sensitivity Test: Kirby Bauer-NCCLS (modified disc agar diffusion technique)
Isolates on NA plates w/ 3mL phosphate buffer solution and turbidity adjusted to .5 McFarland standard MHA plate inoculated with bacterial suspension by streaking in 3 directions and was allowed to dry Antibiotic discs were applied: methicillin, ampicillin, vancomycin, oxacillin, gentamicin, ceftriaxone, pefloxacin and

Collection and Authentication of Parkia biglobosa: leaves, roots and stem bark samples were air dried, ground into powder and was extracted using soxhlet apparatus for methanol: solvent was then removed and methanolic extract was stored in desiccator until used

Methodology
Phytochemical Screening: presence of saponins, tannins, flavonoids, carbohydrates, alkaloids and steroids
Plant chemicals that have protective or disease preventive properties

Antibacterial Activity of Crude Methanolic Extract of Leaves, Roots and Stem Bark of Parkia biglobosa to MRSA isolates:
Overnight broth culture of each isolate was inoculated at MHA medium at 45*C, allowed to set, wells were made using standard cork borer and various concentrations of plant extract were added to each well Plates were allowed to stand at room temp for 1 hr and incubated at 37*C for 24 hrs. Diameter of each zone of inhibition was measured after incubation.

Results

Phytochemical screening

Leaves

Roots

Stem Bark

Anti-staphylococcal properties of Parkia biglobosa

Stem bark showed the greatest activity against MRSA isolates Rich in flavonoids, tannins and saponins
Secondary metabolites that have antibacterial activities
Organic compounds formed near the end of growth so they are nonessential for growth and reproduction Often overproduced Some roles: Protective agents (antioxidants, UV light absorbing, free radical-scavenging) Defend plants against microbes Feeding deterrence: toxicity of phytochemicals

Anti-staphylococcal properties of Parkia biglobosa

Secondary Metabolites found in Parkia biglobosa
Stem bark rich in sterols, triterpenes, tannins, saponosides, anthocyanins, flavonoids, coumarins and reducing compounds Leaf rich in tannins, coumarins, anthocyanins, flavones and reducing compounds Root contains saponins, glycosides, tannins and a trace of alkaloids

These metabolites may be responsible for the observed antibacterial activities

END
(or staph-ylococcus)

References
Microbiology, An Introduction 10th Ed by Tortora, Funke, Case Brock Biology of Microorganisms 13th Ed by Madigan, Martinko Stahl, Clark Phytochemicals: http://www.opc-1-2-3.com/antioxidantphytochemicals.html

Secondary Metabolites: http://advances.nutrition.org/content/2/1/32.full