DISSERTATATION PRESENTATION

2018-2019

Effects Of Moringa oleifera Leaves on

Methicillin Resistant Staphylococcus aureus
(MRSA) Isolated from Rajasthan

SUPERVISOR:- PRESENTED BY:-

Ms. APOORVA RANA SAUMYA SRIMUKHI
BSC BIOTECH III
 CONTENT :-
INTRODUCTION
REVIEW OF LITERATURE
OBJECTIVES
METHODOLOGY
RESULTS
CONCLUSION
SCIENTIFIC PRESENTATION
MRSA (METHICILLIN RESISTANT
Staphylococcus aureus ):-
MRSA is a form of contagious bacrterial infection that is resistant to numerous antibiotics
including methicillin which makes it challenging to treat.

It may begins as a skin sore, pimple or boil before becoming serious , potentially harmful
and sometimes fetal.

Staphylococcus aureus refers to bacterium that commonly results inside the nose and on
human skin .

Around one in three people carry staph in their nose , usually without any iilness. Two in
100 people carry MRSA.

It is caused by bacterium strains that have acquired a resistance to particular antibiotics .

It can spread from person to person and person to object when an individual has active
MRSA or is colonised by the bacteria.
 MORINGA OLEIFERA
Moringa oleifera, native to India , grows in tropical and subtropical regions of the world,
which is commonly known as ‘drumstick tree’.

The leaves are rich in minerals, vitamins and other essential phytochemicals .

The exracts from leaves are used to treat malnutrition , augment, breast milk in lactating
mothers. It is used as potential antioxidant , anticancer and inflammatory , antidiabetic and
antimicrobial agent.

The leaves of Moringa oleifera used for phytochemical screening.

 OBJECTIVES :-
 Isolation and identification of Methicillin Resistant Staphylococcus aureus (MRSA) from
clinical samples
 To evaluate the antibacterial potentials of the crude extracts of leaves of Moringa oleifera
against Methicillin Resistant Staphylococcus aureus (MRSA) isolated from clinical samples
 To screen the phytochemicals of the plant extracts responsible for showing the antibacterial
activity against MRSA isolates
 Extraction of the pure compound and the screening of the phytoconstituent responsible for
the antibacterial potential of Moringa oleifera leaves against MRSA isolates by the method of
Bioautography
 METHODOLOGY :-
Isolation and identification of Methicillin –resistant Staphylococcus aureus(MRSA)

Isolation of bacterial Identification of

isolates from clinical Identification of
S.aureus by
sample of infected MRSA by double
biochemical
patients by streaking disk diffusion test
testing
method

Collection and processing of Moringa oleifera leaves

Processing of Grinding of
Collection of dried leaves in
plant ( washing
Moringa oleifera the form of fine
and shade
leaves powder
drying )
Evaluation of the antibacterial potential of the crude extracts of leaves of Moringa oleifera
against Methicillin – resistant Staphylococcus aureus from clinical sample

Anti-microbial activity
Plant extraction using of crude extracts against
different solvents by soxhlet MRSA by Kirby-baeur
method test

Qualitative and quantitative screening of phytochemicals , extraction of pure compound

and screening of phytoconstituent responsible for the antibacterial potential of medicinal
plants agaainst MRSA isolates
Qualitative
screening of Quantitative Extraction of
phytochemicals from estimation of phytochemical
plant extracts present found in detectable
showing phytochemicals limit
antibacterial
potential

Screening of
phytoconstituent
responsible for the
antibacterial potential
of plant extract by
bioautography
RESULTS
Figure 1:- Isolation of bacterial isolates from clinical samples of infected
patients by streaking method
 Gram’s Staining for Microscopic Identification

Catalase Test for Biochemical Identification Coagulase Test for Biochemical Identification

Figure 2:- Microscopic and Biochemical Identification for Staphylococcus aureus

 Cefoxitin Cefoxitin

Penicillin Penicillin

MNC-3202 C - 191

Figure 3:- Antibiotic susceptibility pattern by Kirby Bauer Method (Disk

Diffusion Test) for MRSA Identification
(For MRSA – Penicillin G ≤ 28 mm and Cefoxitin ≤ 21 mm)
 LEAVES LEAVES

Figure 4:- Washing & Shade drying of

Moringa oleifera Leaves

LEAVES
 Methanolic
Extract of
Moringa leaves

Ethanolic
Extract of
Moringa leaves

Figure 5:- Plant extraction of different solvents using Soxhlet Extraction Method
 Isolate PC 100 mg/ml 200 mg/ml 400 mg/ml NC

S. aureus 32 10 10 11 0

MRSA 27 11 12 13 0

C - 191 35 13 14 15 0

85/30 36 12 13 14 0

C - 2156 34 11 12 13 0

CDC - 519 33 11 13 15 0

MNC - 3202 30 12 13 14 0

CDC - 553 30 10 11 12 0

C - 1954 30 7 9 11 0

CDC - 404 29 7 9 13 0

MSC - 308 20 6 7 8 0

CDC - 361 28 8 10 12 0

71/746 32 7 10 13 0

Mean 30.6±4.4 9.5±2.5 11±2.2 12.7±2 0

Table 1:- Antibacterial Activity of Ethanolic Extracts of Moringa oleifera Leaves

 40

35

30
Mean Zone of Inhibition (mm)

30.6

25

20

15

12.7
10
11
9.5
5

0
0
Positive Control 400 mg/ml 200 mg/ml 100 mg/ml Negative Control
Concentration

Graph 1:- Antibacterial Activity of Ethanolic Extracts of Moringa oleifera Leaves

 Isolate PC 100 mg/ml 200 mg/ml 400 mg/ml NC

S. aureus 32 9 9 10 0

MRSA 27 10 11 12 0

C - 191 35 7 9 11 0

85/30 36 7 10 13 0

C - 2156 34 7 9 11 0

CDC - 519 32 10 11 12 0

MNC - 3202 30 9 11 12 0

CDC - 553 30 10 12 12 0

C - 1954 31 6 7 8 0

CDC - 404 29 7 8 9 0

MSC - 308 19 6 7 8 0

CDC - 361 28 11 12 13 0

71/746 31 7 8 9 0

Mean 30.5±4.5 7.9±1.8 9.5±1.9 10.7±1.9 0

Table 2:- Antibacterial Activity of Methanolic Extracts of Moringa oleifera Leaves

 40

35

30
Mean Zone of Inhibition (mm)

30.5

25

20

15

10
10.7
9.5
7.9
5

0
0
Positive Control 400 mg/ml 200 mg/ml 100 mg/ml Negative Control
Concentration

Graph 2:- Antibacterial Activity of Methanolic Extracts of Moringa oleifera Leaves

Anti-MRSA Activity of Moringa oleifera Anti-MRSA Activity of Moringa oleifera
Leaves – Ethanolic extract Leaves – Methanolic extract

PC PC

NC NC
100 mg 400 mg 100 mg
400 mg

200 mg 200 mg

Figure 6:- Antibacterial activity of Moringa oleifera leaves

 Moringa leaves Moringa leaves (Methanolic
(Ethanolic Extract) Extract)

Steroids + +

Phenols _ _

Phlobatanins _ _

Quinones _ _

Coumarins _ _

Flavonoids + +

Volatile oils + +

Terpenoids _ _

Reducing sugars _ _

Cardiac glycosides _ _

Alkaloids + _

Saponins _ _

Tannins + _

Table 3:- Qualitative Estimation of Phytochemicals in Moringa oleifera Leaves

 Alkaloids Flavonoids Tannins

Figure 7 :- Qualitative Screening of Phytochemicals of Moringa oleifera Leaves

 Total Tannin Content (TTC) in Ethanolic Extract of Moringa oleifera Leaves
0.8
y = 0.0034x + 0.3817
0.75 R² = 0.9495
0.7 TTC = 90.5 mg/ml
0.6995
0.65 0.6895
0.6 0.6005
0.55
0.5
0.476
Absorbance

0.45
0.4 0.4145
0.3795
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0 20 40 60 80 100 120
Concentration (mg/ml)

Graph 3:- Quantitative Estimation of Tannins in Ethanolic Extract of

Moringa oleifera Leaves
Standard – Gallic Acid (Concentration -100 mg/ml)
 Total Flavonoid Content (TFC) in Methanolic Extract of Moringa oleifera Leaves
0.085
0.08
0.075
0.073
0.07
0.065
0.06
y = 0.0008x - 0.0014
0.055
Absorbance

R² = 0.9778
0.05
TFC = 306.75 mg/ml
0.045 0.045
Absorbance = 0.244
0.04
0.035
0.03
0.025
0.02 0.019
0.015
0.01
0.005 0.006
0 0.001
0 20 40 60 80 100 120
Concentration (mg/ml)

Graph 4:- Quantitative Estimation of Flavonoids in Methanolic Extract of

Moringa oleifera Leaves
Standard - Quercetin (Concentration -100 mg/ml)
 Flavonoid Flavonoid Flavonoid
Extraction Extraction Extraction
(Petroleum (Diethyl ether (Ethyl acetate
ether fraction) fraction) from fraction) from
from Moringa Moringa Moringa
Leaves Leaves Leaves

Figure 8 :- Flavonoid Extraction

from Moringa oleifera
Leaves
 Standard – Quercetin
PE – Fraction 1 (Petroleum Ether)
DE – Fraction 2 (Diethyl Ether)
EA – Fraction 3 (Ethyl Acetate)

TLC of
Flavonoid in
Standard PE DE EA Moringa Leaves

Figure 9 :- TLC Plate

Figure 10 :- Bio-autography (Plates

after spraying Tetrazolium salt)
Flavonoid in Moringa Leaves
 CONCLUSION
 The antibacterial potentials of Ethanolic and Methanolic extracts of Moringa
oleifera leaves are (12.7±2) and (10.7±1.9).
 Of the two solvents (Methanol & Ethanol) used for extract preparation, results have
shown that ethanolic extracts show better antibacterial activity.
 As per the quantitative estimation, the total flavonoid content in methanolic extracts
of Moringa oleifera leaves was 306.75 mg/ml.
 The TLC of the Methanolic Extracts of M. oleifera leaves provided compounds
with the Rf values of 0.1, 0.15, 0.22, 0.69, 0.74 and 0.8.
 The agar-overlay bioautography of the isolated flavonoids showed clear zones
thereby confirming the inhibitory activities against selected MRSA isolates.
 Traditional medicines have emerged as a boon in medical sciences as they are
readily available and have almost no side effects.
 The phytoconstituents responsible for the antibacterial potentials can be extracted
from the plant and can lead to the development of novel therapeutic agents capable
of controlling or curbing the problem of drug resistance.
REVIEW OF LITERATURE

 Gopalakrishnan, L., Doriya, K., & Kumar, D. S. (2016). Moringa oleifera: A review on nutritive importance
and its medicinal application. Food Science and Human Wellness, 5(2), 49-56.
 Otarigho, B., & Falade, M. O. (2018). Analysis of antibiotics resistant genes in different strains of
Staphylococcus aureus. Bioinformation, 14(3), 113.
 Joshi, B., Sah, G. P., Basnet, B. B., Bhatt, M. R., Sharma, D., Subedi, K., ... & Malla, R. (2011).
Phytochemical extraction and antimicrobial properties of different medicinal plants: Ocimum sanctum
(Tulsi), Eugenia caryophyllata (Clove), Achyranthes bidentata (Datiwan) and Azadirachta indica
(Neem). Journal of Microbiology and Antimicrobials, 3(1), 1-7.
 Tambekar, D. H., Dhanorkar, D. V., Gulhane, S. R., Khandelwal, V. K., & Dudhane, M. N. (2006).
Antibacterial susceptibility of some urinary tract pathogens to commonly used antibiotics. African Journal
of Biotechnology, 5(17).
 Akhter R, Khan KMA and Hassan F (2009). Isolation and antimicrobial susceptibility pattern of methicillin
resistant and sensitive Staphylococcus aureus. J. Surgery Pak., 14: 161-165.
 Abdul Wadood, Mehreen Ghufran, Syed Babar Jamal, Muhammad Naeem, Ajmal Khan, Rukhsana Ghaffar
and Asnad. Phytochemical Analysis of Medicinal Plants Occurring in Local Area of Mardan. Biochemistry
& Analytical Biochemistry. 2(4).2-4.
 Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H., Acevedo Arozena, A., ... &
Adhihetty, P. J. (2016). Guidelines for the use and interpretation of assays for monitoring
autophagy. Autophagy, 12(1), 1-222.
Participate in poster presentation:-
International conference on Biomedical Engineering, Bioscience,
Bioinformatics, Biochemistry, Cancer Biology, Molecular Biology and
Applied Biotechnology(BCM-2019).