This document appears to be a dissertation submitted to Jawaharlal Nehru Technological University in partial fulfillment of a degree in pharmacognosy. It discusses the development of antibiotic resistance and explores using plant extracts to inhibit efflux pumps in drug-resistant bacteria. The dissertation tests extracts from 6 plants, including Annona squamosa and Talinum triangulare, against resistant strains of bacteria alone and in combination with antibiotics. It finds that some plant extracts combined with doxycycline or ofloxacin were effective against drug-resistant Staphylococcus, Klebsiella, and E. coli strains.

1. Gali Punithkumar11421R0019
KambamVenkata Nikhila 11421R0031
Kempili Anand11421R0034
Seela HemanthKumar 11421R0054
Dissertation Submitted to
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY, ANANTAPUR
ANANTHAPURAMU
In the partial fulfilment for the award of degree of
Under the guidance of
Sri. D. Jamal Basha, M. Pharm.
AssistantProfessor
Department of Pharmacognosy
SRI PADMAVATHI SCHOOL OF PHARMACY
(Affiliated to J.N.T.U.A)

2. Introduction

3. PRE-ANTIBIOTIC ERA – before 20th century
Germ theory of disease
Golden age of antibacterials –
first half of 20th century
Discovery of penicillin
Post golden age – till now
Development of resistance

4. Classification of antibacterial agents
Mode of action
Spectrum of activity
Effect on bacteria
These are classified in several ways

5. Natural sources
– potential and safer antibacterial agents
Chalmoogric oil
Tea tree oil
Berberine
Garlic
And many more….

6. Literature review

7. Antibiotic resistance
Causes
Consequences
Spread
Biological mechanism of resistance Genetic mechanism of resistance

8. Enzymatic inactivation
Decreased permeability
Efflux pumps
Alteration in target site
over production of target molecules
Mechanism of resistance

9. Efflux pumps

10. Inhibitors of efflux pumps as novel
agents for combination therapy for
MDR bacterial infections

11. Plants selected
 Annona squamosa
 Morus alba
 Talinum triangulare
 Tamilnadia uliginosa
 Vigna unguiculata
 Vitex leukoxylon

12. Selection of pathogenic
bacteria
Primary screening of
bacteria
Development of mutants
Screening for resistance
development
Culture of resistance
bacteria
Random selection of plants
Testing of antibacterial
activity
Primary screening of the
phytochemicals for EPI
activity

14.  To detect EPI activity of selected plants
 To detect antimicrobial activity along with EPI activity
 To detect chemical moiety responsible for EPI activity
 To detect the pump which will inhibit by phytochemicals
 To detect the effect of combination of phytochemical
and antibiotic on resistant mutants

18. Mc Farland standard
McFarland standard No. 0.5 1 2 3 4
1% Barium chloride (ml) 0.05 0.1 0.2 0.3 0.4
1% Sulfuric acid 9.95 9.9 9.8 9.7 9.6
Approx. cell density (1X108)
CFU/ml
1.5 3 6 9 12
% Transmmittance 74.3 55.6 35.6 26.4 21.5
Absorbance 0.08 to 0.1 0.256 0.251 0.582 0.669

19. Production of resistant mutants

20. Plant extracts
Name of the
plant
Family Part
used
Solvent
system
Stock solution
concentration
Dilution range in
six concentrations
Annona
squamosa
Annonaceae Leaf Hydroalcoholi
c extract
0.3mg/ml 5 – 100 mcg/ml
Morus alba Moreaceae Leaf Hydroalcoholi
c extract
0.1mg/ml 2 – 25 mcg/ml
Talinum
triangulare
Talinaceae Leaf Hydroalcoholi
c extract
12mg/ml 50 – 4000 mcg/ml
Tamilnadia
uliginosa
Rubiaceae Leaf Hydroalcoholi
c extract
15mg/ml 50 – 5000 mcg/ml
Vigna
unguiculata
Fabaceae Leaf Hydroalcoholi
c extract
0.3mg/ml 5 -100 mcg/ml
Vitex
leukoxylon
Lamiaceae Leaf Hydroalcoholi
c extract
3mg/ml 50 – 1000 mcg/ml

22. Bacterial response for selected antibiotics
and plant extract combination
1 2 3 4 5 6 7 8 9 10 11 12
A R1 R2 R3 R4 R5 R6 R1 R2 R3 R4 R5 R6
B R1+A1 R1+ A2
C R1+ P11 R1+ P22
D R1+ P12 R1+ P21+A1
E R1+ P11+A1 R1+ P22+A1
F R1+ P12+A1 R1+ P21+A2
G R1+ P11+A2 R1+ P22+A2
H R1+ P12+A2 R1+ P22+A2

24. Results and discussion

25. 0
0.5
1
1.5
2
2.5
phytochemical phytochemical with ofloxacin phytochemical with
doxycycline
Opticaldensity
Phytochemical combination
Doxycyline resistance against Annona squamosa
staphylococus strptococcus enterococcus klebsiella psudomonads E.coli
0
0.5
1
1.5
2
2.5
phytochemical phytochemical with
ofloxacin
phytochemical with
doxycycline
Opticaldensity
Phytochemical combination
Ofloxacin resistance against Annona squamosa
staphylococus strptococcus enterococcus klebsiella psudomonads E.coli

26. 0
0.5
1
1.5
2
2.5
3
3.5
phytochemical phytochemical with
ofloxacin
phytochemical with
doxycycline
Opticaldensity
phytochemical combination
Doxycycline resistance against
Talinum triangulare
staphylococus strptococcus enterococcus klebsiella psudomonads E.coli
0
0.5
1
1.5
2
2.5
phytochemical phytochemical with
ofloxacin
phytochemical with
doxycycline
Opticaldensity
Phytochemical combination
Ofloxacin resistance against Talinum
triangulare
staphylococus strptococcus enterococcus klebsiella psudomonads E.coli

27. conclusion

28. Annona squamosa
Antibiotics
Hydroalcoholic leaves extracts of Annona
squamosa
Annona squamosa
and Doxycycline
combination
Annona squamosa and Ofloxacin combination
Ofloxacin resistant
Staphylococcus
Klebsiella
E.coli
Doxycyclin resistant
Staphylococcus
Klebsiella
e.Coli

29. Antibiotic
leaves extracts + Doxycycline in
combination with
combination of
Ofloxacin.
The Hydroalcoholic leaves extracts of
Talinum triangulare
Ofloxacin
resistant
species
Talinum triangulare

32. References
 Keyes K, Lee MD, Maurer JJ. 2003. Antibiotics: Mode of Action, Mechanisms of
Resistance and Transfer. Microbial Food Safetry in Animal Agriculture Current Topics. ME
Torrence and RE Isaacson, eds. Iowa State Press, Ames, Iowa, USA.
 Hare R. 1983. The Scientific Activities of Alexander Fleming, Other Than the Discovery
of Penicillin. Medical History. 27:347-372.
 Gootz TD. 1990. Discovery and Development of New Antimicrobial Agents. Clinical
Microbiology Reviews 3(1)13-31.
 Girija S Singh, Surendra N. Pandeya. Natural products in discovery of potential and
safer antibacterial agents. Opportunity, Challenge and Scope of Natural Products in
Medicinal Chemistry, 2011: 63-101.
 Hare R. The Scientific Activities of Alexander Fleming, Other Than the Discovery of
Penicillin. Medical History. 1983; 27:347-372.
 Chopra I, Roberts M. Tetracycline Antibiotics: Mode of Action, Applications, Molecular
Biology and Epidemiology of Bacterial Resistance. Microbiology and Molecular Biology
Reviews. 2001; 65(2):232-260.