bacterial efflux pump inhibitory activity of some indigenous medicinal plants using 96 well micro...
TRANSCRIPT
Bacterial efflux pump inhibitory activity of some indigenous medicinal plants using 96 well micro titer
based method.
Gali Punith kumar 11421R0019Kambam Venkata Nikhila 11421R0031Kempili Anand 11421R0034Seela Hemanth Kumar 11421R0054
Dissertation Submitted to JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY, ANANTAPUR
ANANTHAPURAMU
In the partial fulfilment for the award of degree of
Under the guidance ofSri. D. Jamal Basha, M. Pharm.
Assistant ProfessorDepartment of Pharmacognosy
SRI PADMAVATHI SCHOOL OF PHARMACY(Affiliated to J.N.T.U.A)
BACHELOR IN PHARMACY
Introduction
PRE-ANTIBIOTIC ERA – before 20th centuryGerm theory of disease
Golden age of antibacterials – first half of 20th century Discovery of penicillin
Post golden age – till nowDevelopment of resistance
Classification of antibacterial agents
Mode of action
Spectrum of activity
Effect on bacteria
These are classified in several ways
Natural sources – potential and safer antibacterial agents
Chalmoogric oilTea tree oilBerberineGarlicAnd many more….
Literature review
Antibiotic resistance
CausesConsequencesSpread
Biological mechanism of resistance
Genetic mechanism of resistance
Enzymatic inactivationDecreased permeability
Efflux pumps Alteration in target siteover production of target molecules
Mechanism of resistance
Efflux pumps
Inhibitors of efflux pumps as novel agents for combination therapy for MDR bacterial infections
Plants selected Annona squamosa Morus alba Talinum triangulare Tamilnadia uliginosa Vigna unguiculata Vitex leukoxylon
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
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
Mc Farland standardMcFarland 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
Production of resistant mutants
Plant extracts
Name of the plant
Family Part used
Solvent system
Stock solution concentration
Dilution range in six concentrations
Annona squamosa
Annonaceae Leaf Hydroalcoholic extract
0.3mg/ml 5 – 100 mcg/ml
Morus alba Moreaceae Leaf Hydroalcoholic extract
0.1mg/ml 2 – 25 mcg/ml
Talinum triangulare
Talinaceae Leaf Hydroalcoholic extract
12mg/ml 50 – 4000 mcg/ml
Tamilnadia uliginosa
Rubiaceae Leaf Hydroalcoholic extract
15mg/ml 50 – 5000 mcg/ml
Vigna unguiculata
Fabaceae Leaf Hydroalcoholic extract
0.3mg/ml 5 -100 mcg/ml
Vitex leukoxylon
Lamiaceae Leaf Hydroalcoholic extract
3mg/ml 50 – 1000 mcg/ml
Bacterial response for selected antibiotics and plant extract combination 1 2 3 4 5 6 7 8 9 10 11 12A 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
Results and discussion
phytochemical phytochemical with ofloxacin
phytochemical with doxycycline
0
0.5
1
1.5
2
2.5Doxycyline resistance against Annona
squamosa
staphylococus strptococcus enterococcus klebsiella psudomonads E.coli
Phytochemical combination
Optic
al d
ensit
y
phytochemical phytochemical with ofloxacin
phytochemical with doxycycline
0
0.5
1
1.5
2
2.5
Ofloxacin resistance against Annona squamosa
staphylococus strptococcus enterococcus klebsiella psudomonads E.coli
Phytochemical combination
Optic
al d
ensit
y
phytochemical phytochemical with ofloxacin
phytochemical with doxycycline
0
0.5
1
1.5
2
2.5
3
3.5
Doxycycline resistance against Talinum triangulare
staphylococus strptococcus enterococcusklebsiella psudomonads E.coli
phytochemical combination
Optic
al d
ensit
y
phytochemical phytochemical with ofloxacin
phytochemical with doxycycline
0
0.5
1
1.5
2
2.5
Ofloxacin resistance against Talinum triangulare
staphylococus strptococcus enterococcus klebsiella psudomonads E.coli
Phytochemical combination
Optic
al d
ensit
y
conclusion
Annona squamosa
Antibiotics
Hydroalcoholic leaves extracts of Annona squamosa
Annona squamosa and Doxycycline combination
Annona squamosa and Ofloxacin combination
Ofloxacin resistantStaphylococcus
KlebsiellaE.coli
Doxycyclin resistantStaphylococcus
Klebsiellae.Coli
Antibiotic
leaves extracts + Doxycycline in combination with
combination of Ofloxacin.
The Hydroalcoholic leaves extracts of Talinum triangulare
Ofloxacin resistant species
Talinum triangulare
HAPPY TO ANSWERSKEPTICS….?
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.
