bacterial efflux pump inhibitory activity of some indigenous medicinal plants using 96 well micro...

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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.

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