Evolution in Bacteria by the use of antibiotic

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Evolution in Bacteria by the use of antibiotic:

Evolution in Bacteria by the use of antibiotic Swarnaprava Behera, 10MS84 Sujeet Kumar Choudhary 10MS54 IISER Kolkata 02-04-2012

Project Overview:

Project Overview Bacterial evolution- Effect of antibiotic – it acts on three parts of the cell Bacterial protein synthesis Nucleic acid replication Cell wall biosynthesis How tetracycline acts : protein synthesis inhibition. T-RNA to M-RNA ribosome complex(30s).

How cells becomes resistant :

How cells becomes resistant When exposed, most dies, some survives having genetic recombination processes. They reproduce & all the descendants will have same resistance power. How bacteria responds to it? 1)enzymatic inactivation(tetracycline) 2)Ribosomal protection 3)Efflux

Key terms:

Key terms E.Coli :- Gram negative, rod shaped. Why we choose ? well developed(lab environment) easily available Life cycle- 20 min. E. coli- efflux & ribosomal protection. Tetracycline :-subclass of polyketides MIC- lowest conc. Of antibiotic which inhibits visible growth after overnight incubation.

Tetracycline :

Tetracycline

Statement of the Problem :

Statement of the Problem Expectation : When bacteria are exposed to harsh condition(bacteria may die) repeatedly, after some generation they can have developed the capability to survive under same situation.

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EXPERIMENTAL PART

Materials Required :

Materials Required Reagents Media – LB and LB Agar Composition : 0.5 % Peptone 0.3 % beef extract/yeast extract 1.5 % agar 0.5% NaCl

Materials Required :

Materials Required Reagents: Antibiotic – Tetracycline(1mg/ml) Bacteria - E. Coli Glass and Plastic Apparatus : Beaker Conical Flask (250 ml) Test Tubes Measuring Cylinder

Materials Required :

Materials Required Glass and Plastic Apparatus : Quartz cuvette Pipette (of diff. range) Others – cotton, aluminum foil, rubber band etc. Electrical Equipments : Spectrophotometer

Materials Required :

Materials Required Electrical Equipments : Incubator (37’c,150 rpm) Autoclave Machine(37’ c.45 min.) Laminar Air Flow

Procedure:

Procedure Day-1 Fresh culture of overnight grown bacteria were taken and OD was measured . 1 OD @ 600 nm ~ 1 X 10 9 CFU/ml Bacteria were grown at 5 different concentration of antibiotics. Concentration of bacteria were kept equal (~ 1 X 10 6 CFU/ml) through out the experiment.

Procedure:

Procedure Each test tubes were duplicated. 10 Petridis plate were prepared with LB Agar media. Fig.1-Test tubes with media only Fig.2- Petridis plates inside Laminar Air Flow

Data set-1:

Data set-1 Media [NB Media] (ml) Antibiotic (Tetracycline) ( ug /ml) Bacterial concentration (E.Coli) (CFU/ml) Sl. No. 5 0.1 1 X 10 6 1 5 0.5 1 X 10 6 2 5 1.0 1 X 10 6 3 5 2.0 1 X 10 6 4 5 5.0 1 X 10 6 5 OD of the fresh culture of Bacteria 1.44

Procedure:

Procedure Day-2 Growth were observed and MIC was determined. MIC = 0.5 ug /ml Fig.3- Grown culture at lowest concentration(0.1 ug /ml)

Procedure:

Procedure OD was measured(0.448 &0.450) of the grown bacteria at lowest antibiotic concentration(0.1 ug /ml). At lowest antibiotic concentration, the previously grown bacteria at 0.1ug/ml(antibiotic concentration) were grown overnight. [Named M1 and P1]. Again the bacterial concentration was ~ 1 X 10 6 CFU/ml

Procedure:

Procedure P1 was the name of duplicate test tube. The amount of media was same for M1 & P1 Fig.4- Grown bacteria in M1 and P1

Procedure:

Procedure Day-3 Bacteria were taken from M1and P1 and OD were observed. Again at lowest concentration it was grown overnight and named it M2 And P2. Like this the same procedure was repeated for 5 days.(For further generations)

Data Set-2:

Data Set-2 Bacteria taken from LB Media (ml) Antibiotic ( ug /ml) Bacteria (CFU/ml) Name Name Day Sl.No . OD of M1 = 0.645 OD of P1 = 0.639 M1 5 0.1 1 X 10 6 M2 P2 3 1 OD of M2 = 0.457 OD of P2 = 0.448 M2 5 0.1 1 X 10 6 M3 P3 4 2 OD of M3 = 1.630 OD of P3 = 1.600 M3 5 0.1 1 X 10 6 M4 P4 5 3 OD of M4 = 1.604 OD of P4 = 1.590

OD of different generations :

OD of different generations

Procedure:

Procedure Bacteria were taken from M4 and OD was measured. Again this bacteria were grown overnight at 5 different concentration of antibiotic. Data set is same as Data Set-1. Next day growth of the bacteria were observed.

Final observation :

Final observation Fig.4 – Growth of the bacteria at higher concentration(0.5 ug /ml)

Pictorial Comparison:

Pictorial Comparison Growth of bacteria before evolution Growth of bacteria after evolution Fig.3 Fig.5 0.1 ug /ml 0.1 Ug /ml 0.1 ug /ml 0.1 ug /ml 0.5 ug /ml 0.5 ug /ml

Observations:

Observations Growth of bacteria at higher concentration of antibiotic. Increment in MIC.(From 0.5 to 1.0 ug /ml) Bacteria has developed resistant power. Increased OD.

Conclusion:

Conclusion Bacteria were evolved over time. From observation we get the expected result. Difference is due to 3 day gap. 2 nd one is for they have developed a high resistance power.

How related to Social issues.:

How related to Social issues. Diseases are not cured. Bacteria getting resistant. Without prescription only 1 dose will do them resistant. 2 or 4 dose according to the Dr. can kill the bacteria(when life time Is known). Super germs. MRSA-Multidrug resistant staphylococcus Aureus .

Limitations :

Limitations Our convention 1 OD @ 600 nm ~ 1 X 10 9 CFU/ml is an approximate value. Many generation were kept for three days. We took only one species of bacteria. Triplication

More Information:

More Information Related readings Evolutionary Biology by Douglas J. Futuyma Useful Web sites www.youtube.com http://en.wikipedia.org

Acknowledgement:

Acknowledgement We are sincerely thankful to Dr. Anuradha Bhat and Dr. Annagiri Sumana,Life Science Department of IISER Kolkata for encouraging us to do this project. We are also thankful to Gregor P. Jose (PhD Student) and Shudhansu Da for their kind help in doing our experiment.

THANK YOU !:

THANK YOU !

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