Effect Of Vancomycin And Kanamycin On Ecoli Biology Essay

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This journal evaluates the combined actions of two antibiotic drugs, Vancomycin and Kanamycin on log phase culture of E.coli and Staph aureus was carried out. Other groups also worked on two other ones, Ampicillin and Cefsoludine. The assay was carried out to find out if their actions will be synergistic, antagonistic, addictive or autonomous. The MIC and MBC of the combined drugs was determined in this assay and FBCs and FICs were calculated. Some were found to be synergistic while others were either antagonistic or addictive.

Modern medicine is dependent on chemotherapeutic agents, chemical agents that are used to treat diseases. Chemotherapeutic agents destroy pathogenic microorganisms or inhibit their growth at concentrations low enough to avoid undesirable damage to the host. Most of these antibiotics, microbial products or their derivatives can kill susceptible microorganisms or inhibit their growth (Rang et al., 2003).

Effectiveness of drugs varies thus making some of them either narrow-spectrum drugs or broad spectrum drugs. An idea of their effectiveness can be obtained from minimal inhibitory concentration (Dawson et al., 2004). Minimal inhibitory concentration (MIC) is the lowest concentration of a drug that prevents growth of a particular pathogen (Underwood, 2007). The minimal lethal concentration (MLC) is the lowest drug concentration (Dawson et al., 2004).

The purpose of this review was to determine the MIC and MBC of drug combinations.

1.2 MATERIALS AND METHODS

Antibiotics A and B of concentrations 500 and 250µg/ml-1 were respectively used. A was Vancomycin while B was Kanamycin. Along with log phase cultures of Staph. Aureus, E.coli, microtitre trays, pipettes, stop watch.

Serial dilutions of drugs were prepared into the microlitre plate. Then 100ul of nutrient broth was dispensed into each well of the microtitre tray, followed with the addition of 100µl of first antibiotic to each well in column 1, thus halving antibiotic concentration. With fresh pipette tip, 100µl was transferred from each well in column 1 into corresponding adjacent well in column 2. The doubling dilution was continued across the plate until column 11 was reached. From each well in column 11, 100ul was removed and discarded off. The first antibiotic was not put in column 12.

Following this was addition of 100ml of the second antibiotic to each row A. The antibiotic was added to well A12 and worked back across the plate to well A1 minimising the risk of transferring the first antibiotic from a well of higher concentration to a lower one.

Now the second antibiotic concentration is halved, making a further concentration of the first antibiotics in this row being halved. Further transfer of 100ul with a fresh pipette tip was made from each well in row A to the corresponding well in row B. This was repeated starting with well A12 and worked back across the plate to well A1 was reached.

Dilution doubling was carried on down the plate as far as row G. Repeated was discarding of 100µl from each well in row G making sure that no second antibiotic was added to row H wells starting from H12 , mixed and 100ul was then discard . Row H wells antibiotic is representing by row H while column 12 is for the second antibiotics. The positive growth control is well H12 which has no antibiotic in it. With fresh pipette tip, E .coli suspension was added to each well in the plate and worked backwards along the rows from column 12 as above. All wells were mixed well throughout the experiment

1.21 DETERMINATION OF FBC

Microtitre tray was left stand for 30 minutes during which 12 nutrient agar plates were each divided into 8 segments and labelled them 1A,1B,1C……1H etc . 10µl drop was taken from each well and placed in the labelled segment on one of the plates.

Tray and plates were incubated at 37oC

1.3 RESULT

After incubation, wells were examined for any bacterial growth and recorded as (+) to indicate growth and (-) if there was no growth. Using the results in row H for first antibiotic and column 12 for second antibiotic, MIC and MBC were calculated, reading the lowest concentration where there is no bacterial growth. A well was identified in another row and recorded as MIC.

Fractional Inhibitory Concentrations (FIC) was calculated for E.coli as:

FIC = Concn of antibiotic1 in this well + Concn of antibiotic2 in this well

MIC of antibiotic1 MIC of antibiotic2

A = Vancomycin 4000µg/ml

B = Kanomycin 250µg/ml

C = Ampicillin 500µg/ml

D = Cetsulodin 125µg/ml

FIC = Concn of antibiotic1 in this well + Concn of antibiotic2 in this well

MIC of antibiotic1 MIC of antibiotic2

MIC A = 1000µg/ml-1 MIC B = 15.63

FIC (E4) = 125 + 7.81 = 0.125 + 0.5 = 0.625

1000 15.63

FIC (D5) = 250 + 7.81 = 0.25 + 0.5 = 0.750

15.63

The value obtained for FIC is between (0.5 - 1), which indicates additivity of the FIC.

E.coli grew in all concentrations of Kanomycin therefore couldn't work out the MBC and FBC in it.

Antibiotic A has a minimum bacterial 1000µg/ml while Antibiotic B is not bactericidal.

Fractional Inhibitory Concentrations (FIC) was calculated for Staph.aureus as:

FIC = Concn of antibiotic1 in this well + Concn of antibiotic2 in this well

MIC of antibiotic1 MIC of antibiotic2

MIC 1 = 12D (31.25) MIC 2 = H2 (1000)

FIC (7F) = 31.91 + 31.25 = 0.25 + 0.03 = 0.28

15.63 1000

FIC (11D) = 15.63 + 1.95 = 1 + 0.00195 = 1.002

15.63 1000

The value obtained for FIC is <0.5 which indicates synergy and the other value obtained is between 1 and 2 which indicates autonomy

3H, minimum bacteria of Staph.aureus MBC for kanomycin for its bactericidal,

Cannot be worked for FBC in this experiment because Vancomycin is stronger (strong bactericidal) inhibiting the growth of Staph.

1.4. DISSCUSSION

An FI(B)C of <0.5 indicates synergis and >2 indicates antagonism.Autonomy (1-2) or additivity (0.5 - 1). From the calculated results, MBC for Vancomycin was 62.5µg/ml and for kanamycin is125µg/ml. That kanamycin doubled that of Vancomycin. While for MIC, Vancomycin 31.25µg/ml doubled that of Kanamycin.

The lowest inhibitory fraction of MICs obtained by the microtitre combination of vancomycin and kamamycin is 1:16. The ratio for the results again E.coli of 1:32 and that against S.aureus is 1.128. The FIC calculated against E.coli and S.areus was >0.5.

The combination of the 2 drugs Vancomycin and Kamamycin can be saib to be synergistic. Other groups used 2 other drugs C, Ampicillin concentration 500µg/ml and D, Cefsoludine concentration = 125µg/ml

Comparative results shows action of the drugs B+D to be synergistic where the FIC is 0.16 and FBC, 0.75 for S.aureus while for same drugs B + D, for E.coli it was found to be additive with FIC as 0.6. Another drug combination of A+B shows antagonistic action at high concentration of FIC as 18 while low with FIC of 0.38. There has been a wide range of different results from other compared groups.

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