A. Antibiotic Concentrations.
1 The solid form of the antibiotics can be added directly to sterilized media that has been cooled to approximately 55°C. If kept at 4°C tetracycline, chloramphenicol, and streptomycin platesare usually good for several months, but kanamycin plates and ampicillin plates may only lastfor several weeks. A sensitive and resistant control should always be included each timeantibiotic plates are used.
2 For liquid media or for a few plates, a stock solution of the antibiotics can be prepared and stored at -20°C. All the antibiotic stock solutions can be prepared in sterile dH2O exceptchloramphenicol which can be dissolved in dimethylformamide and timentin which can bedissolved in 70% ethanol. The stock solution is 300x the concentration of antibiotic required inrich medium. An average petrie dish contains about 30 ml medium, so spread 0.1 ml on richplates.
3 High level expression of ß-lactamase can destroy ampicillin in the medium surrounding the AmpR colonies, allowing Amps satellite colonies to grow. Satellite colonies can be decreasedby using 2x ampicillin when selecting for plasmids. Alternatively, Timentin prevents growth ofsatellite colonies.
4 To select for a KanR gene on a multicopy plasmid use LB + 10 mM Tris HCl pH 7.4 + 250 µg/ml Kanamycin SO4 or Neomycin SO4. High levels of KanR are not expressed if the pH ofthe medium is less than 7.2.
5 Certain strains are only resistant to half this concentration of tetracycline.
6 For streptomycin plates add 1% Nutrient broth to the minimal media (i.e., 1 ml NB/100 ml B. Antibiotic sensitivity and resistance.
Ampicillin is a penicillin derivative that inhibits crosslinking of peptidoglycan chains in the cell
wall of eubacteria. Cells growing in the presence of ampicillin synthesize weak cell walls,
causing them to burst due to the high internal osmotic pressure. AmpR encoded by Mu
derivatives and pBR plasmids is due to a periplasmic ß-lactamase that breaks the ß-lactam ring
of penicillin derivatives.
Chloramphenicol inhibits protein synthesis by binding to the 50s ribosomal subunit and
blocking the peptidyltransferase reaction. CamR encoded by pBR328 is due to a cytoplasmic
chloramphenicol acyltransferase which inactivates chloramphenicol by covalently acetylating it.
Gentamycin is an aminoglycoside antibiotic that inhibits protein synthesis by binding to the
ribosome and preventing translocation Although it is readily transported into many bacteria,
gentamycin does not efficiently cross eukaryotic cytoplasmic membrane. (This contrast is
probably due to differences in membrane potential maintained across the bacterial cytoplasmic
membrane vs the eukaryotic cytoplasmic membrane). GenR is usually due to a plasmid encoded
enzyme that inactivates aminoglycosides by covalent modification
Kanamycin inhibits protein synthesis by binding to the 30s ribosomal subunit and preventing
translocation. KanR is usually due to a cytoplasmic aminoglycoside phosphotransferase that
inactivates kanamycin by covalently phosphorylating it. KanR requires phenotypic expression.
Neomycin is a structural analog of Kanamycin that functions by the same mechanism and is
inactivated by the same mechanism.
Tetracycline inhibits protein synthesis. Tetracycline reversibly binds to the small subunit of
ribosomes and interfere with binding of aminoacyl -tRNA to the Acceptor site. Tetracycline is
bacteriostatic in bacteria. Tetracyclines can also inhibit protein synthesis in eukaryotes, but are
less likely to reach inhibitory concentrations because eukaryotes lack a tetracycline uptake
mechanism. TetR encoded by Tn10 and pBR plasmids is due to a membrane protein that actively
exports tetracycline out of the cell. When Tn10 is present in multiple copies, cells are less
resistant to Tet than when only one copy of Tn10 is present.
Timentin is a mixture of the penicillin derivative Ticarcillin and the ß-lactamase inhibitor,
clavulanic acid. Timentin is useful for selecting for penicillin resistance in cultures of gram
negative bacteria that secrete large amounts of ß-lactamase - resistant cells are protected by ß-
lactamase retained within the periplasm, while exogenous ß-lactamase is inactivated by the
clavulinic acid, allowing the Ticarcillin to inhibit growth of any sensitive cells in the population.
One use of Timentin is to inhibit growth of penicillin-sensitive satellite colonies.
Trimethoprim is an inhibitor of bacterial dihydrofolic acid reductase. Resistance to
trimethoprim can arise by chromosomal mutations that cause overproduction of
dihydrofolate or plasmid encoded trimethoprim-resistant dihydrofolate reductases.
Spectinomycin inhibits protein synthesis by binding to the Sr protein of the 30s ribosomal
subunit and blocking translation. Spectinomycin resistance can arise via a single base
substitution in the chromosomal gene encoding this ribosomal protein (rpsE). Plasmidencoded resistance to both spectinomycin and streptomycin can arise via modification ofthe antibiotics - for example, the aad gene transfers an adenyl group from ATP to theseantibiotics which inactivates the antibiotic.
Streptomycin inhibits protein synthesis by binding to the S12 protein of the 30s ribosomal
subunit and blocking translation. A high level of Strr can result from chromosomal mutations in
the gene for the S12 protein (rpsL) which prevent streptomycin from binding to the ribosome.
Since only mutant ribosomes are StrR, resistance to streptomycin is recessive to streptomycin
sensitivity. StrR requires phenotypic expression.
Foster, T. 1983. Plasmid determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol. Rev. 47:361-409.
Garrett, R., S. Douthwaite, A. Liljas, A. Matheson, P. Moore, and H. Noller. 2000. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. ASM Press,Washington, D.C.
Russell, A., and I. Chopra. 1990. Understanding Antibacterial Action and Resistance. Ellis Zahner, H., and W. Maas. 1972. Biology of Antibiotics. Springer-Verlag, NY.


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