Chloramphenicol: Composition, Management and Safety, Bactericidal Properties, Current Uses

It is a broad-spectrum antibiotic. It is used to treat several bacterial infections.

In the treatment of conjunctivitis is used as an ointment for the eyes.


Molecular weight: 323.1 g / mol

Molecular formula: C11H12Cl2N2O5 Stock

solutions: 10 mg / ml in methanol or up to 35 mg / ml in ethanol and stored at -20 ° C.

Final concentration: 30-35 μg / mL (for plasmids with high copy content)

Management and security

Chloramphenicol is considered dangerous and potentially carcinogenic. Wear gloves and avoid inhaling dust.


Chloramphenicol is soluble in water but not so stable in aqueous solutions and more susceptible to hydrolysis by ultraviolet light. After degradation, the Chloramphenicol solutions turn yellow and a yellow-orange precipitate forms.

Action mode

Bacteriostatic It inhibits protein synthesis by interacting with the 50S portion of the 70S ribosome. At higher concentrations, it can inhibit DNA synthesis.

Resistance mode

Resistance is conferred by the CAT (Chloramphenicol Acetyl Transferase) gene product of Tn9 (in opposition to recombinant plasmids, it is called CATR, CAMR, or CMR).



Chloramphenicol is a broad-spectrum antibiotic effective against most Gram-positive and Gram-negative bacteria strains. Chloramphenicol was derived from the bacterium Streptomyces venezuelae and is now produced synthetically.

In 1950, Chloramphenicol became the first antibiotic to be manufactured synthetically on a large scale.

Chloramphenicol is effective against many microorganisms, but severe side effects, including damage to the bone marrow, cause aplastic anemia in humans.

Chloramphenicol is now only used in humans to treat severe and life-threatening infections (e.g., typhoid fever).

Bactericidal properties

  • Chloramphenicol is bacteriostatic but can be bactericidal at high concentrations.
  • It stops bacterial growth by binding to the bacterial ribosome (blocking Peptidyl transferase) and inhibiting protein synthesis.
  • It is used as a selectable marker in molecular cloning experiments in which a functional chloramphenicol acetyltransferase (CAT) gene will confer resistance to the cells that contain the plasmid. E. coli is grown in media with Chloramphenicol, and only cells transformed with plasmids containing the CAT gene (in plasmids known as CATR, CAMR, or CMR) will survive.
  • Chloramphenicol is soluble in lipids, which allows it to diffuse through the bacterial cell membrane. It then binds reversibly to the L16 protein of the 50S subunit of the bacterial ribosomes. The transfer of amino acids to growing Peptide chains is prevented by decreasing the catalytic rate constant of Peptidyl Transferase.

The suppression of Peptidyl Transferase activity inhibits Peptide bond formation and subsequent protein synthesis.

Due to the enzyme Chloramphenicol Acetyl Transferase activity, the CAT gene encodes the natural resistance to Chloramphenicol in bacteria.

Chloramphenicol Acetyl Transferase catalyzes the transfer of an acetyl group from Acetyl Coenzyme A (Acetyl COA) to the C3 hydroxyl group of the antibiotic.

The product of the reaction, Acetyl Chloramphenicol, does not bind to the Peptidyl Transferase center of Ribosomes the 70S or inhibit Peptidyl Transferase.

There are three mechanisms of resistance to Chloramphenicol:

  1. The reduced permeability of the membrane.
  2. The mutation of the 50S ribosomal subunit.
  3. The expression of the Chloramphenicol Acetyl Transferase (CAT) gene.

Mutations that confer resistance to the 50S ribosomal subunit are rare. It is easy to select to reduce the membrane’s permeability to Chloramphenicol in vitro by the serial passage of bacteria. This is the most common mechanism of low-level resistance of Chloramphenicol.

Strains of Enterobacteriaceae and other Gram-negative bacteria express the Chloramphenicol Acetyl Transferase transported in the plasmids, which confers resistance to the drugs (so they are naturally resistant to Chloramphenicol).

Several variants of the CAT gene have been described, all of which form trimers of identical subunits.

The type I variant encoded by an 1102 bp segment of the Tn9 Transposon is widely used as a reporter gene. A minimal promoter is fused to the variant sequence of type I to measure transient CAT expression.

Most kinetic and structural analyses have been carried out with the type III variant of the CAT gene, which produces crystals suitable for X-ray analysis.

Advances in Bacterial research

In the 1970s and early 1980s, in molecular biology laboratories, Chloramphenicol was often added to growing cultures of moderate and high copy plasmids.

Chloramphenicol inhibits protein synthesis and blocks host DNA synthesis but does not affect the replication of relaxed plasmids (high copy)—the number of copies of simple plasmids increases during the incubation of the bacterial culture in the drug.

Amplification is necessary to achieve high yields of relaxed plasmids, which usually replicate only in moderate amounts in their host bacteria.

In the presence of Chloramphenicol, these vectors containing pMB1 replicon or wild type colE1 continue until 2000, or 3000 copies have accumulated in the cell. After 1982 (e.g., PUC plasmids) contained a modified colE1 replicon and replicated in copy numbers so high that amplification is unnecessary.

Current Uses

Nowadays, treating bacterial cultures with Chloramphenicol can still have some advantages. Since Chloramphenicol blocks bacterial replication, the copy number of the plasmids increases two or three times more in the presence of the drug as the volume and viscosity of the bacterial lysate are reduced, which simplifies the purification of the plasmids.

Many researchers find that adding Chloramphenicol to a growing culture is more convenient than dealing with a highly viscous lysate. Plasmids using the RepA protein as a positive regulator (e.g., PSC101) can not be amplified with Chloramphenicol.

For many years, it was thought that the amplification of plasmids in the presence of Chloramphenicol was adequate only when the host bacterium was cultured in minimal media.

However, a medium rich with Chloramphenicol protocol provides reproducibly high yields with E. coli strains harboring low copy number plasmids carrying the pMB1 or colE1 replicon.

The improved yields of pBR322 and its derivatives have been obtained from cultures treated with low concentrations of Chloramphenicol (10-20 μg / ml) that do not entirely suppress the host protein synthesis.

These results are not understood, but it could be explained if the replication of plasmids carrying the colE1 origin requires an unstable host factor that continues to be synthesized during partial inhibition of protein synthesis.

The active site of Chloramphenicol Acetyl Transferase contains a Histidine residue, and it is postulated that it acts as a general base catalyst in the acetylation reaction.

The two substrates of Chloramphenicol and Acetyl-CoA approach the active site through tunnels located on opposite sides of the molecule.

Because the CAT enzyme has a relatively high content of histidine residues, this would result in a co-purification with His-tagged proteins in Ni NTA columns if the His-tagged protein was expressed in strains of E. coli resistant Chloramphenicol.

Using such bacterial strains to express recombinant His tagged proteins is not recommended.