Ancillary Antibiotics

Chloramphenicol, a remarkably simple halogen-containing antibiotic, was isolated in 1947 from the microbe Streptomyces venezu-elae.1 Once the structure of chloramphenicol had been determined, large-scale industrial production by chemical synthesis followed in 1949. Although common in chemical practice, the nitrobenzene (blue) and the dichloro-acetic acid (red) units of chloramphenicol are unusual for a natural product.2


Chloramphenicol is a wide-spectrum antibiotic, active against 95% of Gram-negative bacteria and a large number of Gram-positive aerobic and anaerobic strains. During the 1950s it was successfully used to quell epidemics of typhoid fever and meningitis. However, because of a serious side effect (blood dyscrasia) in a small fraction of those treated, its application as an oral or intravenous antibiotic was discontinued in the US in the late 1970s. Chloramphenicol is extensively used in poorer countries because of its low cost. In the US it is mainly used to treat ocular infections. Chloramphenicol acts by binding to the large 50S ribosomal subunit and blocks bacterial protein synthesis (see azithromycin, page 134). It also binds to ribosomes in mammalian mitochondria and harms rapidly dividing blood cell precursors in the bone marrow.

Synthetic 2-nitroimidazole (Azomycin) was prepared in 1955 and later shown to be effective against Trichomonas vaginalis, the microbe that causes the vaginal infection trichomoniasis.



Extensive synthesis and evaluation of other imidazoles led to the identification of metronidazole, a 5-nitroimidazole derivative, as the most potent against T. vaginalis both in vivo and in vitro3

Metronidazole (Flagyl™, Pfizer) is effective against a wide range of parasites o2n

Metronidazole (Flagyl1

Metronidazole (Flagyl1

and anaerobic bacteria It is also used to treat acne rosacea, a common facial condition.

Once inside a microorganism, metronidazole accepts an electron to give a very reactive intermediate that attacks DNA and/or proteins and eventually kills the microbe. It has been shown that metronidazole damages the helical DNA structure and thus inhibits its replication.

Cycloserine, a broad-spectrum antibiotic, was first isolated in 1955 from the microbe Streptomyces orchidaceous. It is currently used against tuberculosis (Seromycin™, Lilly) in conjunction with drugs such as rifampicin, ethambutol, and streptomycin, but only when other treatments have failed.

Cycloserine (Seromycin™)

Cycloserine acts by inhibiting the biosynthesis of peptidoglycans that form the bacterial cell wall.4

Bacitracins, cyclopolypeptides isolated from the microbe Bacillus subtilis in 1943, are commonly used as topical antibiotics for the treatment of skin and eye infections. The structure of the most abundant, bacitracin A, is shown below.


Bacitracin A

Bacitracins are often used topically in combination with other antibiotics such as neomycin B and polymyxin B (Neosporin™). Bacitracins inhibit bacterial growth by blocking the formation of dolichol phosphate, a C55-component of membranes that transports intermediates for cell wall biosynthesis.5

1. Adv. Phytochem. 2003, 109-184; 2. Antimicrob. Agents 2005, 925-929; 3. Antimicrob. Agents 2005, 930-940; 4. Antibiotic and Chemotherapy (7th Edition, Churchill Livingstone, 1997); 344-345; 5. Antimicrob. Agents 2005, 377-400; Refs. p. 176



It came as an unpleasant surprise that shortly after the introduction of each of the various classes of antibiotics new pathogenic strains emerged that were no longer susceptible. The emergence of resistant microorganisms is now known to be a predictable consequence of widespread antibiotic use (and misuse). All that is required for this to happen is the survival of genetically modified organisms that can reproduce even in the presence of a particular antibiotic.1 The modifications can come from (1) spontaneous mutations, (2) overexpression of certain genes and overproduction of their gene products or (3) acquisition of genetic material from other microorganisms, for example by plasmid DNA exchange. These genetic changes can confer resistance in the following ways:

1. By an enzyme-catalyzed reaction that renders the antibiotic inactive. There are many examples of this type including hydrolytic cleavage of the fi-lactam ring of penicillins and cephalosporins by [3-lactamases and acety-lation or phosphorylation of aminoglycosides catalyzed by acetyltransferase or phosphotransferase enzymes.

2. Mutations in the organisms can result in changes in the biological target that are protective because they prevent the antibiotic from binding to it. An example of this is the development of bacterial resistance to a tetracycline as a result of mutations that reduces the binding volume of the A site in the 30S subunit of the bacterial ribosome so that it no longer accommodates the tetracycline. Another example is the mutation of vancomycin resistant strains that attenuates hydrogen bonding between the developing cell wall and the antibiotic and reduces vancomycin potency by three orders of magnitude (see page 138).

3. Upregulation of drug efflux from the bacterial cell. Most living cells are endowed with membrane-associated proteins that serve as active transporters of organic substances either into or out of the cell. Some microorganisms have multiple efflux pumps that can handle a variety of molecular types. Usually these pumps are powered by ATP in the sense that the overall hydrolysis of ATP provides the driving force for the conformational changes of the efflux protein that push the ligand molecule out of the cell.

Examples of this process are the efflux pumpmediated removal of tetracyclines or quino-lone antibiotics from microorganisms that have become resistant because of an ability to overproduce efflux pumps. Such mutant microbes grow more slowly since there is a major diversion of energy to the efflux process.

4. Decreased cell-wall permeability by mutation-Induced genetic changes. Such changes contribute to the resistance shown by the microbes responsible for tuberculosis.

5. Genetic changes that alter the metabolic pathways or needs of the microbe so as to circumvent antibacterial actions. An early example of this mechanism for resistance was the finding that sulfanilamide-resistant strains had developed the capability of utilizing folic acid at levels provided by the host organism.

6. Biofilm formation. Bacteria communicate with one another using small organic molecules as signals, for example derivatives of the amino acid homoserine. These messenger molecules can activate the bacterial colony to produce virulent proteins which are toxic to the host or to secrete carbohydrates that join to form tough, protective films, or biofilms. These films resist penetration by antibiotics and consequently function to protect bacteria. Biofilm formation contributes to persistent infection in vivo. It is a major medical problem in device implantation therapy, for instance hip or knee replacements, and is the main reason forfaited procedures.

The development of resistance to drugs also accompanies the application of antiviral drugs to viral infections. Viral resistance is generally due to mutations that change the structure of the protein target of the antiviral drug.

Much information on viral resistance to chemotherapy has emerged from the massive recent studies on the treatment of HIV AIDS. Three major classes of drugs have been employed in medicine over the past decade; (1) nucleoside derivatives as inhibitors of the enzyme reverse transcriptase, which catalyzes the formation of DNA from viral genomic RNA; (2) non-nucleoside reverse transcriptase inhibitors and (3) inhibitors of HIV protease, which cleaves two large precursor proteins into smaller fragments that are essential for the reproduction of HIV.

Resistance to these agents quickly developed when they were used separately. Combination therapy with three or more agents is currently the standard treatment. However, about half of the patients on such therapy develop resistance to at least one of these drugs.

Resistance develops as a result of the ability of HIV to undergo genomic mutations at a surprisingly rapid rate. Consequently, mutant strains evolve with one, two, or even more mutations that render the drugs ineffective because of changes in the structure of the viral target.

Drug resistance is a major problem in cancer chemotherapy. In cancer, as with infectious diseases, the population of cells exposed to a drug is in the billions and so the combination of high mutation rates and large numbers of cells ensures the evolution of survivors and their proliferation by natural selection. Resistance in cancer cells may also involve overexpresslon of efflux pump proteins or acquisition of evasive biochemistry (see page 184 for an overview of cancer).2

Structure and Function of Efflux Pumps adaptor, linking TolC and ArcB when the efflux pump is in its fully assembled functioning form (see image below).3"5







doxycycline doxycycline

This image is a representation of the fully assembled drug efflux pump, connecting the intracellular with the extracellular space. The MexA periplasmatic adaptor fills the gap between AcrB and TolC so there is no opening of the channel into the periplasm. The substrate enters the AcrB assembly and exits into the extracellular space through the opening of the TolC.

A representation of the structures of the three drug efflux pump components (resting state), proteins TolC, MexA and AcrB TolC is barrel-shaped, its upper a-barrel domain is embedded in the outer membrane and forms an exit channel to the extracellular space. The lower (i-barrel domain extends into the periplasmatic space. AcrB is a trimeric protein at the inner membrane that provides entry from the cytosol. MexA is a periplasmatic


Top-side views of the three drug efflux pump components TolC, MexA, and AcrB All three proteins have a cylindrical cavity through which the various drug molecules can pass.

1. Evolution of Microbial Pathogens 2006, 221-241; 2. Nat. Rev. Microbiol. 2006, 4, 629-636; 3. Science 2006, 313, 1295-1298 (2GIF); 4. J. Biol. Chem. 2004, 279, 2593925942 (1VF7); 5. J. Mol. Biol. 2004, 342, 697-702 (1TQQ); Refs, p. 176














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