An antibiotic is any substance produced by a microorganism, i.e. bacteria or fungi, that it sends outside it's cell to harm or kill another microorganism(4). The benefit is easy to see. If an organism is able to produce chemicals that inhibit or kill other nearby organisms, it has an advantage in competing for local resources.
Technically, antibiotics are microbial or fungal products. But we are able to synthesize and mass produce these chemical substances in the laboratory to use against harmfull microorganisms in our environment. There is a distinction between natural and synthetic antibiotics, but in practice most drugs used to combat microbial and fungal infections are grouped under the general heading "antibiotics"(5).
Antibiotics can further be grouped under the broader heading of chemotherapuetic agents, chemical agents used to treat disease. Good chemotherapuetic agents are able to kill or inhibit the target pathogen with out too much damage to the host organism. The basis for this selective toxicity lies in the differences between prokaryotic cells of microorganisms and our own eukaryotic cells. The prokaryotic cells of microorganisms differ in a number of ways from our eukaryotic cells, such as absence of cell walls, different size of ribosomes, and details of metabolism. Thus, the goal of antibiotic therapy is to chose or design drugs that target these differences in host and pathogen cells(5).
The degree of selective toxicity is expressed in terms of :
The ratio of the therapeutic dose to the toxic dose is the therapeutic index. The larger the therapeutic index, the better the chemotherapuetic agent, and the less toxic it is to the host. Drugs often have high therapeutic indexes if the target of the drug is a structure or pathway not found in the host organism. This is why a drug like penicillin is relatively harmless for eukaryotic host cells. Penicillin targets peptidoglycan, a substance found only in prokaryotic cells. Hence, penicillin does not attack our own cells, as we do not have peptidoglycan in our cell walls(5).
On the other hand, drugs that have low therapeutic indexes often inhibit pathways or attack structures also found in the host cells. These undesirable effects on the host cells are termed side effects, and can range from allergic reactions to loss of hearing and renal damage(5).
Antibiotic drugs are classified according to
Bacteria can be classified according to the way they stain with a particular chemical preparation called the Gram Stain. Some bacteria retain a deep purple color even after washing after application of the stain, while other strains retain little or no color at all. Those that keep the purple color are termed Gram positive, and those that loose it or turn light pink are Gram negative. The difference in stain retention is because of differences in cell wall structure. All bacterial cell walls contain peptidoglycan, but in the Gram positive bacteria the peptidiglycan layer is much thicker than in the Gram negative strains. In addition, gram negative bacteria have an outer membrane of lipopoplysacchrides and other materials covering the thin peptidoglycan layer. It is because of the thick peptidoglycan layer in the Gram positive bacteria that the stain is retained(6).
There are various ways in which antibiotics exert their antimicrobial activity. An antibiotic can be bactericidal, killing microorganisms directly, or bacteriostatic, inhibiting growth of microorganisms(5).
Bacterial cell walls are composed of a substance called peptidoglycan. Individual strands of peptidoglycan are composed of alternating N-acetylglucosamine(NAG) and N-acetylmuramic acid(NAM). These strands are linked together by an enzyme called transpeptidase. Transpeptidase acts by cleaving the bond between the two terminal D-alanine residues on the NAM unit of the short peptidoglycan chains, then linking the individual chains of peptidoglycan together via B1-4 linkages to form a rigid structure for support.
Penicillin is one of many antibiotics in a class of antibiotics termed beta-lactam antibiotics. All antibiotics in this class conatain a beta-lactam ring. It is this ring that gives penicillin it's function. The various side chains may add other characteristics that distinguish one beta-lactam antibiotic from another. For example, the side chains can confer traits such as resistance to enzymes that degrade penicillin, changes in half-life of the drug, mechanism of cell wall penetration, and side effects.
Bacteria>cell walls are made in three stages. The third step involves cross linking of peptidoglycan stands by a transpeptidase enzyme to form a rigid cell wall. Penicillin destroys the synthesis of cross linked peptidoglycan by interfering with this final step.
Penicillin has a structural resemblance to the end D-alanyl-D-alanine residues of the small peptides involved in the peptidoglycan cross links. In addition, the beta-lactam ring has stereochemistry identical to that of the peptide bond between the two D-alanine residues that get split in the transpeptidation reaction.
Thus, penicillin acts as a substrate analog, mimicking the substrate of the traspeptidase enzyme by covalently binding to it via the beta-lactam ring, preventing functioning of the enzyme. Consequently, the cell wall is greatly weakened and the cell undergoes lysis. Since human cells do not have peptidoglycan in their cell walls, penicillin has very little toxicity for host cells (7).
The most wide spread cause of resistance to beta-lactam antibiotics, like penicillin, is the production of enzymes called beta-lactamases. (7). Apparently, these enzymes have a 3-D shape similar to the enzyme penicllin binds, transpeptidase. Therefor, it is reasonable to assume that penicillin binds instead to the beta-lactamases rather than the target transpeptidase enzyme. The beta-lactamases then catalyze the hydrolysis of the antibiotic to a biologically inactive form by breaking the labile bond in the beta-lactam ring responsible for penicillin's function.(7).
The genes for these enzymes can be located on plasmids or the bacterial chromosome. Furthermore, these enzymes can be permanent constituents of the cell, or induced by the presence of antibiotics (8).
A notable difference between prokaryotic and eukaryotic cells is the structure of their ribosomes. The difference in ribosomal structure accounts for the selective toxicity of antibiotics that effect protein synthesis. The prokaryotic ribosome is composed of two subunits, a small and a large, or the 30s and 50s ribosome respectively. There are several steps in nucleic acid translation to protein: DNA-->mRNA-->which interacts with the ribosome and tRNA--->protein. Different classes of antibiotics effect different steps in this sequence.
In protein translation, both mRNA and tRNA attach to the 30s ribosome. mRNA is also bound to the 50s subunit. Aminoglycosides inhibit the attachment of mRNA to the 30s subunit, directly inhibiting protein synthesis. The tetracycline group inhibits binding of the 30s subunit, and Chloramphenicol inhibits the attachment of mRNA to the 50s ribosome.
All cells are bound by a cell membrane. And although the membranes of all cells are quite similar, those of bacteria and fungi differ from eukaryotic cells. These slight differences allow for selective action of antimicrobial agents. Certain antibiotics, like polymyxins, act as detergents to dissolve bacterial cell membranes by binding to phospholipids present in the membranes. Other antibiotics, like polyene antibiotics such as amphotericin B bind to particular sterols present in the membranes of fungal cells. Polymixins do not act on fungi, and polyenes have no effect on bacteria (1).
Differences between the enzymes used to synthesize nucleic acids in prokaryotes and eukaryotes provide the means for selective action of antibiotics that take their effect by inhibiting nucleic acid synthesis. Antibiotics of the rifamycin family inhibit RNA synthesis because they bind to RNA polymerase, which is responsible for transcribing bacterial DNA to RNA(1). Antibiotics of the quinolone group interfere with DNA gyrase, the enzyme responsible for unwinding DNA in preparation of replication(2).
An antimetabolite is a substance that prevents a cell from carrying out a metabolic reaction. Antimetabolites function in two ways: 1)by competitive inhibition of enzymes, and 2)by erroneous incorporation into nucleic acids.
In this type of inhibition, an enzyme is inhibited by a substrate that binds to it's active sight but can not react. This slows or completely stops the enzyme function(1).
An example of this type of inhibition can be seen with the antimetabolite sulfanilamide and it's reaction with para-aminobenzoic acid (PABA). In many microorganisms, PABA is the substrate for an enzymatic reaction leading to the synthesis of folic acid. Folic acid in turn functions as a coenzyme for the synthesis of purine and pyrimidine bases of nucleic acids in many amino acids. In the presence of sulfanilamide, the enzyme that normally converts PABA to folic acid combines instead with the sulfanilamide, not it's normal substrate, PABA. Thus, this combination inhibits folic acid synthesis and stops the growth of the microorganism(3).
Some antibiotics have similar structure and stereochemistry to purine and pyrimidines found in microorganisms. In essence, these purine and pyrimidine analogs mimic metabolites in nucleic acid synthesis. And although they are similar to the normal purines and pyrimidines of nucleic acids, when incorporated they can not form the proper base pairs during replication and transcription.
This paper will focus on antibiotic control and regulation as manifested in antibiotic resistance.
Almost all bacteria that were once susceptible to antibiotics are resistant to at least one, if not more antibiotics today. Thought to be miracle drugs when discovered in the earlier part of this century, antibiotics cured many previously fatal infections. We learned how to mass produce these substances in labs, and drug therapy was status quo for treating microbial diseases. However, an over- reliance on these miracle agents soon developed, and we began to treat symptoms normally handled by our body's own immune system with antibiotic drugs. The consequence to this reliance on antibiotic therapy was that bacteria developed ways to resist them. After steady use over long periods of time, bacteria no longer killed or harmed by the drugs were selected for. These strains reproduced and their offspring were also resistant, capable of causing infections not cured by antibiotic drugs (6).
Bacteria are often resistant to certain drugs simply because they do not permit the entrance of the drug. For example, many Gram negative bacteria are penicillin resistant because the structure of their cell walls. These bacteria have an outer membrane of lipopolysacchrides and other materials covering the petidoglycan layer, and penicillin is unable to pass through to the site of peptidoglycan synthesis where the antibiotic functions (6).
Other bacteria are able to resist antibiotic attack by inactivating drugs though chemical modification or through the addition of groups. The best known example of chemical modification is the action of enzymes called Label. These enzymes work on penicillin antibiotics by hydrolysing the beta-lactam ring that gives them their function(6).
Resistance also arises when the antibiotic target is altered as to no longer be susceptible to the drugs. These drugs rely on the shape and stereochemistry of their targets to bind and hence function. Thus, any changes in the targets will alter ability of the antibiotic to function by inhibiting it's ability to even bind to the target. For example, chloramphenicol's action can be resisted by a change in the rRNA in the large ribosomal subunit to which it binds(6).
Antimetabolite effects may also be resisted by bacteria. This is done through alteration of enzymes susceptible to these drugs. For example, in folic acid synthesis of sulfonamide resistant bacteria, the enzyme that uses PABA as a substrate is altered as to have a much lower affinity for the sulfonimide antimetabolite(6).
Bacteria can also resist antimetabolites by using pathways different than those inhibited by the antimetabolite, or by increasing the production of the target metabolite. For example, some bacteria use pre-synthesized folic acid from their surrounding environment. Thus, they have eliminated the need to use the pathway inhibited by sulfonamides. Some strains are able to counteract sulfonamides by increasing the rate of folic acid synthesis, with the responsible enzyme's normal substrate, PABA, outcompeting the sulfonamide drugs(6).
Spontaneous mutations in bacterial chromosomes can lead to drug resistance. These changes in the primary sequence of bacterial DNA result in 3-D changes in the drug receptor sites. Therefore, the drug is unable to bind and therefore can not inhibit.
Bacteria store genetic information in genes on the DNA of their single chromosome. However, bacteria also store genetic information on accessory pieces of DNA separate from the chromosome. These independent, self-duplicating genetic pieces are termed plasmids. Each plasmid can carry up to 300 hundred additional genes, and there may be as many as 1,000 copies of a plasmid per cell. Furthermore, many different plasmids, i.e. those with different genes, can co-exist in a cell(4).
These plasmids contain supplemental genetic information coding for traits not found in the bacteria's chromosomal DNA. It is these plasmids that often contain the "information" for resistance to antibiotics (6). Many have genes that code for enzymes that destroy or alter antibiotic drugs(5).
Plasmids are able to replicate with in a cell, and are subject to mutations involving either the loss or gain of new genes. In addition, plasmids are capable of combining with other plasmids, thus resistance to several antibiotics can reside on one plasmid. Most importantly, bacteria are capable of transferring plasmids from one cell to another through a process termed conjugation. This allows the transfer of plasmids coding for antibiotic resistance among an entire colony of bacterial cells(6).
In conjugation, the bacterial cell containing a plasmid, the doner, passes the plasmid to another cell, the recipient. The doner reaches out to the recipient with a filamentous protein structure called a pilus. Upon contact, the pilus pulls the two cells close together. The doner copies all or a part of it's plasmid or plasmids, and passes it through the pilus to the recipient. Now, both cells have a copy of the plasmid with the resistant gene. In addition, the recipient is now able to act as a doner, continuing the spread of resistant plasmids through more conjugation. This results in an exponential increase in the number of strains with the resistant plasmid(6).
Bacteria can also exchange genes with out the use of plasmids. It is possible for resistance genes to reside on even smaller pieces of DNA called transposons. These pieces of DNA have the capability to "jump" from one piece of DNA, like a plasmid or chromosome, to another and vice versa. This way, a resistant gene can be directly incorporated into host chromosomal DNA and not dependent on plasmid transfer for spread(6).
Bacteriophages are viruses that attack bacteria. They also provide a mechanism to transfer antibiotic resistant genes. Once a phage attaches to a bacterial cell, it injects it's DNA into the host. Once inside, the viral DNA is able to incorporate itself into the host chromosone. Later, upon leaving the host chromosome to replicate, it may take pieces of the host DNA with it. If this host DNA contained information for antibiotic resistance, it will be replicated when the virus replicates. Now, the new viruses have genetic information in their DNA coding for bacterial resistance. When these viruses enter new hosts, they are able to integrate their original DNA as well as the antibiotic resistant genes picked up from the previous host into the new host's chromosome(6).
If the phage has picked up a transposon, this too will be passed to new host cells as the virus invades. Once inside the host cell, the transposon is able to "jump" onto a plasmid or the chromosomal DNA.
1.John C. Brown,1995: Jack's Bugs in the News(on WWW.)
3.Case, C.L., B.R. Funke, and G.J. Tortora. 1993. Microbiology: An Introduction. Benjamin/Cummings Publishing Co., Inc., California.
4, 5. Harley, J.P., D.A. Klein, and L.M. Prescott. 1993. Microbiology. Wm.C.Brown Publishers, Dubuque, Iowa.
6.Levy, Stuart B.,M.D. The Antibiotic Paradox. Plenum Press. New York
7.Mitsuhashi, Susumu. Drug Resistance in Bacteria. Japan Scientific Societies Press, 1982
8.Neuman, Maur. Useful and Harmful Interactions of Antibiotics . CRC Press, Inc. Boca Raton, Florida, 1985 Hugo, W.B. and A.D. Russell. 1987. Parmaceutical Microbiology. Blackwell Scientific Publications, London, Boston.