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Web Review of Todar's Online Textbook of Bacteriology. "The Good, the Bad, and the Deadly"

Tag words: bacterial growth, antibiotic, chemotherapeutic agent, disinfectant, antiseptic, preservative, control of growth, sterilization, pasteurization.









Kenneth Todar currently teaches Microbiology 100 at the University of Wisconsin-Madison.  His main teaching interest include general microbiology, bacterial diversity, microbial ecology and pathogenic bacteriology.

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Control of Microbial Growth (page 5)

(This chapter has 6 pages)

© Kenneth Todar, PhD

Antimicrobial Agents Used in the Treatment of Infectious Disease

The modern era of antimicrobial chemotherapy began following Fleming's discovery in 1929 of the powerful bactericidal substance penicillin, and Domagk's discovery in 1935 of synthetic chemicals (sulfonamides) with broad antimicrobial activity. In the early 1940's, spurred partially by the need for antibacterial agents in WW II, penicillin was isolated, purified and injected into experimental animals, where it was found to not only cure infections but also to possess incredibly low toxicity for the animals. This fact ushered into being the age of antibiotic chemotherapy and an intense search for similar antimicrobial agents of low toxicity to animals that might prove useful in the treatment of infectious disease. The rapid isolation of streptomycin, chloramphenicol and tetracycline soon followed, and by the 1950's, these and several other antibiotics were in clinical usage.

The most important property of a clinically-useful antimicrobial agent, especially from the patient's point of view, is its selective toxicity, i.e., the agent acts in some way that inhibits or kills bacterial pathogens but has little or no toxic effect on the animal taking the drug This implies that the biochemical processes in the bacteria are in some way different from those in the animal cells, and that the advantage of this difference can be taken in chemotherapy.

Antibiotics may have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that are affected by a certain antibiotic is expressed as its spectrum of action. Antibiotics effective against procaryotes which kill or inhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum . If effective mainly against Gram-positive or Gram-negative bacteria, they are narrow spectrum. If effective against a single organism or disease, they are referred to as limited spectrum.

Kinds of Antimicrobial Agents and their Primary Modes of Action

1. Cell wall synthesis inhibitors Cell wall synthesis inhibitors generally inhibit some step in the synthesis of bacterial peptidoglycan. Generally they exert their selective toxicity against eubacteria because human cells lack cell walls.

Beta lactam antibiotics Chemically, these antibiotics contain a 4-membered beta lactam ring. They are the products of two groups of fungi, Penicillium and Cephalosporium molds, and are correspondingly represented by the penicillins and cephalosporins. The beta lactam antibiotics inhibit the last step in peptidoglycan synthesis, the final cross-linking between between peptide side chains, mediated by bacterial carboxypeptidase and transpeptidase enzymes. Beta lactam antibiotics are normally bactericidal and require that cells be actively growing in order to exert their toxicity.

Natural penicillins, such as Penicillin G or Penicillin V, are produced by fermentation of Penicillium chrysogenum. They are effective against streptococcus, gonococcus and staphylococcus, except where resistance has developed. They are considered narrow spectrum since they are not effective against Gram-negative rods.

Semisynthetic penicillins first appeared in 1959. A mold produces the main part of the molecule (6-aminopenicillanic acid) which can be modified chemically by the addition of side chains. Many of these compounds have been developed to have distinct benefits or advantages over penicillin G, such as increased spectrum of activity (e.g.  effectiveness against Gram-negative rods), resistance to penicillinase or  effectiveness when administered orally. Amoxycillin and Ampicillin have broadened spectra against Gram-negatives and are effective orally; Methicillin is penicillinase-resistant.

Clavulanic acid is a chemical sometimes added to a semisynthetic penicillin preparation. Thus, amoxycillin plus clavulanate is clavamox or augmentin. The clavulanate is not an antimicrobial agent. It inhibits beta lactamase enzymes and has given extended life to penicillinase-sensitive beta lactams.

Although nontoxic, penicillins occasionally cause death when administered to persons who are allergic to them. In the U.S. there are 300 - 500 deaths annually due to penicillin allergy. In allergic individuals the beta lactam molecule attaches to a serum protein which initiates an IgE-mediated inflammatory response.

Cephalolsporins are beta lactam antibiotics with a similar mode of action to penicillins that are produced by species of Cephalosporium. The have a low toxicity and a somewhat broader spectrum than natural penicillins. They are often used as penicillin substitutes, against Gram-negative bacteria, and in surgical prophylaxis. They are subject to degradation by some bacterial beta-lactamases, but they tend to be resistant to beta-lactamases from S. aureus.


Chemical structure of some Beta Lactam antibiotics.

Bacitracin is a polypeptide antibiotic produced by Bacillus species. It prevents cell wall growth by inhibiting the release of the muropeptide subunits of peptidoglycan from the lipid carrier molecule that carries the subunit to the outside of the membrane. Teichoic acid synthesis, which requires the same carrier, is also inhibited. Bacitracin has a high toxicity which precludes its systemic use. It is present in many topical antibiotic preparations, and since it is not absorbed by the gut, it is given to "sterilize" the bowel prior to surgery.

2. Cell membrane inhibitors disorganize the structure or inhibit the function of bacterial membranes. The integrity of the cytoplasmic and outer membranes is vital to bacteria, and compounds that disorganize the membranes rapidly kill the cells. However, due to the similarities in phospholipids in bacterial and eucaryotic membranes, this action is rarely specific enough to permit these compounds to be used systemically. The only antibacterial antibiotic of clinical importance that acts by this mechanism is Polymyxin, produced by Bacillus polymyxa. Polymyxin is effective mainly against Gram-negative bacteria and is usually limited to topical usage. Polymyxins bind to membrane phospholipids and thereby interfere with membrane function. Polymyxin is occasionally given for urinary tract infections caused by Pseudomonas that are gentamicin, carbenicillin and tobramycin resistant. The balance between effectiveness and damage to the kidney and other organs is dangerously close, and the drug should only be given under close supervision in the hospital.

3. Protein synthesis inhibitors Many therapeutically useful antibiotics owe their action to inhibition of some step in the complex process of translation. Their attack is always at one of the events occurring on the ribosome rather than the stage of amino acid activation or attachment to a particular tRNA. Most have an affinity or specificity for 70S (as opposed to 80S) ribosomes, and they achieve their selective toxicity in this manner. The most important antibiotics with this mode of action are the tetracyclines, chloramphenicol, the macrolides (e.g. erythromycin) and the aminoglycosides (e.g. streptomycin).

The aminoglycosides are products of Streptomyces species and are represented by streptomycin, kanamycin, tobramycin and gentamicin. These antibiotics exert their activity by binding to bacterial ribosomes and preventing the initiation of protein synthesis. Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria. Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis. Gentamicin is active against many strains of Gram-positive and Gram-negative bacteria, including some strains of Pseudomonas aeruginosa. Kanamycin is active at low concentrations against many Gram-positive bacteria, including penicillin-resistant staphylococci. Gentamicin and Tobramycin are mainstays for treatment of Pseudomonas infections. An unfortunate side effect of aminoglycosides has tended to restrict their usage: prolonged use is known to impair kidney function and damage to the auditory nerves leading to deafness.


The chemical structure of tobramycin.

The tetracyclines consist of eight related antibiotics which are all natural products of Streptomyces, although some can now be produced semisynthetically. Tetracycline, chlortetracycline and doxycycline are the best known. The tetracyclines are broad-spectrum antibiotics with a wide range of activity against both Gram-positive and Gram-negative bacteria. The tetracyclines act by blocking the binding of aminoacyl tRNA to the A site on the ribosome. Tetracyclines inhibit protein synthesis on isolated 70S or 80S (eucaryotic) ribosomes, and in both cases, their effect is on the small ribosomal subunit. However, most bacteria possess an active transport system for tetracycline that will allow intracellular accumulation of the antibiotic at concentrations 50 times as great as that in the medium. This greatly enhances its antibacterial effectiveness and accounts for its specificity of action, since an effective concentration cannot be accumulated in animal cells. Thus a blood level of tetracycline which is harmless to animal tissues can halt protein synthesis in invading bacteria.

The tetracyclines have a remarkably low toxicity and minimal side effects when taken by animals. The combination of their broad spectrum and low toxicity has led to their overuse and misuse by the medical community and the wide-spread development of resistance has reduced their effectiveness. Nonetheless, tetracyclines still have some important uses, such as in the treatment of Lyme disease.


The chemical structure of tetracycline.

Chloramphenicol
has a broad spectrum of activity that exerts a bacteriostatic effect. It is effective against intracellular parasites such as the rickettsiae. Unfortunately, aplastic anemia, which is dose related, develops in a small proportion (1/50,000) of patients. Chloramphenicol was originally discovered and purified from the fermentation of a Streptomyces, but currently it is produced entirely by chemical synthesis. Chloramphenicol inhibits the bacterial enzyme peptidyl transferase thereby preventing the growth of the polypeptide chain during protein synthesis.

Chloramphenicol is entirely selective for 70S ribosomes and does not affect 80S ribosomes. Its unfortunate toxicity towards the small proportion of patients who receive it is in no way related to its effect on bacterial protein synthesis. However, since mitochondria originated from procaryotic cells and have 70S ribosomes, they are subject to inhibition by some of the protein synthesis inhibitors including chloroamphenicol. This likely explains the toxicity of chloramphenicol. The eucaryotic cells most likely to be inhibited by chloramphenicol are those undergoing rapid multiplication, thereby rapidly synthesizing mitochondria. Such cells include the blood forming cells of the bone marrow, the inhibition of which could present as aplastic anemia. Chloramphenicol was once a highly prescribed antibiotic and a number of deaths from anemia occurred before its use was curtailed. Now it is seldom used in human medicine except in life-threatening situations (e.g. typhoid fever).

The chemical structure of chloroamphenicol.

The Macrolides is a family of antibiotics whose structures contain large lactone rings linked through glycoside bonds with amino sugars. The most important members of the group are erythromycin and azithromycin. Erythromycin is active against most Gram-positive bacteria, Neisseria, Legionella and Haemophilus, but not against the Enterobacteriaceae. Macrolides inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit. Binding inhibits elongation of the protein by peptidyl transferase or prevents translocation of the ribosome or both. Macrolides are bacteriostatic for most bacteria but are cidal for a few Gram-positive bacteria.


The chemical structure of erythromycin.

4. Effects on Nucleic Acids Some chemotherapeutic agents affect the synthesis of DNA or RNA, or can bind to DNA or RNA so that their messages cannot be read. Either case, of course, can block the growth of cells. The majority of these drugs are unselective, however, and affect animal cells and bacterial cells alike and therefore have no therapeutic application. Two classes of nucleic acid synthesis inhibitors which have selective activity against procaryotes and some medical utility are quinolones and rifamycins.

Quinolones are broad-spectrum agents that rapidly kill bacteria and are well absorbed after oral administration. Nalidixic acid and ciprofloxacin belong to this group. They act by inhibiting the activity of  bacterial DNA gyrase, preventing the normal functioning of DNA. Bacterial DNA exists in a supercoiled form and the enzyme DNA gyrase, a topoisomerase, is responsible for introducing negative supercoils into the structure. Humans possess DNA gyrase but it is structurally distinct from the bacterial enzyme and remains unaffected by the activity of quinolones. Overuse of these drugs in certain situations is selecting quinolone resistant mutants and these may threaten the long term use of such compounds.

The chemical structure of nalidixic acid.

Some quinolones penetrate macrophages and neutrophils better than most antibiotics and are thus useful in treatment of infections caused by intracellular parasites. However, the main use of nalidixic acid is in treatment of lower urinary tract infections (UTI). The compound is unusual in that it is effective against several types of Gram-negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae and  species which are common causes of UTI. It is not usually effective against Pseudomonas aeruginosa, and Gram-positive bacteria are resistant.  However, a fluoroquinolone, Ciprofloxacin (Cipro) was recently recommended as the drug of choice for prophylaxis and treatment of anthrax.


The chemical structure of ciprofloxacin.

The rifamycins are the products of Streptomyces. Rifampicin is a semisynthetic derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis) and some Gram-negative bacteria. Rifampicin acts quite specifically on eubacterial RNA polymerase and is inactive towards RNA polymerase from animal cells or towards DNA polymerase. The antibiotic binds to the beta subunit of the polymerase and apparently blocks the entry of the first nucleotide which is necessary to activate the polymerase, thereby blocking mRNA synthesis. It has been found to have greater bactericidal effect against M.tuberculosis than other anti-tuberculosis drugs, and it has largely replaced isoniazid as one of the front-line drugs used to treat the disease, especially when isoniazid resistance is indicated. It is effective orally and penetrates well into the cerebrospinal fluid and is therefore useful for treatment of tuberculosis meningitis, as well as meningitis caused by Neisseria meningitidis.

The chemical structure of rifampicin.



5. Competitive Inhibitors The competitive inhibitors are mostly all synthetic chemotherapeutic agents. Most are "growth factor analogs", chemicals which are structurally similar to a bacterial growth factor but which do not fulfill its metabolic function in the cell. Some are bacteriostatic and some are bactericidal.

Sulfonamides were introduced as chemotherapeutic agents by Domagk in 1935, who showed that one of these compounds (prontosil) had the effect of curing mice with infections caused by beta-hemolytic streptococci. Chemical modifications of the compound sulfanilamide gave compounds with even higher and broader antibacterial activity. The resulting sulfonamides have broadly similar antibacterial activity, but differ widely in their pharmacological actions. Bacteria which are almost always sensitive to the sulfonamides include Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli. The sulfonamides have been extremely useful in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of meningococcal meningitis (because they cross the blood-brain barrier). The most useful sulfonamides are sulfanilamide, Gantrisin and Trimethoprim.

The sulfonamides are inhibitors of the bacterial enzymes required for the synthesis of tetrahydrofolic acid (THF), the vitamin form of folic acid essential for 1-carbon transfer reactions. Sulfonamides are structurally similar to para aminobenzoic acid (PABA), the substrate for the first enzyme in the THF pathway, and they competitively inhibit that step. Trimethoprim is structurally similar to dihydrofolate (DHF) and competitively inhibits the second step in THF synthesis mediated by the DHF reductase. Animal cells do not synthesize their own folic acid but obtain it in a preformed fashion as a vitamin. Since animals do not make folic acid, they are not affected by these drugs, which achieve their selective toxicity for bacteria on this basis.

Sulfanilamide is similar in structure to para-aminobenzoic acid (PABA), an intermediate in the biosynthetic pathway for folic acid. Sulfanilamide can competitively inhibit the enzyme that has PABA as it's normal substrate by competitively occupying the active site of the enzyme.

Three additional synthetic chemotherapeutic agents have been used in the treatment of tuberculosis: isoniazid (INH), para-aminosalicylic acid (PAS), and ethambutol. The usual strategy in the treatment of tuberculosis has been to administer a single antibiotic (historically streptomycin, but now, most commonly, rifampicin is given) in conjunction with INH and ethambutol. Since the tubercle bacillus rapidly develops resistance to the antibiotic, ethambutol and INH are given to prevent outgrowth of a resistant strain. It must also be pointed out that the tubercle bacillus rapidly develops resistance to ethambutol and INH if either drug is used alone. Ethambutol inhibits incorporation of mycolic acids into the mycobacterial cell wall. Isoniazid has been reported to inhibit mycolic acid synthesis in mycobacteria and since it is an analog of pyridoxine (Vitamin B6) it may inhibit pyridoxine catalyzed reactions as well. Isoniazid is activated by a mycobacterial peroxidase enzyme and destroys several targets in the cell. PAS is an anti-folate. PAS was once a primary anti-tuberculosis drug, but now it is a secondary agent, having been largely replaced by ethambutol.


The chemical structure of isoniazid.




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