All You Need is a Bacterium and a Dream

Excerpted from "Antimicrobial Agents" one of five books on bacterial infections underwritten by Marion Merrell Dow in 1994. The text has been altered to fit this format.

In l928, chemotherapy's greatest triumph floated through the window of a London Hospital and landed on a plate of growing staphylococci. Bacterial colonies surrounding the migrating mold became transparent, as if they were being lysed or dissolved. Thus, penicillin --a substance molds use to kill bacteria-- came to the attention of Alexander Fleming, Professor of Bacteriology at St. Mary's Hospital Medical School.

Fleming named the "'mold juice" penicillin, described its properties, and cautiously suggested that this newly found natural "antibiotic" could cure bacterial infections in people. However, when Fleming formally announced his findings to the scientific community in l929, it was regarded as an interesting observation and nothing more. Nevertheless, Fleming recognized the biological importance of his finding and preserved the penicillin-secreting mold, Penicillium notatum, for future study.

In any event, Fleming was not the first person to notice that a mold of the genus Penicillium was antagonistic to the growth of bacteria. This finding had already been reported by a number of other investigators, notably John Burden Sanderson in l870, Joseph Lister a year later, and a French medical student, Ernest Duchesne in l896.

But history was on Fleming's side. Scientifically speaking, the nineteenth century was a busy time. The microbiologic basis of infection had been discovered by Louis Pasteur and the highly scientific notion of cause and effect was taking form.

In the latter half of the l870's, Pasteur demolished the concept of "spontaneous creation" and replaced it with the "germ theory" of disease --another seemingly bizarre concept we now take for granted: that illness is caused by an invasion of tiny foreign creatures we can't see.

In l88l, Robert Koch designed methods to study pathogenic microbes. Three years later Koch set down his famous postulates which still serve as a guide for determining the cause of an infectious disease and Joseph Lister was applying the principles of Pasteur's germ theory to prevent infections in his surgical patients.

Pasteur's germ theory of disease has given rise to many practices in modern medicine, including quarantine and disinfection. It also triggered the search for antimicrobial medications on the premise that if the cause is known a cure can be found.

This is not to say that chemicals weren't already being used to treat infections: two of the most popular remedies were mercury for syphilis and quinine for malaria. The Chinese were applying moldy soybean curd to carbuncles, boils, and other infections more than 2500 years ago. And a prehistoric Tyrolean man, discovered frozen to death in an Austrian glacier a few years ago, was carrying antibiotics on his ill-fated journey. Pieces of an antibiotically active fungus, fastened to a leather band, were found among the possessions this "ice man" who has been dated at between 5,300 and 5,400 years old.

But such therapies resulted from trial and error; they worked a lot of the time but no one knew exactly why.

Cause and Cure Replace Hit or Miss

By the l920s, when Alexander Fleming walked into his laboratory and stumbled upon Penicillium notatum fighting for its life in a dish of multiplying Staphylococcus aureus, bacteriology was an important scientific discipline. Thus, he was prepared for the fortuitous (but by no means uncommon) event. Not only was Fleming an acute observer who was intrigued by the mold's ability to kill staphylococci, he also appreciated the implication of what was happening.

Fleming showed that his "mold juice" was active against several types of Gram-positive bacteria. He also showed that the penicillin-containing broth was no more harmful to white blood cells than ordinary broth. This was of paramount importance to Fleming who, like his boss, the pioneering immunologist Sir Almroth Wright, was certain that the only way to fight disease was to help the body help itself. (Sir Almroth investigated the destruction of bacteria by white blood cells and helped demonstrate the presence of "opsonins," substances such as antibody that help the process proceed.)

Fleming's penicillin proved nontoxic to rabbits and was even used on a few patients. Between l930 and l932 Fleming and one of his former medical students, C.G. Paine, successfully treated people infected with pneumococcus, staphylococcus, and gonococcus with topically applied crude penicillin.

But Fleming wasn't a chemist and purification of enough penicillin for clinical trials was beyond his capability. As a consequence, Fleming put the study of penicillin aside and continued work on another important antimicrobial substance he had discovered, lysozyme.

Meanwhile in Germany, scientists at the I.G. Farbenindustrie were examining a large number of chemical dyes in hopes of finding antimicrobial activity. Their search was inspired by Paul Ehrlich's synthesis in Frankfort of salvarsan (the "magic bullet") and neosalvarsan, antisyphilitic arsenic agents in l912.

Two decades and thousand of compounds after Ehrlich's landmark achievement, Gerhard Domagk, research director of Farbenindustrie, reported that a sulfonamide-containing red dye known as prontosil protected mice against experimentally induced streptococcal infections and even cured patients. One of Domagk's first human subjects was his own child, a daughter who had developed a severe streptococcal abscess after a needle stick.

By l936, sulfonamide drugs were a clinical reality. But the antibiotic age didn't begin in earnest, however, until the penicillins became clinically available in the l940s.

The Allies Mount an Organized Attack

Microbial antagonism in nature was well-studied by l938 when Howard W. Florey and Ernest B. Chain, working at the Sir William Dunn School of Pathology in Oxford, began a systematic survey of antibacterial substances microorganisms manufacture to war among themselves. Fleming had characterized the properties of penicillin so well that when Chain read Fleming's l929 paper, penicillin became the first substance the two scientists chose to investigate.

In l941, the Oxford group, which now included a chemist, Dr. E.P. Abraham, succeeded in purifying enough penicillin to conduct a small clinical trial. The antibiotic proved life saving, but still so difficult to prepare that it had to be recovered from the urine of treated patients and used over and over again.

But we were at war and the enemy had sulfonamides. The value of an effective method to treat battle wounds did not escape the attention of government officials in Britain and the U.S. They collaborated, supporting strenuous pharmaceutical-industry efforts to produce enough penicillin for military use. The Anglo-American alliance paid off: A higher-yielding strain of penicillin-secreting mold was discovered (and Penicillium chrysogenum replaced Penicillium notatum for manufacturing purposes); an improved growth medium was developed; and deep fermentation was substituted for surface methods. The outcome was a penicillin yield thousands of times greater than that obtained in the research laboratories at Oxford.

The success of penicillin triggered a vast international search for other naturally occurring antimicrobial substances. A great number were found. Most were too toxic for human use and soon discarded. Others, such as bacitracin, proved only marginally useful. Some, like the erythromycins, tetracyclines, and aminoglycosides proved to have enduring clinical value. By l950 the "golden age" of antimicrobial chemotherapy was well underway.

Bacterial Targets of Opportunity

Bacterial cells offer a limited number of targets; thus despite their apparent diversity most antibiotics kill or disable prokaryotic cells by one of five basic mechanisms:

1) Disruption of cell wall synthesis. (Penicillins, cephalosporins, vancomycin, teicoplanin, cycloserine and isoniazid bind to enzymes needed by bacteria to build and maintain their cell walls, sabotaging structural integrity.)

2) Inhibition of protein synthesis. (Tetracyclines, erythromycins, and aminoglycosides infuse into a bacterium, attach to ribosomes, and disable the organism's ability to produce proteins. The rifamycins interfere with the ability of DNA to direct protein synthesis via messenger RNA.)

3) Interference with DNA. (Ciprofloxacin and other quinolones target DNA gyrase or topoisomerase IV, enzymes necessary for DNA coiling and uncoiling.)

4.) Inhibition of metabolic enzymes. (Sulfonamides and p-aminosalicylic acid, block a bacterium's ability to metabolize nutrients and dispose of its waste products.)

5.) Alteration of cell-membrane permeability. (Polymyxins damage the integrity of the microbe's membranous coat enabling vital cellular components leak out.)

Bacteria Fight Back

Microbes have been fighting one another for billions of years, they're good at war. Single cells generally live in crowded, competitive places, like soil ‚one gram, which easily fits on a fingertip, harbors over a million regular bacteria, another million actinomyces, almost as many fungi, close to a million protozoa, and up to 10,000 algae-- each struggling to earn a living. Little wonder that successful bacteria are endowed with highly efficient defensive as well as offensive competitive strategies. Moreover, they seem to easily distinguish between conditions favorable for rapid growth and those requiring a protective response.

Bacteria are clearly inventive when it comes to survival. Confronted with antibiotics, they can elude destruction by taking on new traits either by altering their permanent genetic material (chromosomal DNA) or acquire new genes from other microbes (extra chromosomal DNA). Such changes enable a bacterium to:

1. produce the sorts of enzymes that inactivate the threatening antibiotic;
2. prevent the antibiotic from reaching its target site(s); and/or
3. change a target site altogether.

The finding that bacteria can easily defeat antibiotics isn't new. Abraham and Chain noted in 1940 that Escherichia coli quickly destroyed their Oxford penicillin with an antibiotic-inactivating substance they named "penicillinase." Since then bacteria have quickly disabled almost every new antimicrobial with great skill; developing new pathways, new proteins, and new strategies for survival even more ingenious than those devised by humans to destroy them. Tricking bacteria into sustained susceptibility requires more than just developing new antibiotics. It requires human strategies every bit as clever as those of the bacteria we fight. Some are simple common sense solutions like frequent hand washing. Others are not so simple and include sophisticated global resistance monitoring, fast and accurate laboratory testing, use of narrow-spectrum targeted antibiotics instead of shotgun treatment and probably most important of all, using antibiotics only when necessary. Importantly, this mandates restriction of antibiotic use in agriculture as well with the necessary attention to good, old fashioned farming methods.

Links

1. The Centers for Disease Control and Prevention (CDC)
   http://www.cdc.gov
2. The American Society for Microbiology
   http://www.asmusa.org
3. The Food and Drug Administration
   http://vm.cfan.fda.gov

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