~~~ What the Heck is Penicillin? ~~~

This article is kind of long.. <grin>. There is a bunch of interesting stuff (to me anyway) about penicillin - the history of discovery and some other stuff. So, try to sit back and enjoy the information... but, don't yell at me too much if you don't like it... <grin>

Penicillin was the first naturally-occurring antibiotic discovered - and the first to be used therapeutically. An antibiotic - (Greek anti, against and bio life) is any substance produced by a microorganism which can kill or inhibit the growth of a different microorganism. We now call such substances, and any similarly-acting substances which humans design, chemotherapeutic agents. If you'd like, please see What the Heck is an Antibiotic?

Before we begin to talk specifically about penicillin, it will be useful to kind of place you in the life and times of people in the 1800's. During this time, no one had any idea about anesthetics (pain-killers), and only limited ideas of how diseases were generated. There were no antiseptics, there was no such thing as sterile conditions - instruments used in one surgery were simply re-used without any particular cleaning for the next surgery. Although Pasteur and Koch had clearly shown the connection between disease and bacteria, and by 1800 people were already receiving vaccinations against smallpox (Jenner), not very many people placed too high of an opinion on this work. One person did - Joseph Lister - credited for introducing antiseptic surgical procedures. Lister based his work on studies by Semmelweiss. Semmelweis documented association of sepsis (meaning "putrefaction") in hospitals - particularly birth mothers - with certain surgeons. Back then the more prominent the surgeon, the more filthy the lab coat the surgeon wore (blood, fluids, etc., were left on the coat) - was a mark of distinction, since the reasoning was that if a surgeon's lab coat was covered with dried blood and the like, then the more surgeries (operations) this person performed; then, in order to be so busy to get such a filthy lab coat, he had to have many patients, and the more patients meant the better he must be at surgery, and therefore the more prominent. Personally, I'll take a Mercedes over a filthy lab coat as a measure of surgical expertise, any day.

Semmelweiss noticed that if certain "prominent" surgeons delivered babies (these were all-around kind of physicians back then), the birth mother had a good chance of dying from infection (sepsis). Unlike his peers, Semmelweiss thought that these infections were being transmitted from the physician to the patient - that there must be some kind of infectious agent on the hands, lab coats, instruments, etc., which could be given to the birth mothers; so, Semmelweiss always routinely cleaned his hands and instruments with a phenol solution - none of his patients ever acquired infections. Finally, after much debate and many more deaths, the hospital adopted his cleaning procedures. Lister published his studies on antiseptic surgery around 1867 and established the procedures to maintain sterile conditions during any kind of surgery.

OK - it's around 1890 or so, and we now we have a few people beginning to make the connection between disease and unseen things. Along comes a 21 year-old medical student in France, who chances upon what will later become (about 40 years) a revolutionary treatment of disease - the discovery of penicillin. Like many discoveries in science, the discovery of penicillin was actually a re-discovery, and partly due to the fact that chance usually favors the "prepared mind". The earliest record of a substance produced by a mold which could apparently kill bacteria was made in 1896 or so, by a French medical student named Ernest Duchesne. However, his work was largely ignored, and eventually forgotten. Then, in 1928 the Scottish physician Alexander Fleming was working on staphylococci bacteria ("staph") isolated from wounds, and was culturing them (growing them) on nutrient agar (agar is a semi-solid, porous substance made from seaweed - when the agar is heated it is a liquid, but upon cooling the agar becomes kind of like the consistency of "Jello" - if nutrients are added to the agar in liquid form, when the agar gels the nutrients will be dispersed inside).

Fleming had been working during and after World War I trying to find ways to kill bacteria isolated from infected wounds in order to find a way to treat wounds and prevent infections. Like most wars up to that time, infections of wounds rather than the wound itself caused the vast majority of deaths. One way to do these studies was to get a swab of the wound, and wipe the swab onto some agar containing nutrients. The agar was inside what is called a Petri dish (named after the person who worked with Koch while Koch was establishing the bacterial agar culture system and the association of bacteria with disease). If bacteria were present on the swab, they would be transferred to the surface of the agar - then, the bacteria would feed on the nutrients within the agar and would grow atop the agar (the identical procedure used today to isolate and identify bacteria). These bacteria could later be isolated and studied further.

As the story goes, one day in 1928 Fleming left the lid off the top of one of his Petri dishes for a little too long, and a fungal spore landed on it (the culture became contaminated). After returning from vacation, Fleming noticed that his Staphylococcus culture was contaminated with this fungus - but - instead of throwing the Petri dish away, he carefully examined it first. Fleming noticed that there was a zone around the fungus which was completely devoid of bacteria - whereas elsewhere on the agar, the bacteria were growing just fine. If you pretend that the little white discs are fungal growth, and ignore the label, Fleming's Petri dish might have looked something like the Petri plates shown below:
Fleming correctly reasoned that this growing fungus must be producing some sort of diffusible substance which killed the bacteria. He then identified the fungus as Penicillium notatum, isolated the material which killed the bacteria and named the substance, penicillin. Although finding that this substance killed many different kinds of bacteria and should be highly useful to treat infections, he also found that the substance appeared very unstable - many times would not work - so - he quit doing any research on the substance and published his last work on penicillin around 1931.

Time passes, it's around 1935 or so, and still no major breakthroughs with respect to treatment of bacterial diseases - except for the discovery of a human-made substance, sulfanilamide. Now, nobody went out and said - "I know, I'll just make some chemical compounds which kill bacteria - how about sulfanilamide - that ought to work." Instead, there was a big testing effort - a screening effort - to examine human-made substances to see if these substances could kill bacteria, and at the same time not hurt an animal or a person. Sulfanilamide was originally part of a leather dye compound - dyes were screened for toxicity to animals, and also for their potential ability to kill bacteria - one of these dyes was found to kill bacteria and to be relatively nontoxic - research on this dye led to the discovery of the body's action on it which resulted in the formation of sulfanilamide. When the dye is broken-down inside the body (our body and animals in general have the capacity to alter chemicals, and to often - but not always - render them harmless), it is converted biochemically to the compound, sulfanilamide. More time passes to about 1939 - still no further breakthroughs on treatment of infections.

We now turn to Oxford, and the scientists Howard Florey, Ernst Chain, and Norman Heatley. Chain was working in Florey's laboratory (studying infectious diseases) and had read Fleming's papers. Chain wrote Fleming and obtained the Penicillium notatum fungus - Chain then began to prepare penicillin isolated from the fungus for testing against all kinds of bacteria. Heatley, a biochemist, designed all of the experimentation for isolation, purification, and characterization of penicillin, and after quite a lot of work, these scientists purified penicillin for testing in mice. Mice given streptococci and staphylococci were also treated with penicillin - and all of the mice survived. Then, in 1940 these test results were published, and the first human clinical trial to test penicillin was generated - the test as we all know, was successful - It is of some interest to note that the amount of penicillin tested in the entire clinical trial was less than one receives in a single shot of penicillin, today! With the successful testing of penicillin, the age of the use of naturally-occurring substances for treatment of infectious disease was born. Although the original purification and testing of penicillin occurred in England, the United States became the place for large-scale production of this antibiotic - remember that by now, World War II had begun, and Great Britain was in the midst of this war - almost all of Great Britain's industrial capacity was devoted to the war effort - and because of the danger from bombing raids, the entire process for penicillin production was moved to the United States.

Here follows a warm, human interest anecdote concerning Ernst Chain, provided by Rob Carter, whose father-in-law, Jack Judah, tells of his early days in Oxford as a medical student.

"Visiting your pages while looking for information on Ernest Duchesne and various scientific ideas that seem to arrive ahead of their time put me in mind of a nice anecdote my father-in-law, Jack Judah, tells of his early days in Oxford when he was a medical student.

It was wartime and some pretty interesting things were happening in Oxford. Ernst Chain was a Jewish immigrant and a friend. He was a little unworldly and having plenty of more important things to think about did not care too much about his appearance. One day he went to my father-in-law's sister, a very stylish lady who regarded Ernst as beyond sartorial help, and asked her advice about acquiring a tuxedo and an appropriate pair of shoes. The advice was duly given and only later did she ask my father-in-law, "Jacky, what in the world did Ernst Chain want with a dinner jacket?" "Oh," Jack said. "That would be for his trip to Stockholm." It dawned on her a little later that he was intending to go pick up his Nobel prize, but he had said nothing to that effect. He was a lovely man without a self-promoting bone in his body, and I like to think of him and the millions of lives his work has saved."

Rob Carter

We will now get to exactly what penicillin is - and how it works... Here is the structure of penicillin G - the compound many different species of the fungus genus Penicillium produces - the same one that was killing bacteria during Duchesne's work in 1896, Fleming's work in 1928, and Florey, Chain and Heatley's work in 1939 - and the same one we get in a shot (hypodermic) form.

The Chemical Structure of Penicillin G

Penicillin G with R-group = Phenyl-CH2-     The # equals a double-bond

                           O           S    CH3
                           #          / \  /
                Phenyl-CH2-C-NH-CH---CH  C--CH3
                                |    |   |  
                      ----->    |    |   |
Beta-lactam ring ___ /          C----N---CH--C#O
                                #            |
                                O            OH

R-group =   Phenyl-CH2-       Penicillin G
R-group =   Phenyl-(O-CH3)2   Methicillin
R-group =   Phenyl-CH2-       Ampicillin
What in General Does Penicillin Do?
Penicillin works by preventing a VERY important part of a bacterial cell from forming - the cell wall. As you can see, penicillin is a pretty complicated chemical compound. If you have ever had a moldy piece of bread lying around, smell it - you will smell a "musty" kind of odor - is the penicillin that is being produced..... the mold itself will be kind of greyish-green in color on the bread. The cell wall of all bacteria is critical to maintenance of the integrity of the cell - so - if the cell wall cannot be made for any reason, the cell will rupture and die. Bacteria reproduce by a process called binary fission - meaning that a single cell divides to produce two daughter cells. During this cell-division process (every 20 minutes or so for many bacteria), the individual cell will enlarge to about twice the usual size - cytoplasmic substances will be made, and the single chromosome made of DNA will be replicated. The two chromosomes will separate and move apart from one another as the cell enlarges... then, the brand-spanking-new cell wall being synthesized will begin to grow together, to finally join and the two new cells will separate and begin the cell-division process all over again. Penicillin works at this particular stage - the synthesis of new cell wall. Penicillin will not harm any cell wall already made - kind of like won't hurt a brick wall, but will prevent any more bricks from being laid down to make more wall. The cell wall is incredibly strong, and prevents the cell from bursting, for the following reasons:

The Need for a Cell Wall
The bacterial cell is kind of like a little balloon filled with all kinds of important things necessary for the cell to stay alive, and to reproduce. However, this balloon skin - the cytoplasmic membrane which surrounds all of the cell's contents (cytoplasm) is kind of like a real balloon in that the skin is not very strong. If water were to come in, and keep coming in, the bacterial cell would eventually swell and the membrane would burst - just like a real balloon would burst if you kept pumping water into it.

The cytoplasmic membrane (skin of the balloon) of a bacterium is semi-permeable - meaning that the membrane is "open" _only_ to water and gases by simple diffusion - and _EVERYTHING_ else must be specifically carried across the membrane - can't just float across. Now, when there is such a membrane, and if inside the balloon there are many chemicals at concentrations greater than the concentration of these chemicals on the outside (other side of the balloon's skin), water will rush inside - there is a chemical "pressure" for the concentrations of things _to be the same_ on both sides of such a membrane. A bacterial cell is like this - the concentration of stuff on the inside is much, much greater than the concentration of the same stuff on the other side of the membrane (towards the environment). A bacterium can literally pack itself full of chemicals... So, if a bacterium did not have a special structure - called a CELL WALL - then the water rushing in would cause the cell to fill with more and more water - with the membrane stretching and stretching, until finally - the membrane would rupture and the cell would explode...

What the Heck is a Cell Wall?
The cell wall is made of two different kinds of sugar molecules hooked together (covalent bond) and some other molecules (amino acids) linked together which kind of dangle down from one of the sugars. There are many, many sugars connected to one another to make a long string - and many, many strings which encircle the spherical membrane- and every-other sugar has these amino acids attached to it - all of these individual sugars connected to one another - like a string of beads - is called a polymer....AND, these strings of polymer are CONNECTED to one another - to form cross-links.. kind of like having 10 individual, long strings, and then using little pieces of additional string to tie EACH of the long strings to one another in lots of places - all around and all over the cell.... completely surrounds the membrane - like a metal mesh grid wrapped around an air-compressor tank (a safety feature of some air-compressors - in case the metal casing of the air-storage tank ruptures because of all the air pressure inside - the mesh grid keeps the pieces of casing from flying out to hurt someone)

What Penicillin Does
In order for these long strings of polymers to get connected to one another (the cross-linking step), a special enzyme is required... PENICILLIN BLOCKS THE ACTION OF THIS ENZYME - the name of the enzyme is: transpeptidase... Now, the long polymer strings will still be made - they just cannot be cross-connected to one another in the presence of penicillin. Remember the ß-lactam ring structure - well - this structure reacts with the place on the enzyme which catalyzes the chemical reaction of linking amino-acids together... this place - the active site of the enzyme - is irreversibly blocked. Penicillin forms an irreversible connection to this site (covalently connects). So, as the bacterial cell is growing (making new cell wall) and dividing, new cell wall must be made continuously to completely surround each of the dividing cells - if penicillin is around, the cell wall cannot be cross-linked - the result is that the cell wall is very weak in places - and now as water comes in, the membrane which isn't protected by cross-linked cell wall, will squeeze out and rupture... this rupturing of the membrane will cause the cell to die.

A Little Diagram of the Cell Wall of Bacteria
Here is kind of how the cell wall is made, and how penicillin stops the cell wall from being completed: There are many, many of these polymer strings - we'll look at only two of them...

       |     |     |     |    two individual polymer strings
       |     |     |     |    made of two different sugars (a) --- and, 
       |     |     |     |    (b) xxx;  and, amino acids | connected to xxx
       |\    |\    |\    |\   and other amino acids \ connected to |
         \     \     \     \    

2.  ---xxx---xxx---xxx---xxx---  
       |     |     |     |       
       |     |     |     |       Now, the transpeptidase enzyme connects
       |\    |\    |\    |\      | in one string to | in a _different_ string
	 \     \     \     \     like this.....

1.  ---xxx---xxx---xxx---xxx--- 
       |     |     |     |       
       |     |     |     |       
       |\    |\    |\    |\        Polymer strings 1 and 2, connected....
         \     \     \     \
2.  ---xxx---xxx---xxx---xxx---  
       |  \  |  \  |  \  |  \    
       |   \ |   \ |   \ |   \ 
       |    \|    \|    \|    \      etc.....   

If penicillin is around, the cross-connections cannot be made....

Here is something you can try... get some balloons, and blow them up until they burst.... Now, get some panty-hose.... put the balloon inside the panty-hose and try to do the same thing... YOU WILL NOT BE ABLE TO CAUSE THE BALLOON TO BURST.. the panty-hose is like the cell wall - it is made of lots and lots of individual strings - but - each string is connected to another string, cross-wise... is cross-linked... this cross-linking makes the panty-hose very, very strong..... This panty-hose, balloon thing idea, was from a friend and colleague of mine - Dr. Laurence Draper... it is a great idea and shows pretty much exactly how the cell wall prevents a bacterial cell's cytoplasmic membrane from bursting.... Take a look at some pictures - and a movie to boot - on the effect of penicillin on bacteria: James A. Sullivan's CELLS ALIVE!

From Where Do We Get the Penicillin We Use?
Huge fermentation vats (great big containers) filled with liquid and nutrients necessary for molds to grow are used to grow large amounts of Penicillium chrysogenum - one of the species of Penicillium which can be grown in stirred fermenters - for large-scale production. Now, Penicillium chrysogenum if given high amounts of all of the nutrients necessary for growth, will mostly just grow - and will produce only small amounts of penicillin. Why is that? Well, genetics plays an important role here... penicillin production by this fungus provides a nice advantage if the fungus is competing with bacteria for limited food available. if there isn't much food around, the genetics of this fungus allows for the synthesis of penicillin - which will result in the killing of any nearby bacterial competitors. On the other hand, if there is plenty of food around, the mold will not make much penicillin - a waste of uneccessary energy - living things have regulation of genes which respond to these environmental differences by making different things which are useful only under very precise conditions - like the absence of a certain kind of necessary food. So, knowing how genes work, and how this fungus responds to food availability - commercial firms (pharmaceutical firms) induce Penicillium chrysogenum to make lots of penicillin by limiting the amount of food available within the fermenter where the mold is growing - the genes inside the fungal cells don't know that there aren't any bacteria out there - are only responding to the decreased availability of food (which if in the wild, could result in elimination of bacterial competitors - and at the same time provide nutrients made available from the dead bacteria). Once the penicillin is released from the fungal cells, the compound is isolated from the fermenter's contents and purified by special biochemical processes. Such commercial production results in more than 100,000,000 pounds of penicillin production per year!

Other Forms of Penicillin
Penicillin G is not stable - just as Fleming discovered back in 1928 - As it turns out, the chemical structure of penicillin makes the structure of this compound very unstable in the presence of acid (is therefore said to be: acid-labile). Since our stomach has lots of hydrochloric acid (common name: muriatic acid) in it (the pH can be around 2.0), if we were to ingest penicillin G, the compound would be destroyed in our stomach before it could be absorbed into the bloodstream, and would therefore not be any good to us as a treatment for infection somewhere in our body. It is for this reason that penicillin G must be taking by intramuscular injection - to get the compound in our bloodstream - which is not acidic at all. However, there are all kinds of what are called semi-synthetic derivatives of penicillin - like Ampicillin, Penicillin V, Carbenicillin, etc... These compounds consist of the basic Penicillin G structure, but have been purposefully modified in the laboratory to make the drug acid-stable (Ampicillin, Carbenicillin) - or - more acid-stable than Penicillin G (Penicillin V) - or - to make the compound more effective against different bacteria (Ampicillin - works against Gram+ as well as Gram- bacteria) - or - more resistant to an enzyme which can destroy penicillin (Methicillin). This penicillin-destroying enzyme is called, penicillinase (ß-lactamase).

What is Penicillinase
This enzyme is produced by certain penicillin-resistant bacteria... ooops!... bugs fight back against fungi! Unfortunately, if we get a bacterium which causes disease (a pathogen) and if this bacterium can make penicillinase, then, giving penicillin or _any_ of the semi-synthetic derivatives of penicillin G (except for methicillin which is more resistant to breakdown by this enzyme), penicillin will not harm these bacteria. So, we turn to the use of a specific inhibitor of penicillinase - called clavulanic acid. This compound will irreversibly bind to penicillinase and prevent the enzyme from working. So, if we are worried about a particular bacterium being resistant to penicillin, sometimes clavulanic acid is given along with one of the semi-synthetic penicillins. - block penicillinase in the resistant bacterium, allow penicillin to work - kill the bacterium - remove the infection. We also have other things to fight infection which are also somewhat like penicillin - same ß-lactam structure, but more resistant to penicillinase.

Another member of the ß-lactam antibiotics, but quite different than the penicillins (except for the ß-lactam ring and the identical mechanism of action, e.g.,inhibition of transpeptidase) is called cephalosporin (produced by the fungus, Cephalosporium), and like the various penicillin derivatives, there are many different ones available. One of note is called Cefoxitin. As it turns out, the cephalosporins tend to be resistant to the action of the Beta-lactamase enzyme, and, are also used in place of penicillin if a person is allergic to penicillin. There are acid-stable and acid-labile semi-synthetic derivatives available. The reasons why cephalosporin derivatives are not given as often as penicillin are partly due to cost, but mostly due to the attempt to avoid overuse of _this_ ß-lactam antibiotic, too. The over-use of penicillin has led to the appearance of more and more penicillin-resistant strains (selection of those bacteria which can produce Beta-lactamase). If there are 100,000 bacterial cells capable of causing an infection, and only one cell in this population can make penicillinase, then treating the infection with penicillin will allow only this cell to survive - and to therefore reproduce - now - we can have 100,000 penicillin-resistant disease-causing bacteria! It is for this reason - the potential to select for antibiotic-resistant bacteria, that careful and limited use of antibiotics is necessary. Also, because of the appearance of so many antibiotic-resistant pathogenic bacteria, there is an on-going search for natural substances produced by plants, other recently identified bacteria, and other microorganisms for their ability to kill bacteria while at the same time causing no harm to us.

Book: Don't Touch That Doorknob!

Copyright John C. (Jack) Brown, July, 2001
My special thanks to Raymond Lin in Singapore for his kind permission to use the antibiotic sensitivity-plate image.
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