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.
"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
|
NH2
What in General Does Penicillin Do?
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...
1.---xxx---xxx---xxx---xxx---
| | | | 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.
Cephalosporins
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!