Penicillin blocks the action of the enzyme transpeptidase and forms an irreversible connection to the active site (covalently connects). As the bacterial cell is growing (making a new cell wall) and dividing, penicillin prevents the cross-linking of small peptide chains in the cell wall of the bacteria. Pre-existing walls are unaffected, but all newly produced cells grow abnormally, unable to maintain their wall rigidity, and are susceptible to osmotic lysis.
The production of penicillin can be used to illustrate the principle of an industrial fermenter. Huge fermentation vats filled with liquid and nutrients necessary for moulds 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.
Repeated steps of mutation and selection have led to the development of the strains of Penicillium chrysogenum used today, which produces penicillin at a concentration of about 30,000 units per cm³.
Penicillium chrysogenum if given in small amounts of all of the nutrients necessary for growth, will mostly just grow and will produce only small amounts of penicillin. Penicillin production by this fungus provides a nice advantage if the fungus is competing with bacteria for limited food available. If there is not 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 mould will not make much penicillin – a waste of unnecessary 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.
Industrially, Penicillin chrysogenum is grown in large fermenters but initially the fungus is grown in the laboratory on a small scale to produce an inoculum, which is used to inoculate the fermenter. Commercial firms (pharmaceutical firms) induce Penicillium chrysogenum to make lots of penicillin by limiting the amount of food available within the fermenter where the mould is growing. The genes inside the fungal cells don’t know that there are not any bacteria out there and are only responding to the decreased availability of food.
A supply of oxygen is required, as the growth of the fungus and the production of penicillin require aerobic conditions. The production of penicillin is stimulated by the addition of phenylacetic acid, but the concentration is critical as it is toxic to the fungus. Once the penicillin is released from the fungal cells, the compound is isolated from the fermenter’s contents and purified by special biochemical processes. The process of extraction, purification and subsequent chemical modification of penicillin is referred to as downstream processing. The penicillin is extracted firstly by filtration, which separates fungal material from the medium, then by using solvent extraction to isolate the penicillin. The pH is reduced to between 2.0 and 2.5 and penicillin is extracted into an organic solvent such as amyl acetate. Penicillin is then re-extracted back into the aqueous buffer of pH 7.5, concentrated and then crystallised. Penicillin produced in this way is known as penicillin G, which may be converted into semi-synthetic penicillin’s as means of overcoming the problems of penicillin-resistant strains of bacteria (see Antibiotic Resistance section). Such commercial production results on more than £100,000,000 of penicillin production each year.
Although more than 50 years has passed since penicillin was first produced in volume, the biochemical engineer of today would immediately recognise the production plant and equipment used then: the stirred aerated reactor is still the standard; air is still sterilised by filtration; and the product is recovered by solvent extraction. Penicillin was the first important commercial product by an aerobic, submerged fermentation. In the 1930’s, citric acid was produced by aerobic fermentation, but by surface fermentation in shallow trays and not by submerged fermentation in agitated deep tanks.
The instability of the penicillin molecule under acidic conditions and its low concentration in the fermentation broth required the development of extraction equipment that could efficiently contact the aqueous penicillin – containing broth with water – extraction solvent, and then rapidly separate the two phases. The application of freeze drying of penicillin opened the door to drying and preservation of sensitive biological products in general. Although techniques of genetic engineering are of use, they have supplemented rather than replaced the “classical” methods applied so effectively to Penicillium chrysogenum and many other organisms since.
Methods of comparing relative efficiency of antibiotics have developed considerably over the years. The minimum inhibitory concentration (MIC) is a basic measure of the activity of antibiotics. The MIC is the lowest concentration of the antibiotic that results in inhibition of visible growth (i.e. colonies on a plate or turbidity in broth culture) under standard conditions (See Figure 1). For an antibiotic to be effective the MIC must be able to be achieved at the site of the infection. The pharmalogical absorption and distribution of the antibiotic will influence the dose, route and frequency of administration of the antibiotic in order to achieve an effective dose at the site of infection.
In clinical laboratories a more common method of comparing the relative efficiency of antibiotics is a disk diffusion test (see Figure 2).
Antibiotic Resistance
Antibiotic resistance is not a new phenomenon. This problem was recognised soon after the natural penicillin’s were introduced for disease control, and bacterial strains held in culture collections from before “the antibiotic era” have also been found to harbour antibiotic-resistance genes.
Antibiotic resistance is the ability of a bacterial cell to resist the harmful effect of an antibiotic. This resistance may be represented by several different systems and a given bacterial cell may have one or more of these systems available:
- The bacterium may have a system that prevents entry of the antibiotic into the cell;
- The bacterium may have a system that destroys the antibiotic if the antibiotic gains entry into the cell;
- The bacterium may have a system that associates with the antibiotic inside the cell and therefore blocks the action of the antibiotic;
- The bacterium may have a system that pumps the antibiotic back out of the cell before the antibiotic can act within the cell.
Bacteria most easily acquire resistance to a given antibiotic when bacteria are in a limited geographic environment with routine, consistent exposure to antibiotics or within a single individual on long-term antibiotic therapy. If antibiotics are used extensively, there is an increased likelihood of selection of a bacterial cell that is resistant to the effects of a given antibiotic.
Another situation that encourages selection for antibiotic resistance is indiscriminate usage of antibiotics. If an antibiotic is not needed to treat a disease, such as a disease caused by a virus (antibiotics only work on bacteria), the use of antibiotics in this situation increases the opportunity to select a bacterial strain that is resistant to that antibiotic.
There is increasing awareness of what we are doing to increase the presence of antibiotic-resistant strains of disease-causing bacteria – both in the medical community and in the food industry. The issue of antibiotic-resistant bacteria is a world-wide problem and continuous efforts are being made to reduce this threat to human health. If action is not taken soon to find ways of combating this problem we may find that in years to come people will die of diseases that we are successfully treating today.