Bacteriophages (also called phages) are viruses that specifically infect bacteria.
They were first described by an English researcher, Frederick Twort, in 1915. Two years later, and independently, Felix d’Hérelle, a Franco-Canadian researcher, also discovered bacteriophages. He immediately considered their use to eliminate pathogenic bacteria and treat infections. It is thus considered as the precursor of phagotherapy (phage therapy). Since these viruses are capable of killing a species of bacteria, why not use them to kill a pathogenic bacterium? This is the basis of phagotherapy which is currently used in many Eastern countries.
Each type of phage recognizes a species, see a subspecies of bacteria. A bacteriophage will not infect and eliminate all bacteria but only those that will be recognized by the phage and allow its multiplication. Because of its high specificity, a bacteriophage can never infect one of our own cells.
Present throughout the biosphere, bacteriophages are the most widespread and diverse biological entity on Earth. We estimate their number at 10 31 on the planet. They play an essential environmental role, in particular by regulating bacterial growth but also by contributing to the genetic evolution of many microorganisms. The study of bacteriophages has contributed to the development of our knowledge of life and to the development of molecular biology. Currently, with the emergence of bacterial strains resistant to antibiotics, phages are considered a more promising alternative to conventional antibiotics.
When a bacteriophage is in the presence of a host bacterium, it will bind to specific receptors located on the surface of the bacteria: it is adsorption. The virus will then inject its DNA into the bacteria. It will divert the bacterial machinery that will become a factory to manufacture bacteriophages. A hundred viruses will be produced by the bacteria that will eventually lyse to let out bacteriophages. These will be able to infect other host bacteria. This is called the lytic cycle.
Some particular types of bacteriophages are also able to integrate their genetic material into the genome of the host bacterium. We then speak of lysogenic phages. The host bacterium will not be lysed but its genome will contain and express phage genes. These can code for dangerous toxins. Some strains of E. coli or Vibrio cholera, for example, become pathogenic through the DNA of a phage integrated into their genome. In unfavorable conditions for the bacterium, the lysogenic phage becomes lytic again.
The proposed experiment will make it possible to identify bacteriophages in the environment capable of lysing a strain of E. coli. coli. Pedagogical resources (links at the bottom of the protocol) will allow the teacher to go further in the discussion with his class by talking about the importance and the abundance of bacteriophages in the environment as well as phagotherapy as a complement antibiotics.
Where there are bacteria, there are bacteriophages! The easiest way is to take a sample from different rivers (Arves, Rhône, other rivers or fountains near the school). There is no need for a heroic harvest in the middle of the stream. The bacteria and therefore the phages, diffuse widely in the water and a sample from the bank works very well. The more “dirty” the stream, the more likely it is to have bacteriophages. The Arve at the junction is easy to access and contains many bacteriophages. A sample downstream of the outlet of a treatment plant is also possible.
Immerse a 50 ml tube in water and fill it.
Close the tube and note the source of the sample on the tube.
2) Removal of bacteria from the sample
The bacteria do not pass through the filters with a porosity of 0.45 μm. On the other hand, the smaller bacteriophages pass through.
Filter about 20 ml of the water sample into a new 50 ml tube (noted F). Be careful to work sterilely.
For this It is important to remove the piston before putting the filter so as not to tear it. Then the filter is added to the end of the syringe. The water is then poured into the syringe and the plunger inserted gently. Press gently to filter the water. Too much pressure may catch the filter.
3) Enrichment with bacteriophages
- Take 9 ml of the filtrate (tube F) and transfer to a new 50 ml tube (noted E). The rest can be used by another pair. Keep the remaining ml of filtrate (tube F) at 4 ° C. They will be used later.
The 9 ml no longer contain bacteria but may contain bacteriophages against E. coli.
- Add 1 ml of concentrated culture medium (10x LB) to 9 ml of filtered water.
With the help of a sterile loop take a little E. coli and resuspend it in the 10 ml solution.
- Close the tube
- Incubate the tube at 37 ° C 24h
E. coli will multiply thanks to the culture medium. If one or more phages able to infect E. coli are present in the 9 ml of the water sample, they will infect and multiply (enrichment). After 24 hours a sufficient number of bacteriophages can be highlighted.
After this incubation period, the tube can be kept 1 week at 4 ° C
4) Demonstration of bacteriophages
Filter the enrichment medium gently as described in point 2) by recovering the filtrate in a 15 ml tube (noted EF).
This step makes it possible to eliminate E. coli which has not been lysed by the possible bacteriophages.
The filtrate (EF tube) can be kept at 4 ° C. Bacteriophages are relatively stable and the preparation can be kept for at least one year under these conditions
- Make a suspension of E. coli (the same strain used for phage enrichment (point 3 of the protocol) as performed in point 2 but this time resuspending E. coli in 5 ml of sterile physiological saline
- Soak a sterile swab with the suspension. Spin lightly against the tube wall and rub the swab onto a petri dish. It is important to make tight streaks to deposit bacteria on the whole box in large quantities. Repeat the operation by turning the box one-third to streak in another direction. Repeat the operation a third time, turning the box a third more.
This manipulation is identical to the spreading carried out for experiment No 15: antibiotics . This is to obtain a “grass” bacterial.
- Observe the lysis pads
- Conclude about the presence or absence of bacteriophages against E. coli
- If E. coli was a pathogenic strain, discuss the concept of phagotherapy
Arve: water of the filtered Arve (without enrichment); C-: sterile physiological water; T4: T4 phage suspension; 1 to 4: Different water samples after enrichment: 1) Arve water (junction); 2 water of the Arve (Quai E.- Ansermet); 3) Arve water (Carouge); 4) Rhone water (junction). No phage has been detected in the Rhône water.