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Good day to all listeners thirsty for knowledge.
Lecture on bacteriophages: their discovery history, structure, morphological classification, lytic vs. lysogenic life cycles, and applications like phage therapy and typing.
Key Takeaways
- Bacteriophages are highly specific viruses that infect only particular bacterial species or strains.
- Phage therapy has a long history, was overshadowed by antibiotics, and is being revisited due to antibiotic resistance.
- Phages have diverse structures and life cycles, with lytic and lysogenic modes shaping their ecological and medical roles.
What the video covers
- Bacteriophages were independently discovered in 1915 (Twort) and 1917 (d'Herelle), with earlier observations by Hankin (1896) and Gamaleya (1897).
- Félix d'Herelle first proposed phage therapy; George Eliava is considered its founder, treating bacterial dysentery with phages.
- Penicillin's discovery in 1928 shifted research away from phages, which regained interest as bacterial antibiotic resistance grew from the 1960s.
- Phage structure is explained using T4 (E. coli): an icosahedral (cubic-symmetry) capsid head and a helical-symmetry tail with baseplate, spikes, and tail fibers for receptor recognition.
- The baseplate contains lytic enzymes that degrade the bacterial cell wall so the nucleic acid can be injected into the cytoplasm.
- Phages are classified morphologically by tail/sheath type and by nucleic acid (mostly double-stranded DNA; few RNA phages).
- Taxonomically, phages belong to the orders Caudovirales and Ligamenvirales, with 13 families, about 140 genera, and over 5,300 species.
- Virulent phages follow the lytic cycle, killing the host cell, while temperate phages can integrate as prophages and enter the lysogenic cycle.
- Phage typing differentiates bacteria, useful for diagnosing nosocomial infections and epidemiological analysis; phage titer is determined by serial dilution.
- Phages can indicate water contamination: phages with E. coli suggest fresh fecal contamination, while phages alone suggest older contamination.
Chapters
- 00:00Introduction and discovery of bacteriophages
- 02:16Phage therapy and the history of penicillin
- 10:40Structure of the bacteriophage (T4 example)
- 17:58Morphological classification and nucleic acid types
- 22:22Taxonomy: orders and families
- 24:40Life cycles: lytic and lysogenic
- 31:10Phage typing and diagnostic applications
- 36:40Phage titer determination and environmental indicators
Full Transcript — Download SRT & Markdown
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Today we are talking about bacteriophages.
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These are viruses capable of selectively infecting bacterial cells.
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The phenomenon of bacterial lysis caused by then-unknown infectious agents was observed back in 1896 by British chemist Ernest Hankin.
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Following him, the next year, similar phenomena were observed by bacteriologist and epidemiologist Nikolay Gamaleya.
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But bacteriophages, as independent infectious agents, were discovered twice: in 1915 and 1917, more or less independently.
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The first discoverer was British microbiologist Frederick William Twort.
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The second was a Belgian who worked at the Pasteur Institute in Paris.
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His name was Félix d'Herelle.
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It should be noted that although Frederick William Twort began to seriously research bacteriophages and described them, he even managed to publish his studies in a very famous general medical journal, yet he did not continue his research for various reasons.
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Primarily, this is related to World War I.
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But the second scientist, Félix d'Herelle, found the opportunity and inspiration to continue studying and describing bacteriophages.
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And it was this scientist who first proposed using them for phage therapy, that is, for treating infections caused by bacteria.
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However, it is still considered that the founder of phage therapy was George Eliava.
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He is a Georgian scientist.
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Uh-huh.
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Thanks to this scientist, they began to treat bacterial dysentery with specific bacteriophages.
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And by the time of 1930, seven preparations based on bacterial viruses were already known.
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But we know that after the 1930s, bacteriophages began to be gradually forgotten.
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And this is due to the fact that in 1928, Alexander Fleming discovered penicillin.
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That is, it was the most significant discovery in human history.
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And all scientists switched to studying this specific antibiotic and to searching for antibiotic producers among fungi and bacteria.
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And that is why bacteriophages were slightly forgotten.
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But due to the fact that starting from the 1960s, when bacteria began to acquire resistance to penicillin, bacteriophages began to be remembered once again.
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This means we are currently forced to return to the pre-penicillin era and continue studying bacteriophages.
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It should also be noted that Nikolay Bulgakov, the brother of the famous writer Mikhail Afanasyevich Bulgakov, was involved in the study of bacteriophages.
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Few people know, but Nikolay Bulgakov was a bacteriologist.
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He studied bacteriophages.
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And, by the way, he is the co-discoverer of the streptococcus bacteriophage.
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But let us first familiarize ourselves with the structure of a bacteriophage using the example of the most famous E. coli bacteriophage, T4.
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It should be noted that bacteriophages are unique in that they have a mixed type of symmetry.
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The head has a cubic type of symmetry, while the tail (rod) has a helical type of symmetry.
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The head consists of a capsid.
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This is the protein shell of bacteriophages, consisting of proteins that are made of repeating morphological subunits called capsomers.
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And inside this capsid is the nucleic acid of the bacteriophage, either RNA or DNA.
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An appendage extends from the head, inside of which is a hollow rod.
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And when a bacteriophage infects a bacterial cell, this rod contracts.
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And the bacteriophage's nucleic acid moves along this core into the bacterial cell.
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It should be noted that there is a collar on this appendage.
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There is also a sheath.
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The sheath ends at the bottom with a baseplate and spikes.
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It should be noted that the spikes and the baseplate are necessary for the bacteriophage to attach itself.
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They function as an anchor, attaching to the bacterial cell wall.
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Furthermore, the baseplate contains special lytic enzymes that help break down the bacterial cell wall so the core can enter the bacterial cell, that is, the cytoplasm.
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Tail fibers are attached to the bacteriophage's baseplate.
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It is with these tail fibers that the bacteriophage recognizes specific receptors on the bacterial cell.
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You see these tail fibers, they somewhat resemble the legs of a bacteriophage.
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It lands on the bacterial cell like a little spider and recognizes specific receptors on that cell.
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Because a bacteriophage is a sniper.
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It will destroy a cell only of a certain species or even a certain strain.
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As for the length of the bacteriophage, it can be said that its height is approximately 200 nm.
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In the photograph below, you can see a bacterial cell infected by dozens of bacteriophages.
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They use their tail fibers to recognize specific receptors on the bacterial cell wall and then proceed to infect it, if it is specific to that bacteriophage.
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Next, let's look at the classification of bacteriophages by morphology.
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This is a very popular and widespread classification.
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It should be noted that bacteriophages can have a contractile sheath, a long non-contractile sheath, or a short non-contractile sheath.
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It should be noted that the first three morphological groups of bacteriophages contain a double-stranded DNA molecule.
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You see, double-stranded DNA.
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This means that double-stranded DNA is contained inside the capsid of these bacteriophages.
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Furthermore, bacteriophages can be without a sheath and have capsomeres.
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They contain single-stranded DNA.
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You see, single-stranded DNA, they can be without a sheath of capsomeres.
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They contain single-stranded RNA.
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And there are filamentous bacteriophages, which are represented by a single-stranded DNA molecule.
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So, as you can see, if we consider all bacteriophages, the most numerous group is DNA-containing bacteriophages.
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There are very few RNA-containing bacteriophages.
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Regarding the taxonomic position of bacteriophages, it should be noted that they belong to two orders.
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These are Caudovirales and Ligamenvirales.
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These two orders include 13 families of bacteriophages, which encompass about 140 genera containing more than 5,300 species of bacteriophages.
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On this slide, you can see the names of these bacteriophage families.
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Myoviridae, Siphoviridae, Leviviridae.
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These are the most numerous bacteriophage families.
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Let's examine a few representatives from the Myoviridae family, which belongs to the order Caudovirales.
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These are double-stranded DNA-containing bacteriophages, also known as tailed bacteriophages.
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So, as you can see, even within the same family, the morphology of the capsid, sheath, and tail appendages can vary greatly.
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This photo also shows types of bacteriophages belonging to specific families: Siphoviridae, Podoviridae, Myoviridae, and Leviviridae.
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Bacteriophages have a very, very interesting life cycle.
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They are divided into virulent ones, which cause lysis of the bacterial cell—that is, cell death resulting from the fact that after infection by the bacteriophage, new phage particles form, the bacterial cell bursts, and thus new bacteriophages are released into the environment to seek out their hosts.
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And there are temperate bacteriophages, also called symbiotic bacteriophages.
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They are also capable of adsorbing to the cell.
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The bacteriophage genome ends up in the cytoplasm of the bacterial cell.
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But this genome, the bacteriophage DNA, is capable of integrating into the bacterial chromosome.
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And in this latent, non-infectious form, the bacteriophage is able to remain there for a long time.
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And then it is called a prophage.
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So, remember, yes, a prophage is a bacteriophage that is integrated into the chromosome of a bacterial cell.
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And bacterial lysis does not occur during this process.
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Meaning the bacterial cell does not die.
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And it is interesting that during bacterial division, it passes the bacteriophage's genetic material from generation to generation.
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And bacteria containing this temperate phage are called lysogenic.
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Let's take a closer look at these life cycles.
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The lytic cycle, where bacterial death occurs, and the lysogenic cycle, where the bacteriophage—or rather its genome—integrates into the bacterial chromosome.
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Let's look at it in order.
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Th
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spread it with a Drigalski spatula— this is called lawn seeding—and then place a drop of the specific bacteriophage in the center and tilt the Petri dish slightly, you see, so that the drop spreads out. After that, wait 20 minutes for this material to be
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slightly absorbed into the meat-peptone agar. Close the Petri dish, turn it over, and place it into an incubator, into a thermostat for incubation at a temperature of 37 ° C. And after 18 hours, or one day, we can see the
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result. If there is a transparent zone, a zone of lysis, it means that yes, the bacteriophage showed specificity for this bacterium. Which led to its lysis.
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And then we can accurately identify this pathogen. An equally important method is phage typing. This is a method of differentiating bacteria using bacteriophages. It is of great importance, especially for the diagnosis of nosocomial infections and epidemiological analysis. With this
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method, we can determine the phagovar. A phagovar is a list of typical phages that lyse a certain bacterium. This method is very simple to reproduce. It is necessary to inoculate the bacterium , a pure culture, as a lawn onto a
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Petri dish. On the bottom side, we can take a marker and outline sectors so that we don't get confused about which part we are applying which phage to.
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And after the bacteriophage is applied, the Petri dish is closed, turned over, and placed in an incubator, just like in phage identification. And the next day, we evaluate the results. If we have seen lysis zones like these in a
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specific sector, it means the bacteriophage has shown specificity for this bacterium. And in this way, we can determine the phage type of a particular microbe. Well, for example, let’s look at the second photograph.
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Here we have the plating of Staphylococcus aureus, golden staph, and Phage Type 3B 5381. As you can see, the bacteriophage only showed specificity and lysed bacterial cells in three sectors. To determine at which bacteriophage dilution we still observe
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bacterial cell lysis, we can use the Appelman phage titration method. This method involves using a liquid medium.
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An equal amount of meat-peptone broth, for example, 4.5 ml, is poured into sterile test tubes. Into the first tube , we add 0.5 ml of a bacteriophage specific to a certain bacterial culture . Mix thoroughly, then take 0.5 ml from
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the first tube and add it to the second . Mix from the second into the third, from the third into the fourth, and so on for 10 tubes. Well, here you see six test tubes shown. In the last tube, of
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course, the volume will be 0.5 ml greater. And so, after mixing, we remove 0.5 ml and discard it into a disinfectant solution. In this way, all tubes have the same volume of nutrient medium, but a different quantity and
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concentration of bacteriophages. After that, we inoculate an equal amount of bacterial culture, 0.2 ml of the culture suspension, into all tubes.
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Well, as studies have shown, the concentration of bacterial cells does not matter. But most often, a suspension of 1x10 to the 8th power cells per milliliter is prepared. After which, the tubes are incubated in a thermostat. The following day, we
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record the results. The last tube where there is no turbidity, meaning we do not observe bacterial growth, will be the phage titer. In this example, your phage titer is 10 to the minus 5th, because at 10 to the minus 6th the tube
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is already cloudy. Bacterial growth is observed there; presumably, the concentration of bacteriophages was too low. However, it should be noted that to perform the Appelman method, it is essential to include controls: a phage control and a bacterial control. Only
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after analyzing the control samples do we proceed to record our experimental results. Also very interesting and important is the Gratia method, which allows for titrating bacteriophages on a solid nutrient medium. Titrating bacteriophage using the Gratia method allows for an accurate calculation of
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the preparation's activity. What does this mean? It means we can clearly determine the number of phage particles in a specific volume of the test liquid . As a rule, this is per 1 ml. On this slide, you are presented with the
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detailed methodology for how to replicate this method. Furthermore, results can be counted after 8 hours of incubation in a thermostat at 37 ° C.
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And what do we need to do? Count the number of negative colonies, each of which resulted from the replication of a single phage particle. And so, if we see on the Petri dish, for example, seven colonies, we must multiply this
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number by the dilution of the bacteriophages. For example, the dilution was 10 to the power of negative X. And thus, we get a phage titer of 7 times 10 to the power of negative X phage particles per 1 ml of
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undiluted substrate. Why are these studies so important? In the previous lecture, we talked about how bacteriophages are an important indicator of the epidemiological safety of drinking water. Bacteriophages present in water can even indicate how long ago fecal contamination occurred.
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For example, if both bacteriophages and E. coli are found in water, this indicates fresh fecal contamination. If , for example, only bacteriophages are present, it means the contamination happened a long time ago, several months ago, because bacteriophages are
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able to remain viable in water for several months, despite the fact that the number of E. coli is at a low level . And this is very, very important.
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Bacteriophages can control the number of not only E. coli in water, but even the cholera vibrio. In the polluted rivers of India, primarily in the Ganges River, cholera vibrio is often detected, but cholera outbreaks are very rare. This is due to the fact that
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when the number of cholera vibrio starts to increase, the number of bacteriophages specific to the cholera vibrio increases in parallel. And that is why the bacteriophages in this river are called guardian angels. So, we have finished. Thank you all for your
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attention and see you next time.
Topics:bacteriophagesphage therapylytic cyclelysogenic cycleprophageT4 bacteriophagephage typingantibiotic resistancevirologymicrobiology
