Bacteriophages: Discovery, Detection, and Applications

By Guest Author •  Created: 05/21/22 •  5 min read

Bacteriophages are viruses that infect bacteria and archaea. They have a simple structure with genetic materials covered in a protein coat, just like typical viruses. Bacteriophages have recently been exploited for various purposes, prompting intensive research into their understanding.

Bacteriophage
Diagram of the T4 bacteriophage. Source; The phage blog

History of Scientists Discovering Bacteriophages

Bacteriophages were discovered in 1915 by William Twort, and their ability to kill bacteria was found in 1917 by Felix d’Herelle. These viruses were ignored entirely in the Western Hemisphere after discovering antibiotics. Phage appeared to be less effective and time-consuming to prepare compared to antibiotics. Bacteriophage research and use continued in some countries at the time, such as Georgia and other USSR members, where they were used as a standard form of medication to treat various bacterial diseases.

This turbulent discovery, followed by a loss of interest, has resulted in a patchy understanding of phage biology. However, after scientists discovered that phages control many aspects of Bacterial/Archaeal biological evolution, things had to change and can be used as a last resort for antimicrobial resistance. These discoveries have sparked renewed interest in bacteriophages, prompting many scientists to conduct extensive and in-depth research on these organisms.

Bacteriophage replication

Bacteriophages have two replication cycles: Lysogenic and Lytic cycles. In the lysogenic process, the phage invades a bacterium and then integrates itself into the host genome, allowing the host to survive but carrying its genome. Bacteriophages are also known as prophages once integrated into the host genome. And during the lytic replication process, a phage attaches to a susceptible host bacterium, inserts its genetic material cell cytoplasm, and manufactures its protein molecules using the host’s ribosomes. When the viral proteins become too numerous and assemble themselves, they cause host cell rupture and thus released into the extracellular environment, ready to attack their next prey.

Where to find phages?

Phages, like their “prey,” are present almost anywhere. The basic principle is that the presence of bacteria determines the presence of these viruses. As a result, if one needs to isolate phage for a specific bacteria, one must look for a location where such bacteria is common. Not to mention that bacteriophages are more numerous than bacteria, and a single bacteria can be susceptible to multiple types of phages.

How to grow Bacteriophages from the Environment

Given the information we have so far, it is clear that the environment is the most likely source of phages. If you want to isolate phages for any use or host bacteria, the procedures are the same, with minor variations depending on the growth characteristics of the bacteria. E. coli will be used as a model organism in the following procedure, so the growth conditions listed are specific to this bacteria. Phages can be isolated in two ways: with and without sample pre-enrichment. Pre-enrichment helps multiply phages so that their presence in a sample is easily detected (we do the same thing with bacteria, using broth media as a nutrient during isolation).

Procedure

  1. Sample collection in a sterile container. (Sewage samples are recommended for E. coli and other enteric bacteria.)
  2. Sample transportation following a standard procedure. 
  3. Sample centrifugation and filtration. (You can use a syringe filter or compartment/chamber filter of 0.22 µm)
  4. Sample enrichment (Adding an overnight bacterial culture, E. coli culture in this sense) to the pre-filtered sample and incubating in an incubator shaker for 18-24 hours at 170 RPM.
  5. Centrifugation of pre-enriched sample.
  6. Filtration of the pre-enriched sample (same as step 3)
  7. Spot the filtered pre-enriched sample on a bacterial lawn. (Made using the double-layer agar method)
  8. Incubate for 18-24 hours at 37ºC.
  9. Observe the plaques.

Consider the image below: AE/ED and AE/ED2 are positive samples, while the rest are harmful. If you get a double clearance, also known as phage plaque halo, it’s because of the effect of the enzymes released by bacteriophages.

A plate with a positive and negative spot assay test, with areas with transparent patches indicating bacteriophage activity on susceptible host bacteria. Taken by Raphael Hans Lwesya at Makerere University

This procedure is for detecting bacteriophages in a sample; the plaque you’re looking at could have been induced by more than one phage type. Individual phage isolates are required for better characterization of the phage. The phage filtrate is serially diluted (Read this article on plaque assay) and surface poured on a solid media to create a double layer of agar during the isolation steps.

* This is optional, but it produces better and more consistent results.

Uses of bacteriophages

Bacteriophages, which are considered safe for humans, have been assigned to various uses. They have remained one of the most widely beneficial viruses today, either through genetic engineering or through their natural life cycle. Bacteriophages are used in the following ways.

  • Vaccine production
  • Drug delivery (especially filamentous phages due to their large sizes)
  • As a biomarker agent
  • Phage therapy (for bacterial diseases)
  • Monitoring infections
  • Diagnosis of bacterial diseases
  • Decontaminating surfaces

And many others. Despite having a wide range of applications, some of them, particularly those involving therapy, are still in the early stages of development and are only used in emergencies where antibiotics have failed.

About the Author

Raphael Hans Lwesya is a young scientist, phage enthusiast, and passionate about driving bacteriophage development for application to combat communicable and non-communicable diseases. He is also the creator of The Phage blog, an online hub for providing phage technology updates and the possible alternative to antibiotic use. Raphael found the booming AMR cases in recent years to be of concern, hence decided to dig deep into Antibiotics’ promising alternatives. Contact LinkedIn, Twitter, and Facebook

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