Diagnostics
entire bacterial species; in other cases, it would be a certain strain within a species.
In each case, there is a structure on the surface of the phage that binds to something unique on the surface of the bacterial cell.
“If we can detect these specific phage infection events, it’s a way of letting us know what type of bacterial cell is present in a sample,” says Dr Cath Rees, professor of microbiology at the University of Nottingham. “It means you don’t have to grow the bacteria to levels where you can use traditional microbiological methods to detect them.”
Bacterial detection This isn’t an altogether new idea. In fact, phages have long been used in epidemiology as a means of identifying bacterial strains. After taking a bacterial sample, you apply a panel of phages known to infect that species. The sample will show a pattern of susceptibility and resistance, which can be matched up with a given strain. Phage detection is also commonly used to determine levels of water pollution – if a certain phage is present, that suggests its host is present too. Since phages replicate much faster than bacteria, this can be a quick way to detect contaminants. Another line of research involves genetically modifying the phages, so that they produce a luminescent molecule when they infect their host. Researchers can track the resulting signal, helping them identify bacteria in a sample. “In my lab, we are detecting the increase of phage replication using molecular diagnostics,” says Dr Thomas Edwards, a post-doctoral research assistant at the Liverpool School of Tropical Medicine. “It’s very hard to detect a single bacterium in a blood sample using molecular testing, but we are finding it easier to detect 100 phages becoming 1,000 as they replicate through that cell.” Ultimately, he thinks phage-based diagnostics could be a game changer. As well as being time efficient, these methods are highly sensitive and specific. They can also distinguish between live or dead bacteria. This means a lower risk of false positives, as they won’t flag up an infection that has already cleared.
“Assays to sensitively detect bacterial bloodstream infections, without the 24–48-hour delay posed by blood cultures, could revolutionise clinical microbiology,” he says. “Using phages in this manner only requires a one-hour incubation, followed by a PCR-based assay.” His team have also used phages to detect antibiotic resistance. Since a phage will not replicate in a dead bacterial cell, the presence of phages may indicate that the antibiotics aren’t doing their job.
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The difficulties with diagnostics Given their many noted advantages, it is perhaps surprising that phages haven’t been more widely exploited within diagnostics. To date, very few phage- based tests have reached the point of clinical usage. As Rees explains, these types of tests have been in development for more than 30 years. However, since each phage, host and application is different, progress has been slow. Each research group is effectively starting from scratch, without that pool of shared knowledge to draw from. “There have only been small numbers of scientists who have focused on phages, which are specific for each of the different types of bacteria,” she says. “In comparison, PCR-based methods use the same basic technique to detect a whole range of different bacteria, so there have been hundreds of different groups and companies working on developing the methods. This has moved the science on very quickly.”
On top of that, bacteria have evolved a wide range of defence mechanisms to guard against phage infection. “To that end, many groups are working towards better mapping these defences, developing inhibitors, and also evolving phages to have increased host range,” says Edwards. Even once you get past these basic science barriers, getting the funding to commercialise new tests is often tricky. The regulatory process can be complicated too, given that the phages are biological organisms.
“The financial returns could be great being the first, but it could also prove to be a flop, and investors risk losing all their money! It’s much easier to make a financial case if others have come before you,” says Rees.
The Actiphage test Together with fellow researchers at the University of Nottingham, and their spin-off company PBD Biotech, Rees is working on a new phage-based diagnostic called Actiphage. This test targets mycobacterial cells – the bacteria that cause TB in humans, and bovine TB or Johne’s disease in cattle.
“It’s very hard to detect a single bacterium in a blood sample using molecular testing, but we are finding it easier to detect 100 phages becoming 1,000 as they replicate through that cell.” Dr Thomas Edwards
“Mycobacteria are very different to other commonly known bacteria that cause infections, like Staphylococcus aureus, Salmonella or E. coli,” says Rees. “The cell walls of mycobacteria are coated in a waxy material. This makes them very
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Bacteria and Bacteriophage Lytic cycle
Prokaryotic cell
1: Attachment and penetration
Bacteriophage
2: Transcription
3: Biosynthesis
4: Maturation
5: Lysis
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