The first successful attempt at the creation of synthetic bacteriophages was achieved by scientists from the CEB – Centre of Biological Engineering, Universidade Do Minho, Braga in Portugal. The synthetic bacteriophages against Pseudomonas aeruginosa bacteria were created to contain a shortened genome. Researchers hope that this advancement may be the first step in creating a new antibacterial approach to the battle against the constantly evolving antibiotic-resistant threat.
Although natural bacteriophages are being looked at as an alternative antibiotic therapeutic treatment, and in some countries are already being used for many years; some scientists are looking into synthetic biology and its capabilities when working with bacteriophages for treatments. There is still a lot of the regions in the bacteriophage genome that is yet to be understood, as well as about bacteriophage biology. Bacteria and phages are constantly adapting to one another in order to survive in their battle against one another. Using the whole genome of a bacteriophage does not leave much room for further manipulation, for instance; to add genes that are needed for therapeutic purposes. Additionally, the removal of genes whose functions are still not known would only leave the genes that have been studied and whose functions have been understood, creating room for new ones.
Synthetic bacteriophage against Pseudomonas aeruginosa
The creation of the first synthetic bacteriophage focuses on a phage that infects the Pseudomonas aeruginosa bacteria, which is categorized under the priority 1 critical group of highly antibiotic-resistant bacteria by the World Health Organization. The bacteriophage, named PE3, which infects P. aeruginosa, was isolated from wastewater. The scientists began testing the natural abilities of the bacteriophage and it was observed that from 28 P. aeruginosa bacteria samples obtained from patients, only 7 were infected by the phage. The genome of the bacteriophage was sequenced and images were captured of the bacteriophage using transmission electron microscopy (TEM).
The genome of the PE3 bacteriophage consisted of 43.5 thousand nucleotide bases of double-stranded DNA. Based on computer analysis, the researchers suggested that there are 55 protein-coding sequences in the genome. The genome sequencing and the TEM concluded that the bacteriophage PE3 belonged to the Autographiviridae family, part of the Caudovirales order. This family of bacteriophages includes phages similar to Bacteriophage T7, with genomes composed of linear terminally redundant dsDNA encoding an RNA polymerase. In addition, the genome analysis showed that the bacteriophage PE3 was probably a virulent phage, as it lacked the genes that are associated with lysogeny.
The scientists suggested two gene modules; from the first to the fifth (gp1-gp5) and from the sixth to the twelfth (gp6-gp12), which probably encoded proteins, could be removed from the genome of bacteriophage PE3. Three variants of the synthetic genome were created; in two of the synthetic phages one of the modules was removed, and in the third synthetic phage both were removed. With the use of PCR, the remaining genes were amplified and were linked in yeast cells, to an artificial yeast chromosome. The resulting synthetic bacteriophage genome was isolated and introduced to the host P. aeruginosa bacteria, in order to test the ability of hereditary information of the bacteriophage to start the assembly of the viral particles. The experiment was a success; resulting in visible bacteriophage plaques that were formed where the virus damaged the P. aeruginosa bacteria on the petri dishes.
To get a better understanding of how the gene removal influences the bacteriophage, the scientists compared the phage plaques with the original PE3 bacteriophage. Firstly, the original PE3 bacteriophage showed a decrease in the size of the phage plaques. Secondly, not all synthetic bacteriophages had the ability to infect the same P. aeruginosa bacteria strains as their predecessor. Only synthetic bacteriophages with gp6-gp12 removed had a significant effect on 7 of the 28 specimens of P. aeruginosa bacteria samples obtained from patients. The rest of the synthetic bacteriophages infected only 4 strains. Thirdly, synthetic bacteriophages with gp6-gp12 removed, revealed a growth rate similar to the original PE3 bacteriophage, while the synthetic bacteriophage with gp1-gp5 removed was five minutes behind and gp1-gp12 removed was fifteen minutes behind.
Antibacterial efficiency of the synthetic bacteriophages
The antibacterial efficiency of the synthetic bacteriophages was unaffected by the alterations made to the genome. This had been observed from the in vitro experiments, where the phages were added to the bacterial cultures during the exponential growth phase. The ratio of viral particles to cells was 1:5. All the bacteriophages had shown the same efficiency, with no significant differentiation. After two hours of introducing the bacteriophages, the number of cells decreased by five orders of magnitude in all samples compared to the control group. However, after the living cells began to grow and within 24 hours from the start of the experiment, it hardly differed from the control group. This experiment helped demonstrate the rapid development of bacteriophage-insensitive mutants (BIMs). BIMs in a short period of time post bacteriophage infection is common within P. aeruginosa bacteria, and have been studied in depth. However, the main focus of the experiment and the results, show that the performance of bacteriophages was unaffected by genome knock-outs, and ultimately, the synthetic bacteriophages performed equally as the original PE3 bacteriophage.
Therapeutic efficiency of the synthetic bacteriophages
The therapeutic efficacy of the synthetic bacteriophages was also tested in vivo on the Great wax moth, G. mellonella. After the insects were injected with the bacteria, injections containing bacteriophage solutions were administered. After 24 hours the control group had a survival rate of 20 percent, after 48 hours the control group had a survival rate of 13 percent and after 72 hours the control group had a survival rate of 10 percent. The group that was treated with the bacteriophage solutions showed better results; after 24 hours the survival rate was 50 percent, after 48 hours and 72 hours the survival rate was 30 percent. It was also observed that both the synthetic bacteriophages and the original PE3 bacteriophages, succeeded equally well with the task.
Synthetic bacteriophages can play a crucial role against highly-resistant bacteria. They can introduce many opportunities in developing treatments using specialized bacteriophages against many bacterial species. Bacteriophages have shown positive results when being used in a range of treatments; natural phages, a combination treatment of bacteriophages with antibiotics, genetically modified phages, and now even synthetic phages. All of which shows the extensive flexibility of phages and the range of techniques that are now available for treatments.
Designing P. aeruginosa synthetic phages with reduced genomes