Browsing by Subject "Bacteriophages"

Antibiotic resistance of pathogenic bacteria has increased in recent years. When antibiotics do not work, alternative therapies are developed to prevent major bacterial epidemics. Phage therapy is one of the alternative possibilities to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds or even thousands of phages are required to cover the various bacterial pathogens. For a reliable selection process, high-throughput rapid host range screening of phages is needed to cover the future demands. In addition, collaboration between laboratories is highly important, as the collections of phages of individual laboratories are not broad enough. Thus, the transportation of phages between laboratories is one of the key elements to provide successful phage therapy for patients. The aim of the study was to use gel-based products as protective matrix in phage host-range screening and transportation. The optimal conditions were selected to set a baseline for high-throughput rapid host range screening process, and to set up a ready-to-screen plate assay for phage transportation. In addition, the purpose of the study was to evaluate whether hydrogels could be used as a long-term storage matrix for phages and future product development. Fourteen Escherichia coli phages were used to optimize the liquid culture assay for the E.coli strains. The hydrogel based assays were conducted with two Escherichia and two Staphylococcus phages. For long-term storage, phages were mixed with different consistencies of hydrogels and stored in three different conditions for up to six months at +4oC. The transportation experiments were conducted with phages stored with optimized hydrogel consistencies. The phage viability was measured using liquid culture method. Results show that liquid culture method on microtiter plate is a convenient way to screen bacteriophages in high-throughput assay and that phages can be stored reliably in hydrogel format. When stored in microcentrifuge tubes, phage stability was shown to last for at least six months. When stored as drops on microtiter plate, the phages retained their viability for up to two months. These plates can be used as a robust means for phage transportation.

The gut microbiota has a major impact on the health and early life development in humans. Viruses infecting prokaryotes, called bacteriophages, are the most abundant group of the gut virome that shapes the prokaryotic community. They have been shown to directly interact with the human host or indirectly by interfering with the gut bacterial community. While in the recent years many studies have explored the human gut virome, the field is currently under active investigation, but no standardised protocols for creating high-throughput virome extractions or bioinformatic pipelines for sequences analyses is available. The first aim of this study was to (1) compare the most promising methods for viral particle concentration (dithiothreitol (DTT) and polyethylene glycol (PEG)), DNA extraction afterwards and scaling the methods for high-throughput procedure. The second aim was to (2) compare four bioinformatics tools: Centrifuge, MetaPhlAn, Gut Virome Database (GVD) and a combination of Centrifuge, MetaPhlAn, VirFinder and Blast (Consensus) by analysing shotgun metagenome sequencing results of infant’s stool samples at three time points: 1, 6 and 12 months. The adjustments for high-throughput DNA extraction, resulted in five protocols. The highest yield of DNA was achieved for 1- and 12-months samples with the PEG method. On the other hand, the DTT method was the best for 6-month samples. The infant’s age was the only significant factor driving the viral composition differences on family level for MetaPhlAn (p = 0.004), Centrifuge (p = 0.001) and Consensus (p = 0.001) methods. However, the number of annotated reads and the virome composition depended exclusive on the software used (p = 0.001). All the methods identified phage families: Siphoviridae, Podoviridae and Myoviridae. GVD was the only method that annotated up to 90% of reads to viruses. In conclusion, our results suggest that the PEG extraction method may be best suited for large-scale virome enrichment, as it allowed to obtain the highest DNA yield, was suitable for high-throughput extractions and allowed to create a virome with a high variability in phage representation. For the novel virus identification, GVD method would be used further as it annotated most of the reads to phages.