Q130293559
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- MeSH
- antibiotická rezistence * účinky léků MeSH
- bakteriální infekce farmakoterapie MeSH
- bakteriofágy izolace a purifikace klasifikace růst a vývoj MeSH
- fágová terapie * dějiny metody MeSH
- lidé MeSH
- profágy patogenita MeSH
- průmyslová mikrobiologie trendy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Phage therapy could offer a safe and effective alternative to antibiotic treatment of infections caused by Gram-positive bacterium Staphylococcus aureus that have emerged as a significant threat in hospital and community environment and is attracting growing interest among clinicians. The legislation process of approving the phage therapeutics by pharmaceutical authorities requires rapid analytical techniques for assessment of phage activity. Here, we present a three-step method for on-line monitoring the phage effect on bacterial cells dynamically adhered from microliter volumes of high conductivity matrix onto the inner surface of fused silica capillary with a part etched with supercritical water. Phage K1/420 particles of the Kayvirus genus generated by propagation on the host S. aureus cells together with the uninfected cells were concentrated, separated and detected using capillary electrophoretic methods. The phage interactions with selected S. aureus strains exhibiting differences in phage susceptibility were compared. The method allowed determination of the phage burst size and time of phage latent period in analyzed strains. Apart from enumeration of bacteriophages by the plaque assays, the proposed method is suitable for phage activity testing.
- MeSH
- antibakteriální látky MeSH
- bakteriofágy * MeSH
- lidé MeSH
- oxid křemičitý MeSH
- stafylokokové infekce * MeSH
- Staphylococcus aureus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A method for the fast isolation, propagation, and characterization of very low count bacteriophages active against pathogenic bacterial strains is described in this study. Bacteriophages with a count of 102 phage particles were dynamically adhered from the maximum 10 mL blood plasma sample onto the nanostructured part of the fused silica capillary. One-step propagation of phage particles of genus Kayvirus inside the etched capillary on 104Staphylococcus aureus host cells increased their number to 6 × 104 phage particles. Phage particles were concentrated online and separated by capillary electrophoretic methods. No phage replication occurred when the phage-resistant S. aureus or Escherichia coli cells were used. Two-step phage propagation in the capillary allowed an increase in the total virion count to up to 6 × 105 phage particles and subsequent off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the phage zone collected after capillary electrophoresis. Relative standard deviations of the phage peak area were at most 2.3%. We expect that the method of isolating bacteriophages from blood plasma and their simultaneous identification will facilitate clinical studies of phage preparations and contribute to pharmacokinetics studies during phage therapy. This approach is also suitable for capturing and enriching new phages from the environment when a susceptible indicator strain is available.
- MeSH
- lidé MeSH
- methicilin rezistentní Staphylococcus aureus * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- stafylokokové bakteriofágy genetika MeSH
- stafylokokové infekce * MeSH
- Staphylococcus aureus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10 μL of the prepared sample at the optimized flow rate of 6.5 μL min-1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 = 0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (±) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract.
Phages infecting Staphylococcus aureus can be used as therapeutics against antibiotic-resistant bacterial infections. However, there is limited information about the mechanism of genome delivery of phages that infect Gram-positive bacteria. Here, we present the structures of native S. aureus phage P68, genome ejection intermediate, and empty particle. The P68 head contains 72 subunits of inner core protein, 15 of which bind to and alter the structure of adjacent major capsid proteins and thus specify attachment sites for head fibers. Unlike in the previously studied phages, the head fibers of P68 enable its virion to position itself at the cell surface for genome delivery. The unique interaction of one end of P68 DNA with one of the 12 portal protein subunits is disrupted before the genome ejection. The inner core proteins are released together with the DNA and enable the translocation of phage genome across the bacterial membrane into the cytoplasm.
- MeSH
- bakteriofágy genetika MeSH
- buněčná membrána genetika MeSH
- cytoplazma genetika MeSH
- DNA virů genetika MeSH
- genom virový genetika MeSH
- Staphylococcus aureus genetika MeSH
- virion genetika MeSH
- virové plášťové proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phage therapy is increasingly put forward as a "new" potential tool in the fight against antibiotic resistant infections. During the "Centennial Celebration of Bacteriophage Research" conference in Tbilisi, Georgia on 26-29 June 2017, an international group of phage researchers committed to elaborate an expert opinion on three contentious phage therapy related issues that are hampering clinical progress in the field of phage therapy. This paper explores and discusses bacterial phage resistance, phage training and the presence of prophages in bacterial production strains while reviewing relevant research findings and experiences. Our purpose is to inform phage therapy stakeholders such as policy makers, officials of the competent authorities for medicines, phage researchers and phage producers, and members of the pharmaceutical industry. This brief also points out potential avenues for future phage therapy research and development as it specifically addresses those overarching questions that currently call for attention whenever phages go into purification processes for application.
- MeSH
- Bacteria genetika virologie MeSH
- bakteriální infekce mikrobiologie terapie MeSH
- bakteriofágy fyziologie MeSH
- fágová terapie * metody MeSH
- lidé MeSH
- mikrobiologie životního prostředí MeSH
- potravinářská mikrobiologie MeSH
- znalecký posudek MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- kongresy MeSH
- práce podpořená grantem MeSH
Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages.
- MeSH
- biologické přípravky izolace a purifikace MeSH
- chemická frakcionace metody MeSH
- lidé MeSH
- replikace viru MeSH
- shluková analýza MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody MeSH
- stafylokokové bakteriofágy klasifikace metabolismus MeSH
- Staphylococcus aureus virologie MeSH
- virové proteiny analýza chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
