The bacteriophage K1/420 is a member of genus Kayvirus that was extensively studied as an alternative treatment to combat bacterial infections caused by antibiotic-resistant Staphylococcus aureus strains. Despite the promise of phage therapy, the development of clinical applications of phages is facing regulatory and technical hurdles before it can receive acceptance in the Western World. Suitable simple and accurate diagnostic techniques to control the quality of the phage, which would satisfy the requirements of regulatory authorities are still being discussed. Here, we present the conditions for the simultaneous separation and detection of phage K1/420 and S. aureus by CZE and by CIEF were found, and the phage isoelectric point was determined to be 3.6. After removing the cell debris, the phage was successfully purified from the crude phage lysate and pre-concentrated by preparative isoelectric focusing. Its zone was localized by the positions of colored pI markers in the cellulose bed. The phage from the harvested zone had a decreased ability to infect its host. However, it was suitable for its separation, detection and identification by capillary electrophoretic methods, MALDI-TOF MS and electron microscopy.
Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated φB166 and φB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.
- MeSH
- DNA virů chemie genetika MeSH
- DNA viry genetika izolace a purifikace MeSH
- epidemický výskyt choroby MeSH
- exfoliatiny genetika MeSH
- fylogeneze MeSH
- genom virový * MeSH
- impetigo epidemiologie mikrobiologie MeSH
- infekce spojené se zdravotní péčí epidemiologie MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- novorozenec MeSH
- otevřené čtecí rámce MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- pořadí genů MeSH
- porodnice MeSH
- přenos genů horizontální MeSH
- profágy klasifikace genetika izolace a purifikace MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie MeSH
- shluková analýza MeSH
- stafylokokové bakteriofágy klasifikace genetika izolace a purifikace MeSH
- stafylokokové infekce epidemiologie mikrobiologie MeSH
- Staphylococcus aureus izolace a purifikace virologie MeSH
- syntenie MeSH
- transdukce genetická MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
We have isolated and characterized two distinct types of exfoliative toxin A (ETA)-converting bacteriophages originating from Staphylococcus aureus strains responsible for massive outbreaks of pemphigus neonatorum in the Czech Republic. Three induced phages designated as ph iB531, phi B557 and phi B122 were found to be capable of transferring the eta gene into the prophageless non-toxigenic S. aureus strain and converting it into an ETA producer. Comparisons of the phage sequences derived from 12 selected genes and 2 genomic segments (polymorphic P2 and conserved C4) revealed that phi B531 and phi B557 were identical each other, but phi B122 differed from them in 5 gene sequences, the xis gene content and the virion protein profile. Thus, phi B122 represents a new type of still undescribed ETA-converting phage. This study highlights not only the conclusive genomic diversity of eta gene-positive phages, but also their virulence implications in impetigo S. aureus strains.
- MeSH
- epidemický výskyt choroby MeSH
- exfoliatiny genetika metabolismus MeSH
- genetická variace MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- pemfigus epidemiologie mikrobiologie MeSH
- přenos genů horizontální MeSH
- stafylokokové bakteriofágy genetika izolace a purifikace metabolismus MeSH
- stafylokokové infekce epidemiologie mikrobiologie MeSH
- Staphylococcus aureus genetika metabolismus virologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Magnetic microspheres P(HEMA-co-EDMA) were used for PCR-ready phage DNA isolation from lysogenic strains of Staphylococcus aureus, including two new clinical isolates. The conditions of phage particle lysis were optimized. The quality of eluted phage DNA was evaluated by PCR. It was demonstrated that PCR-ready phage DNA can be isolated from small volumes of phage lysates (150 microl) by magnetic microspheres. The reported method is very expeditious without using toxic compounds such as phenol or chloroform. It can be used for phage identification and phage gene detection.
- MeSH
- DNA virů genetika izolace a purifikace MeSH
- extrakce na pevné fázi metody MeSH
- financování organizované MeSH
- genetické techniky MeSH
- magnetismus MeSH
- mikrosféry MeSH
- polymerázová řetězová reakce MeSH
- stafylokokové bakteriofágy genetika izolace a purifikace MeSH
- Staphylococcus aureus virologie MeSH
- MeSH
- DNA primery MeSH
- DNA virů analýza chemie izolace a purifikace MeSH
- genom virový MeSH
- molekulární sekvence - údaje MeSH
- profágy genetika izolace a purifikace klasifikace MeSH
- stafylokokové bakteriofágy genetika izolace a purifikace MeSH
- Staphylococcus aureus klasifikace virologie MeSH
- techniky in vitro MeSH
- MeSH
- koagulasa MeSH
- lidé MeSH
- stafylokokové bakteriofágy izolace a purifikace MeSH
- Staphylococcus MeSH
- Check Tag
- lidé MeSH
- MeSH
- antibakteriální látky terapeutické užití MeSH
- Bacteria MeSH
- bakteriologie trendy MeSH
- lidé MeSH
- lyzogenie * fyziologie imunologie účinky léků MeSH
- stafylokokové bakteriofágy * imunologie izolace a purifikace metabolismus MeSH
- stafylokokové infekce diagnóza klasifikace komplikace MeSH
- Staphylococcaceae enzymologie fyziologie genetika imunologie MeSH
- Staphylococcus aureus izolace a purifikace metabolismus MeSH
- virion izolace a purifikace metabolismus MeSH
- Check Tag
- lidé MeSH
