A novel lytic phage with a broad host range was isolated from pig faeces and the complete genome was subsequently sequenced. The phage was found to lyse Staphylococcus hyicus, S. pseudintermedius, S. schleiferi and S. warneri, generating approximately 27 PFU per infected S. hyicus cell. The phage has an isometric head of 42 ± 2 nm in diameter and a noncontractile tail of 114 ± 9 nm long. The genome is 53,660 bp in size and consists of 79 predicted ORFs and one tRNAArg gene. The phage has been classified within the Caudoviricetes, specifically the Chaseviridae family. Its broad host range and absence of harmful genes make it suitable for use in phage therapy. In addition, a novel temperate phage was discovered that was spontaneously released from a S. hyicus isolate Pel11 from a pig with exudative epidermitis. This novel temperate phage differs from the known temperate phages in S. hyicus strains NCTC10350, MM2101 or 83/7-1B, representing a novel pathogenicity element in the S. hyicus genome.
- Klíčová slova
- Alternative to antibiotics, Exudative epidermitis, Lytic phage, Temperate phage,
- MeSH
- feces virologie MeSH
- fylogeneze MeSH
- genom virový MeSH
- hostitelská specificita MeSH
- otevřené čtecí rámce MeSH
- prasata MeSH
- stafylokokové bakteriofágy * genetika izolace a purifikace klasifikace ultrastruktura fyziologie MeSH
- Staphylococcus * virologie genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Staphylococcus epidermidis is a leading opportunistic pathogen causing nosocomial infections that is notable for its ability to form a biofilm and for its high rates of antibiotic resistance. It serves as a reservoir of multiple antimicrobial resistance genes that spread among the staphylococcal population by horizontal gene transfer such as transduction. While phage-mediated transduction is well studied in Staphylococcus aureus, S. epidermidis transducing phages have not been described in detail yet. Here, we report the characteristics of four phages, 27, 48, 456, and 459, previously used for S. epidermidis phage typing, and the newly isolated phage E72, from a clinical S. epidermidis strain. The phages, classified in the family Siphoviridae and genus Phietavirus, exhibited an S. epidermidis-specific host range, and together they infected 49% of the 35 strains tested. A whole-genome comparison revealed evolutionary relatedness to transducing S. aureus phietaviruses. In accordance with this, all the tested phages were capable of transduction with high frequencies up to 10-4 among S. epidermidis strains from different clonal complexes. Plasmids with sizes from 4 to 19 kb encoding resistance to streptomycin, tetracycline, and chloramphenicol were transferred. We provide here the first evidence of a phage-inducible chromosomal island transfer in S. epidermidis Similarly to S. aureus pathogenicity islands, the transfer was accompanied by phage capsid remodeling; however, the interfering protein encoded by the island was distinct. Our findings underline the role of S. epidermidis temperate phages in the evolution of S. epidermidis strains by horizontal gene transfer, which can also be utilized for S. epidermidis genetic studies.IMPORTANCE Multidrug-resistant strains of S. epidermidis emerge in both nosocomial and livestock environments as the most important pathogens among coagulase-negative staphylococcal species. The study of transduction by phages is essential to understanding how virulence and antimicrobial resistance genes spread in originally commensal bacterial populations. In this work, we provide a detailed description of transducing S. epidermidis phages. The high transduction frequencies of antimicrobial resistance plasmids and the first evidence of chromosomal island transfer emphasize the decisive role of S. epidermidis phages in attaining a higher pathogenic potential of host strains. To date, such importance has been attributed only to S. aureus phages, not to those of coagulase-negative staphylococci. This study also proved that the described transducing bacteriophages represent valuable genetic modification tools in S. epidermidis strains where other methods for gene transfer fail.
- Klíčová slova
- Staphylococcus epidermidis, antibiotic resistance, bacteriophages, horizontal gene transfer, pathogenicity islands, transduction,
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- genomové ostrovy genetika MeSH
- lidé MeSH
- plazmidy genetika MeSH
- stafylokokové bakteriofágy klasifikace účinky léků genetika MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus epidermidis účinky léků virologie MeSH
- transdukce genetická * MeSH
- virulence MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
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.
- Klíčová slova
- Capillary electrophoretic methods, Kayvirus, MALDI-TOF MS, Pre-concentration, Preparative IEF, Purification, Separation, Staphylococcus aureus,
- MeSH
- isoelektrická fokusace metody MeSH
- lidé MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- stafylokokové bakteriofágy klasifikace izolace a purifikace fyziologie MeSH
- Staphylococcus aureus virologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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.
- Klíčová slova
- Kayvirus, MALDI-MS, Staphylococcus, Viral proteins, bacteriophages, phage therapy,
- 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
- Názvy látek
- biologické přípravky MeSH
- viron MeSH Prohlížeč
- virové proteiny MeSH
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 epidemiologie MeSH
- Názvy látek
- DNA virů MeSH
- exfoliatiny MeSH
The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococcus aureus. While SCCmec is generally regarded as a mobile genetic element, the precise mechanisms by which large SCCmec elements are exchanged between staphylococci have remained enigmatic. In the present studies, we observed that the clinical methicillin-resistant S. aureus (MRSA) isolate UMCG-M4 with the sequence type 398 contains four prophages belonging to the serological groups A, B and Fa. Previous studies have shown that certain serological group B bacteriophages of S. aureus are capable of generalized transduction. We therefore assessed the transducing capabilities of the phages from strain UMCG-M4. The results show that some of these phages can indeed transduce plasmid pT181 to the recipient S. aureus strain RN4220. Therefore, we also investigated the possible involvement of these transducing phages in the transmission of the large SCCmec type V (5C2&5) element of S. aureus UMCG-M4. While no transduction of the complete SCCmec element was observed, we were able to demonstrate that purified phage particles did contain large parts of the SCCmec element of the donor strain, including the methicillin resistance gene mecA. This shows that staphylococcal phages can encapsulate the resistance determinant mecA of a large SCCmec type V (5C2&5) element, which may lead to its transfer to other staphylococci.
- Klíčová slova
- Phage, SCCmec, ST398, Staphylococcus aureus, Transduction,
- MeSH
- bakteriální geny * MeSH
- methicilin rezistentní Staphylococcus aureus genetika virologie MeSH
- plazmidy MeSH
- profágy genetika fyziologie MeSH
- rezistence na methicilin MeSH
- sestavení viru * MeSH
- stafylokokové bakteriofágy klasifikace genetika MeSH
- transdukce genetická * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Conserved genomic sequences distinctive of Staphylococcus aureus phage types 3A, 11, 77, 187 and Twort, representative of phage serogroups A, B, F, L and D, were identified and characterized. PCR primers designed for the above sequences were used for development of a multiplex PCR assay which enabled us not only to classify all phages of the International Typing Set plus 16 additional phages, but also to detect prophages in S. aureus genomes. One to four different prophages were unambiguously detected in experimentally lysogenized S. aureus strains, and substantial variation in prophage content was found in 176 S. aureus clinical strains of different provenance. In addition, by using a comparative genomics approach, all the prophages in the S. aureus genomes sequenced to date could be revealed and classified.
- MeSH
- DNA primery MeSH
- DNA virů analýza chemie izolace a purifikace MeSH
- genom virový MeSH
- lyzogenie MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce MeSH
- profágy klasifikace genetika izolace a purifikace MeSH
- sekvenční analýza DNA MeSH
- stafylokokové bakteriofágy klasifikace genetika izolace a purifikace MeSH
- Staphylococcus aureus virologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA primery MeSH
- DNA virů MeSH
On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb.
- MeSH
- DNA sondy MeSH
- DNA virů genetika izolace a purifikace MeSH
- fagotypizace * MeSH
- kruhová DNA genetika izolace a purifikace MeSH
- lyzogenie * MeSH
- proviry klasifikace genetika izolace a purifikace MeSH
- restrikční endonukleasy typu II MeSH
- restrikční mapování MeSH
- shluková analýza MeSH
- stafylokokové bakteriofágy klasifikace genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA sondy MeSH
- DNA virů MeSH
- GTYRAC-specific type II deoxyribonucleases MeSH Prohlížeč
- kruhová DNA MeSH
- restrikční endonukleasy typu II MeSH
Ninety-five percent of 782 culture collection strains, as well as hospital strains of Staphylococcus aureus subsp. aureus of different provenance and 43% of 89 culture collection strains of different coagulase-negative species of the genus Staphylococcus, were found to be sensitive to the polyvalent phage phi 812 or to at least one of its host-range mutants or to the polyvalent phages SK311, phi 131, and U16. Thus sensitivity to the polyvalent staphylococcal phages seems to be one of the common features of S. aureus subsp. aureus strains. The adsorption kinetics and one-step growth characteristics of the phages phi 812 and SK311 were estimated. Restriction genomic maps of the phages phi 812 (146.5 kb) and SK311 (141.1 kb) were constructed by use of the restriction endonucleases AvaII, PstI, KpnI, SacI, SmaI, and XhoI. The host-range mutations of the phage phi 812 were localized on this map. Comparison of restriction patterns of the phages phi 812 and SK311 with those of the polyvalent phages U16 and phi 131 suggests that all these phages are closely related. Their genomes differ from each other mostly by some deletions, insertions (1-3 kb), or inversions. Evidence was given that the phage phi 812 together with SK311, phi 131, and U16 belongs in the phage species Twort, the description of which is substantially supplemented with the data on the phage phi 812 reported in this paper.
- MeSH
- adsorpce MeSH
- DNA virů MeSH
- kinetika MeSH
- lidé MeSH
- mutace MeSH
- restrikční mapování MeSH
- stafylokokové bakteriofágy klasifikace genetika patogenita ultrastruktura MeSH
- Staphylococcus aureus metabolismus virologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA virů MeSH
Several Staphylococcus aureus strains were lysogenized by the phages of serological group B (phages phi 53, phi 85) as well as by some of serological group F (phages phi 77, phi 84) and macrorestriction fragment patterns of genomic DNA were estimated in the lysogenized, non-lysogenic and delysogenized (cured of prophages) strains. It was shown that the integration of phage DNA into chromosome of S. aureus leads to specific changes in restriction fragment pattern in all the lysogenized strains. These changes correlate well with the SmaI restriction map of S. aureus NCTC 8325 since they concern the restriction fragments defined in this map. Phages phi 53 and phi 85 integrate into SmaI fragment B. On the other hand, phages phi 77 and phi 84 integrate into SmaI fragment E of the S. aureus restriction map. The prophages of strain NCTC 8511 have their integration sites, as follows: the phage designated by us phi M integrates in fragment A, whereas the integration site for phage phi J lies in fragment E. Phage phi M was estimated to be genetically related to phages of serological group A and phage phi J to those of serological group F. Evidence was given that lysogenization of S. aureus strains by at least four prophages does not cast any doubt upon the estimation of their genetic relatedness based on their similarity in restriction pattern.
- MeSH
- DNA bakterií genetika izolace a purifikace MeSH
- DNA virů genetika izolace a purifikace MeSH
- druhová specificita MeSH
- genetická variace MeSH
- lyzogenie genetika MeSH
- místa připojení (mikrobiologie) genetika MeSH
- pulzní gelová elektroforéza MeSH
- restrikční mapování MeSH
- sérotypizace MeSH
- stafylokokové bakteriofágy klasifikace genetika MeSH
- Staphylococcus aureus genetika virologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA bakterií MeSH
- DNA virů MeSH