Mastitis in dairy cows is generally considered to be the most expensive disease for dairy farmers worldwide. The overuse of antibiotics is a major problem in the treatment of bovine mastitis, and bacteriophage therapy is expected to provide an alternative treatment. The primary aim of this study was to evaluate the efficacy of a phage cocktail against mastitis in a mouse model. First, a Staphylococcus aureus strain was isolated from milk samples taken from mastitis cows from dairy farms in Xinjiang, China, and it was designated as Sau-XJ-21. Next, two phages (designated as vBSM-A1 and vBSP-A2) with strong lytic activity against Sau-XJ-21 were isolated from mixed sewage samples collected from three cattle farms in Xinjiang. Phages vBSM-A1 and vBSP-A2 were identified as members of the Myoviridae and Podoviridae families, respectively. The two phages exhibited a wide range of hosts, especially phage vBSM-A1. To evaluate the effectiveness of the two phages in the treatment against mastitis, female lactating mice were used 10-14 days after giving births. The mice were divided into six groups; one group was kept as healthy control, while the remaining five groups were inoculated with the isolated S. aureus strain to induce mastitis. Four hours after bacterial inoculation, mice in these groups were injected with 25 μL phosphate buffer saline (negative control), ceftiofur sodium (positive control), or phage, either individually or as a cocktail. The mice were sacrificed 20 h later, and the mammary glands were removed and subjected to further analysis, including the quantitation of colony-forming units (CFU), plaque-forming units (PFU), and gross macroscopic as well as histopathology observation. Mice with induced mastitis exhibited significantly improved mastitic pathology and decreased bacterial counts after they had been given phage treatments, with the phage cocktail being more superior than either phage alone. Furthermore, the cocktail treatment also maintained the highest intramammary phage titer without spreading systemically. The effectiveness of the phage cocktail was comparable to that produced by ceftiofur sodium. According to the data obtained for the mouse model of mastitis, phage therapy could be considered as an innovative alternative to antibiotics for the treatment of bovine mastitis.
- MeSH
- bakteriofágy fyziologie MeSH
- fágová terapie metody veterinární MeSH
- mastitida skotu mikrobiologie terapie MeSH
- mléko mikrobiologie MeSH
- Myoviridae fyziologie MeSH
- myši MeSH
- Podoviridae fyziologie MeSH
- skot MeSH
- stafylokokové infekce mikrobiologie veterinární MeSH
- Staphylococcus aureus fyziologie virologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- Geografické názvy
- Čína MeSH
Staphylococcus aureus may be a highly virulent human pathogen, especially when it is able to form a biofilm, and it is resistant to antibiotic. Infections caused by these bacteria significantly affect morbidity and mortality, primarily in hospitalized patients. Treatment becomes more expensive, more toxic, and prolonged. This is the reason why research on alternative therapies should be one of the main priorities of medicine and biotechnology. A promising alternative treatment approach is bacteriophage therapy. The effect of the anti-staphylococcal bacteriophage preparation Stafal® on biofilm reduction was assessed on nine S. aureus strains using both sonication with subsequent quantification of surviving cells on the catheter surface and evaluation of biofilm reduction in microtiter plates. It was demonstrated that the bacteriophages destroy planktonic cells very effectively. However, to destroy cells embedded in the biofilm effectively requires a concentration at least ten times higher than that provided by the commercial preparation. The catheter disc method (CDM) allowed easier comparison of the effect on planktonic cells and cells in a biofilm than the microtiter plate (MTP) method.
- MeSH
- antiinfekční látky * MeSH
- bakteriologické techniky MeSH
- biofilmy * MeSH
- lidé MeSH
- methicilin rezistentní Staphylococcus aureus růst a vývoj izolace a purifikace virologie MeSH
- mikrobiální viabilita MeSH
- počet mikrobiálních kolonií MeSH
- stafylokokové bakteriofágy fyziologie MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus růst a vývoj izolace a purifikace virologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Lytic bacteriophages are valuable therapeutic agents against bacterial infections. There is continual effort to obtain new phages to increase the effectivity of phage preparations against emerging phage-resistant strains. Here we described the genomic diversity of spontaneous host-range mutants of kayvirus 812. Five mutant phages were isolated as rare plaques on phage-resistant Staphylococcus aureus strains. The host range of phage 812-derived mutants was 42% higher than the wild type, determined on a set of 186 methicillin-resistant S. aureus strains representing the globally circulating human and livestock-associated clones. Comparative genomics revealed that single-nucleotide polymorphisms from the parental phage 812 population were fixed in next-step mutants, mostly in genes for tail and baseplate components, and the acquired point mutations led to diverse receptor binding proteins in the phage mutants. Numerous genome changes associated with rearrangements between direct repeat motifs or intron loss were found. Alterations occurred in host-takeover and terminal genomic regions or the endolysin gene of mutants that exhibited the highest lytic activity, which implied various mechanisms of overcoming bacterial resistance. The genomic data revealed that Kayvirus spontaneous mutants are free from undesirable genes and their lytic properties proved their suitability for rapidly updating phage therapeutics.
- MeSH
- bakteriální léková rezistence MeSH
- bakteriofágy genetika MeSH
- délka genomu MeSH
- genom virový MeSH
- genomika MeSH
- jednonukleotidový polymorfismus MeSH
- methicilin farmakologie MeSH
- mutace * MeSH
- Staphylococcus aureus růst a vývoj virologie MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
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
The transduction mediated by bacteriophages is considered to be one of the primary driving forces in horizontal gene transfer in staphylococci, which is crucial to their adaptation and successful evolution. For a transduction to be effective, it is generally accepted that the recipient strain should be susceptible to the transducing phage. In this study, we demonstrate that the plasmid DNAs are effectively transduced into the recipient Staphylococcus aureus strains in spite of their insensitivity to the lytic action of the transducing phage, provided that these phages adsorb effectively to the bacterial cells. The tetracycline and penicillinase plasmids were transduced to insensitive laboratory and clinical strains by bacteriophages ϕ29, ϕ52A and ϕ80α as well as by prophage ϕ53 and naturally occurring prophages induced from donor lysogenic strains. Comparable frequencies of transduction were achieved in both phage-sensitive and phage-insensitive recipient strains. We have demonstrated that such mechanisms as the restriction of DNA and lysogenic immunity which are responsible for insensitivity of cells to phages may not be a barrier to the transfer, maintenance and effective spread of plasmids to a wider range of potential recipients in the staphylococcal population.
- MeSH
- antibakteriální látky farmakologie MeSH
- lyzogenie MeSH
- mnohočetná bakteriální léková rezistence MeSH
- penicilinasa genetika MeSH
- plazmidy * MeSH
- profágy genetika fyziologie MeSH
- stafylokokové bakteriofágy genetika fyziologie MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus genetika virologie MeSH
- tetracyklin farmakologie MeSH
- transdukce genetická * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bacteriophages from the family Myoviridae use double-layered contractile tails to infect bacteria. Contraction of the tail sheath enables the tail tube to penetrate through the bacterial cell wall and serve as a channel for the transport of the phage genome into the cytoplasm. However, the mechanisms controlling the tail contraction and genome release of phages with "double-layered" baseplates were unknown. We used cryo-electron microscopy to show that the binding of the Twort-like phage phi812 to the Staphylococcus aureus cell wall requires a 210° rotation of the heterohexameric receptor-binding and tripod protein complexes within its baseplate about an axis perpendicular to the sixfold axis of the tail. This rotation reorients the receptor-binding proteins to point away from the phage head, and also results in disruption of the interaction of the tripod proteins with the tail sheath, hence triggering its contraction. However, the tail sheath contraction of Myoviridae phages is not sufficient to induce genome ejection. We show that the end of the phi812 double-stranded DNA genome is bound to one protein subunit from a connector complex that also forms an interface between the phage head and tail. The tail sheath contraction induces conformational changes of the neck and connector that result in disruption of the DNA binding. The genome penetrates into the neck, but is stopped at a bottleneck before the tail tube. A subsequent structural change of the tail tube induced by its interaction with the S. aureus cell is required for the genome's release.
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
Staphylococcus aureus is a serious human and veterinary pathogen in which new strains with increasing virulence and antimicrobial resistance occur due to acquiring new genes by horizontal transfer. It is generally accepted that temperate bacteriophages play a major role in gene transfer. In this study, we proved the presence of various bacterial genes of the S. aureus COL strain directly within the phage particles via qPCR and quantified their packaging frequency. Non-parametric statistical analysis showed that transducing bacteriophages φ11, φ80 and φ80α of serogroup B, in contrast to serogroup A bacteriophage φ81, efficiently package selected chromosomal genes localized in 4 various loci of the chromosome and 8 genes carried on variable elements, such as staphylococcal cassette chromosome SCCmec, staphylococcal pathogenicity island SaPI1, genomic islands vSaα and vSaβ, and plasmids with various frequency. Bacterial gene copy number per ng of DNA isolated from phage particles ranged between 1.05 × 10(2) for the tetK plasmid gene and 3.86 × 10(5) for the SaPI1 integrase gene. The new and crucial finding that serogroup B bacteriophages can package concurrently ccrA1 (1.16 × 10(4)) and mecA (1.26 × 10(4)) located at SCCmec type I into their capsids indicates that generalized transduction plays an important role in the evolution and emergence of new methicillin-resistant clones.
- MeSH
- bakteriální chromozomy genetika MeSH
- bakteriální geny * MeSH
- bakteriální proteiny genetika MeSH
- bakteriofágy genetika metabolismus MeSH
- DNA bakterií genetika MeSH
- frekvence genu MeSH
- genetické lokusy MeSH
- klonování DNA MeSH
- penicilinasa genetika MeSH
- plazmidy genetika MeSH
- polymerázová řetězová reakce MeSH
- přenos genů horizontální MeSH
- rezistence na methicilin genetika MeSH
- rozptýlené repetitivní sekvence * MeSH
- sekvenční analýza DNA MeSH
- sestavení viru MeSH
- Staphylococcus aureus genetika fyziologie virologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Given the great biological importance and high diversity of temperate Staphylococcus aureus bacteriophages, a method is needed for the description of their genomic structure. Here we have updated a multiplex PCR strategy for the complex characterization of S. aureus phages of the family Siphoviridae. Based on the comparative genomic analysis of the available phage sequences, a multilocus PCR strategy for typing the major modules of the phage genome was designed. The genomic modules were classified on the basis of the genes for integrase (10 types), anti-repressor (five types), replication proteins polA, dnaC and dnaD (four types), dUTPase (four types), portal protein (eight types), tail appendices (four types) and endolysin (four types) corresponding to the integrase locus, lysogeny control region, and modules for DNA replication, transcription regulation, packaging, tail appendices and lysis respectively. The nine PCR assays designed for the above sequences were shown to be capable to identify the bacteriophage gene pool present both in the phage and bacterial genomes and their extensive mosaic structure. The established multiplex PCR-based multilocus diagnostic scheme is convenient for rapid and reliable phage and prophage classification and for the study of bacteriophage evolution.
- MeSH
- DNA virů genetika MeSH
- genom virový MeSH
- multilokusová sekvenční typizace MeSH
- polymerázová řetězová reakce metody MeSH
- profágy klasifikace genetika MeSH
- Siphoviridae klasifikace genetika MeSH
- srovnávací genomová hybridizace MeSH
- Staphylococcus aureus genetika virologie MeSH
- virové proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
