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
- Drug Resistance, Bacterial MeSH
- Bacteriophages genetics MeSH
- Genome Size MeSH
- Genome, Viral MeSH
- Genomics MeSH
- Polymorphism, Single Nucleotide MeSH
- Methicillin pharmacology MeSH
- Mutation * MeSH
- Staphylococcus aureus growth & development virology MeSH
- Base Composition MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In Staphylococcus aureus, generalized transduction mediated by temperate bacteriophages represents a highly efficient way of transferring antibiotic resistance genes between strains. In the present study, we identified and characterized in detail a new efficiently transducing bacteriophage of the family Siphoviridae, designated ϕJB, which resides as a prophage in the meticillin-resistant S. aureus (MRSA) strain Jevons B. Whole-genome sequencing followed by detailed in silico analysis uncovered a linear dsDNA genome consisting of 43 ,12 bp and comprising 70 ORFs, of which ∼40 encoded proteins with unknown function. A global genome alignment of ϕJB and other efficiently transducing phages ϕ11, ϕ53, ϕ80, ϕ80α and ϕNM4 showed a high degree of homology with ϕNM4 and substantial differences with regard to other phages. Using a model transduction system with a well-defined donor and recipient, ϕJB transferred the tetracycline resistance plasmid pT181 and a penicillinase plasmid with outstanding frequencies, beating most of the above-mentioned phages by an order of magnitude. Moreover, ϕJB demonstrated high frequencies of transferring antibiotic resistance plasmids even upon induction from a lysogenic donor strain. Considering such transducing potential, ϕJB and related bacteriophages may serve as a suitable tool for elucidating the nature of transduction and its contribution to the spread of antibiotic resistance genes in naturally occurring MRSA populations.
- MeSH
- Virus Activation MeSH
- Drug Resistance, Bacterial MeSH
- DNA, Viral chemistry genetics MeSH
- Phylogeny MeSH
- Genome, Viral MeSH
- Lysogeny MeSH
- Methicillin-Resistant Staphylococcus aureus virology MeSH
- Molecular Sequence Data MeSH
- Open Reading Frames MeSH
- Plasmids MeSH
- Gene Order MeSH
- Gene Transfer, Horizontal MeSH
- Prophages genetics isolation & purification ultrastructure MeSH
- Sequence Analysis, DNA MeSH
- Sequence Homology MeSH
- Siphoviridae genetics isolation & purification ultrastructure MeSH
- Synteny MeSH
- Transduction, Genetic * MeSH
- Microscopy, Electron, Transmission MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
It may be possible to regulate the cell colonization of biodegradable polymer nanofibrous membranes by plasma treatment and by the density of the fibers. To test this hypothesis, nanofibrous membranes of different fiber densities were treated by oxygen plasma with a range of plasma power and exposure times. Scanning electron microscopy and mechanical tests showed significant modification of nanofibers after plasma treatment. The intensity of the fiber modification increased with plasma power and exposure time. The exposure time seemed to have a stronger effect on modifying the fiber. The mechanical behavior of the membranes was influenced by the plasma treatment, the fiber density, and their dry or wet state. Plasma treatment increased the membrane stiffness; however, the membranes became more brittle. Wet membranes displayed significantly lower stiffness than dry membranes. X-ray photoelectron spectroscopy (XPS) analysis showed a slight increase in oxygen-containing groups on the membrane surface after plasma treatment. Plasma treatment enhanced the adhesion and growth of HaCaT keratinocytes on nanofibrous membranes. The cells adhered and grew preferentially on membranes of lower fiber densities, probably due to the larger area of void spaces between the fibers.
- MeSH
- Cell Adhesion physiology MeSH
- Cell Line MeSH
- Keratinocytes cytology physiology MeSH
- Humans MeSH
- Membranes, Artificial * MeSH
- Nanofibers chemistry ultrastructure MeSH
- Bandages MeSH
- Tensile Strength MeSH
- Compressive Strength MeSH
- Plasma Gases chemistry MeSH
- Electroplating MeSH
- Surface Properties MeSH
- Cell Proliferation physiology MeSH
- Materials Testing MeSH
- Hardness MeSH
- Skin, Artificial * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Nanocrystalline diamond (NCD) films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination) or oxygen atoms (O-termination). Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers - type I collagen, alkaline phosphatase, and osteocalcin - was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix mineralization, and this is promising for better osteoconductivity of potential bone implant coatings.
- MeSH
- Alkaline Phosphatase genetics metabolism MeSH
- Cell Differentiation * MeSH
- Diamond chemistry MeSH
- Extracellular Matrix metabolism MeSH
- Fluorescent Antibody Technique MeSH
- Collagen Type I metabolism MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Humans MeSH
- Microscopy, Atomic Force MeSH
- Microscopy, Electron, Scanning MeSH
- Osteoblasts cytology metabolism MeSH
- Cell Proliferation * MeSH
- Spectrum Analysis, Raman MeSH
- Wettability MeSH
- Tissue Engineering * MeSH
- Calcium metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Chromosomes, Bacterial genetics MeSH
- Genes, Bacterial * MeSH
- Bacterial Proteins genetics MeSH
- Bacteriophages genetics metabolism MeSH
- DNA, Bacterial genetics MeSH
- Gene Frequency MeSH
- Genetic Loci MeSH
- Cloning, Molecular MeSH
- Penicillinase genetics MeSH
- Plasmids genetics MeSH
- Polymerase Chain Reaction MeSH
- Gene Transfer, Horizontal MeSH
- Methicillin Resistance genetics MeSH
- Interspersed Repetitive Sequences * MeSH
- Sequence Analysis, DNA MeSH
- Virus Assembly MeSH
- Staphylococcus aureus genetics physiology virology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.
- MeSH
- Drug Resistance, Bacterial MeSH
- Bacteriophages genetics MeSH
- Humans MeSH
- Methicillin-Resistant Staphylococcus aureus genetics isolation & purification virology MeSH
- Plasmids MeSH
- Gene Transfer, Horizontal MeSH
- Prophages genetics MeSH
- Staphylococcal Infections microbiology MeSH
- Transduction, Genetic MeSH
- Check Tag
- Humans MeSH
- Publication type
- Letter MeSH
- Research Support, Non-U.S. Gov't MeSH
