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.

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.

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.

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 wJB, 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 43 012 bp and comprising 70 ORFs, of which ,40 encoded proteins with unknown function. A global genome alignment of wJB and other efficiently transducing phages w11, w53, w80, w80a and wNM4 showed a high degree of homology with wNM4 and substantial differences with regard to other phages. Using a model transduction system with a well-defined donor and recipient, wJB 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, wJB demonstrated high frequencies of transferring antibiotic resistance plasmids even upon induction from a lysogenic donor strain. Considering such transducing potential, wJB 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.