Jana Kahánková

Multilocus PCR typing strategy for differentiation of Staphylococcus aureus siphoviruses reflecting their modular genome structure

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.

Genome rearrangements in host-range mutants of the polyvalent staphylococcal bacteriophage 812.

Mutations extended the host range of the polyvalent bacteriophage 812 of the familyMyoviridae in up to 95 % ofStaphylococcus aureus strains and 43 % of strains of different coagulase-positive and -negativeStaphylococcus species. Mutational changes in the genome of several host-range mutants of phage 812 were identified. Host-range mutant 812F1 harbors a deletion in endolysin gene that arose together with intron excision. Four mutants (812i, 812b, 812p, 812F3) harbor deletion in the structural geneorf8 that results from a genome rearrangement associated with intron insertion. This rearrangement was also detected in the genome of the closely related phages U16 and ϕ131. Another intron was discovered in therecA812 gene in these four mutants. An insertion was found in a non-coding region of the restriction fragmentPstI-O of three mutants (812b, 812F3, 812g) and phages U16 and ϕ131. The above results contribute to the explanation of genetic factors affecting the host range of polyvalent staphylococcal bacteriophages.

Extraction of PCR-ready DNA from Staphylococcus aureus bacteriophages using carboxyl functionalized magnetic nonporous microspheres

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.