Roman Pantůček

Identification of bacteriophage types and their carriage in Staphylococcus aureus.

Summary.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.

Genomic Variability of Staphylococcus aureus and the Other Coagulase-Positive Staphylococcus Species Estimated by Macrorestriction Analysis Using Pulsed-Field Gel Electrophoresis

The genomic DNAs of 95 culture collection and hospital Staphylococcus aureus subsp. aureus strains of various origins, as well as the genomic DNAs of other coagulase-positive Staphylococcus species, were cleaved with restriction endonuclease SmaI and subjected to pulsed-field gel electrophoresis. The levels of similarity of the SmaI restriction patterns of the S. aureus subsp. aureus strains varied from 30 to 100%, which is considered characteristic of this species; thus, these organisms belonged to the same species restriction group. Within this range of similarity values 13 S. aureus intraspecies restriction groups were identified, and each group consisted of strains whose levels of similarity ranged from 65 to 100%. S. aureus subsp. aureus CCM 885T (T = type strain) belonged to the major intraspecies restriction group that comprised 39% of the S. aureus strains which we studied. The strains of the other coagulase-positive staphylococci, including Staphylococcus aureus subsp. anaerobius, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus schleiferi subsp. coagulans, clustered with their type strains in separate restriction groups. S. aureus subsp. aureus exhibited almost no similarity to these species. We found 44-kb SmaI fragments in all of the S. aureus subsp. aureus and S. aureus subsp. anaerobius strains studied, and these fragments are considered characteristic of the species S. aureus. The high level of homology of these fragments was confirmed by the results of DNA hybridization experiments in which we used representatives of individual intraspecies restriction groups. Of the other staphylococci studied, only Staphylococcus epidermidis and one strain of S. hyicus contained these fragments. However, the levels of homology between these fragments and the fragments of S. aureus were found to be very low.

Bacteriophages of Staphylococcus aureus efficiently package various bacterial genes and mobile genetic elements including SCCmec with different frequencies

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.

Efficient transfer of antibiotic resistance plasmids by transduction within methicillin-resistant Staphylococcus aureus USA300 clone.

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.

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.

Molecular diagnostics of clinically important staphylococci

Bacterial species of the genus Staphylococcus known as important human and animal pathogens are the cause of a number of severe infectious diseases. Apart from the major pathogen Staphylococcus aureus, other species until recently considered to be nonpathogenic may also be involved in serious infections. Rapid and accurate identification of the disease-causing agent is therefore prerequisite for disease control and epidemiological surveillance. Modern methods for identification and typing of bacterial species are based on genome analysis and have many advantages compared to phenotypic methods. The genotypic methods currently used in molecular diagnostics of staphylococcal species, particularly of S. aureus, are reviewed. Attention is also paid to new molecular methods with the highest discriminatory power. Efforts made to achieve interlaboratory reproducibility of diagnostic methods are presented.

Multiplex PCR for Detection of Three Exfoliative Toxin Serotype Genes in Staphylococcus aureus

Rapid and specific detection of exfoliative toxin (ET)-producingStaphylococcus aureus strains by multiplex polymerase chain reaction (PCR) was used for identification of exfoliative toxin genes in a diverse set of 115 clinicalS. aureus strains isolated in 14 Czech cities between 1998 and 2004. Fifty-nine wild-type ET-positive isolates of which 40 strains were the causative agents of toxic epidermolysis in neonates were classified into 4 PCR types. The genes coding for ETA, ETB or ETD were not detected in any of non-ET-producing isolates. The PCR method using the multiplex and specific primer set was shown to be reliable in rapid identification of the exfoliative toxin producingS. aureus and can be used as a convenient tool for hospital epidermolytic infection control.

Identification of Staphylococcus spp. using (GTG)5-PCR fingerprinting

A group of 212 type and reference strains deposited in the Czech Collection of Microorganisms (Brno, Czech Republic) and covering 41 Staphylococcus species comprising 21 subspecies was characterised using rep-PCR fingerprinting with the (GTG)₅ primer in order to evaluate this method for identification of staphylococci. All strains were typeable using the (GTG)₅ primer and generated PCR products ranging from 200 to 4500 bp. Numerical analysis of the obtained fingerprints revealed (sub)species-specific clustering corresponding with the taxonomic position of analysed strains. Taxonomic position of selected strains representing the (sub)species that were distributed over multiple rep-PCR clusters was verified and confirmed by the partial rpoB gene sequencing. Staphylococcus caprae, Staphylococcus equorum, Staphylococcus sciuri, Staphylococcus piscifermentans, Staphylococcus xylosus, and Staphylococcus saprophyticus revealed heterogeneous fingerprints and each (sub)species was distributed over several clusters. However, representatives of the remaining Staphylococcus spp. were clearly separated in single (sub)species-specific clusters. These results showed rep-PCR with the (GTG)₅ primer as a fast and reliable method applicable for differentiation and straightforward identification of majority of Staphylococcus spp.

Structure and genome release of Twort-like Myoviridae phage with a double-layered baseplate

Significance Resistance to antibiotics is widespread among pathogenic bacteria, including Staphylococcus aureus, which cause serious human diseases. Bacteriophages from the Twort-like genus of the family Myoviridae infect and kill pathogenic bacteria, and therefore are used to treat bacterial diseases. Detailed knowledge of the interactions of phages with bacterial cells is a prerequisite for the effective and safe use of phages for medical purposes. However, the molecular details of the processes regulating infections by these phages are not well understood. We used cryo-electron microscopy and tomography to describe the series of structural changes of a bacteriophage phi812 virion required to deliver its genome into the S. aureus cell. 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.

Staphylococcus microti sp. nov., isolated from the common vole(Microtus arvalis)

Two strains of Gram-positive cocci were isolated from viscera of common voles (Microtus arvalis Pallas) with generalized Brucella microti infection in the Czech Republic. Biochemical features and phylogenetic analysis based on 16S rRNA gene sequences showed that the strains are representatives of the genus Staphylococcus and assigned Staphylococcus muscae as the nearest relative. A detailed characterization done by ribotyping, rpoB and hsp60 gene sequencing, whole-cell protein analysis and rep-PCR using the (GTG)(5) primer differentiated the two strains from all described staphylococci. DNA-DNA hybridization with the type strain of S. muscae demonstrated that the two strains should be considered as members of a novel species (26.8 % reassociation). The two analysed strains were found to be coagulase-negative, novobiocin-susceptible, oxidase-negative cultures, phenotypically close to one another, but showing differences in ribotype profiles. The major fatty acids were iso-C(15 : 0), iso-C(17 : 0), anteiso-C(15 : 0), C(18 : 2 )omega6,9c/anteiso-C(18 : 0), C(18 : 0) and C(18 : 1) omega9c. MK-7 was the predominant isoprenoid quinone, with minor amounts of MK-6 and MK-8. The polar lipid profile was composed of the major lipids diphosphatidylglycerol and phosphatidylglycerol and several unknown lipids. These results proved that the two isolates represent a novel staphylococcal species. The name proposed for this novel taxon is Staphylococcus microti sp. nov.; the type strain is 4005-LJ(m)(T) (=CCM 4903(T) =CCUG 55861(T) =DSM 22147(T)).