Temperate bacteriophages play an important role in the pathogenicity of Staphylococcus aureus, for instance, by mediating the horizontal gene transfer of virulence factors. Here we established a classification scheme for staphylococcal prophages of the major Siphoviridae family based on integrase gene polymorphism. Seventy-one published genome sequences of staphylococcal phages were clustered into distinct integrase groups which were related to the chromosomal integration site and to the encoded virulence gene content. Analysis of three marker modules (lysogeny, tail, and lysis) for phage functional units revealed that these phages exhibit different degrees of genome mosaicism. The prevalence of prophages in a representative S. aureus strain collection consisting of 386 isolates of diverse origin was determined. By linking the phage content to dominant S. aureus clonal complexes we could show that the distribution of bacteriophages varied remarkably between lineages, indicating restriction-based barriers. A comparison of colonizing and invasive S. aureus strain populations revealed that hlb-converting phages were significantly more frequent in colonizing strains.Staphylococcus aureus asymptomatically colonizes the anterior nares of humans but also causes a wide spectrum of acute and chronic diseases. Most of the dissimilarity between S. aureus strains is due to the presence of mobile genetic elements such as plasmids, bacteriophages, pathogenicity islands, transposons, and insertion sequences (2,14,19,23). Many virulence factors are encoded on such mobile elements (3,6,17,26,27,35). In particular, bacteriophages play an important role in the pathogenicity of S. aureus either by carrying accessory virulence factors such as Panton-Valentine leukocidin (PVL) (encoded by the luk-PV operon), staphylokinase (encoded by sak), enterotoxin A (encoded by sea), and exfoliative toxin A (encoded by eta) or by interrupting chromosomal virulence genes such as those for -hemolysin (hlb) and lipase (geh) upon insertion. Additionally, phages are the primary vehicle of lateral gene transfer between S. aureus strains, providing the species with the potential for broad genetic variation. We could show that phages increase the genome plasticity of S. aureus during infection, facilitating the adaptation of the pathogen to various host conditions (11,12).Despite the obvious importance of phages for the biology of S. aureus, epidemiological data on the prevalence of phages in this species are limited (28, 33). More than 80 genome sequences of staphylococcal bacteriophages and prophages are available in the public genome databases. Most published S. aureus phages belong to the Siphoviridae family of temperate, tailed bacterial viruses. Traditionally, S. aureus phages were characterized according to their lytic activity, morphology, and serological properties (1, 28). Today, the temperate phages in clinical S. aureus isolates can by identified with a multiplex PCR strategy, which is based on sequence differences between viral genes codin...
Sequencing of at least 13 Staphylococcus aureus isolates has shown that genomic plasticity impacts significantly on the repertoire of virulence factors. However, genome sequencing does not reveal which genes are expressed by individual isolates. Here, we have therefore performed a comprehensive survey of the composition and variability of the S. aureus exoproteome. This involved multilocus sequence typing, virulence gene, and prophage profiling by multiplex PCR, and proteomic analyses of secreted proteins using 2-DE. Dissection of the exoproteomes of 25 clinical isolates revealed that only seven out of 63 identified secreted proteins were produced by all isolates, indicating a remarkably high exoproteome heterogeneity within one bacterial species. Most interesting, the observed variations were caused not only by genome plasticity, but also by an unprecedented variation in secretory protein production due to differences in transcriptional and post-transcriptional regulation. Our data imply that genomic studies on virulence gene conservation patterns need to be complemented by analyses of the extracellular protein pattern to assess the full virulence potential of bacterial pathogens like S. aureus. Importantly, the extensive variability of secreted virulence factors in S. aureus also suggests that development of protective vaccines against this pathogen requires a carefully selected combination of invariably produced antigens.
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.
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.
Ninety-five percent of 782 culture collection strains, as well as hospital strains of Staphylococcus aureus subsp. aureus of different provenance and 43% of 89 culture collection strains of different coagulase-negative species of the genus Staphylococcus, were found to be sensitive to the polyvalent phage phi 812 or to at least one of its host-range mutants or to the polyvalent phages SK311, phi 131, and U16. Thus sensitivity to the polyvalent staphylococcal phages seems to be one of the common features of S. aureus subsp. aureus strains. The adsorption kinetics and one-step growth characteristics of the phages phi 812 and SK311 were estimated. Restriction genomic maps of the phages phi 812 (146.5 kb) and SK311 (141.1 kb) were constructed by use of the restriction endonucleases AvaII, PstI, KpnI, SacI, SmaI, and XhoI. The host-range mutations of the phage phi 812 were localized on this map. Comparison of restriction patterns of the phages phi 812 and SK311 with those of the polyvalent phages U16 and phi 131 suggests that all these phages are closely related. Their genomes differ from each other mostly by some deletions, insertions (1-3 kb), or inversions. Evidence was given that the phage phi 812 together with SK311, phi 131, and U16 belongs in the phage species Twort, the description of which is substantially supplemented with the data on the phage phi 812 reported in this paper.
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.
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.
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.
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