Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.

• Keywords
• Kayvirus, Staphylococcus aureus, abortive infection, bacteriophage evolution, bacteriophage therapy, bacteriophages, cell death, lysogeny, phage resistance, phage therapy,
• MeSH
• Humans MeSH
• Lysogeny MeSH
• Membrane Proteins genetics   MeSH
• Prophages * genetics   MeSH
• Staphylococcus Phages genetics   MeSH
• Staphylococcal Infections * microbiology   MeSH
• Staphylococcus aureus genetics   MeSH
• Staphylococcus MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH

Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages.

• Keywords
• Kayvirus, MALDI-MS, Staphylococcus, Viral proteins, bacteriophages, phage therapy,
• MeSH
• Biological Products isolation & purification   MeSH
• Chemical Fractionation methods   MeSH
• Humans MeSH
• Virus Replication MeSH
• Cluster Analysis MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * methods   MeSH
• Staphylococcus Phages classification   metabolism   MeSH
• Staphylococcus aureus virology   MeSH
• Viral Proteins analysis   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biological Products MeSH
• viron MeSH Browser
• Viral Proteins MeSH

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

• Keywords
• Endolysin, Endopeptidases, Enzybiotics, Src homology domains, Staphylococcal infections, Staphylococcus bacteriophage,
• MeSH
• Endopeptidases genetics   isolation & purification   metabolism   MeSH
• Host Specificity * MeSH
• Mutant Proteins genetics   isolation & purification   metabolism   MeSH
• Myoviridae enzymology   genetics   physiology   MeSH
• Peptidoglycan metabolism   MeSH
• Protein Domains MeSH
• Sequence Deletion * MeSH
• Staphylococcus virology   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• endolysin MeSH Browser
• Endopeptidases MeSH
• Mutant Proteins MeSH
• Peptidoglycan MeSH

Staphylococcus sciuri is a bacterial pathogen associated with infections in animals and humans, and represents a reservoir for the mecA gene encoding methicillin-resistance in staphylococci. No S. sciuri siphophages were known. Here the identification and characterization of two temperate S. sciuri phages from the Siphoviridae family designated ϕ575 and ϕ879 are presented. The phages have icosahedral heads and flexible noncontractile tails that end with a tail spike. The genomes of the phages are 42,160 and 41,448 bp long and encode 58 and 55 ORFs, respectively, arranged in functional modules. Their head-tail morphogenesis modules are similar to those of Staphylococcus aureus ϕ13-like serogroup F phages, suggesting their common evolutionary origin. The genome of phage ϕ575 harbours genes for staphylokinase and phospholipase that might enhance the virulence of the bacterial hosts. In addition both of the phages package a homologue of the mecA gene, which is a requirement for its lateral transfer. Phage ϕ879 transduces tetracycline and aminoglycoside pSTS7-like resistance plasmids from its host to other S. sciuri strains and to S. aureus. Furthermore, both of the phages efficiently adsorb to numerous staphylococcal species, indicating that they may contribute to interspecies horizontal gene transfer.

• MeSH
• Genes, Bacterial * MeSH
• Phospholipases metabolism   MeSH
• Genome, Viral MeSH
• Genomics methods   MeSH
• Host Specificity MeSH
• Metalloendopeptidases metabolism   MeSH
• Plasmids genetics   MeSH
• Gene Transfer, Horizontal MeSH
• Virus Attachment MeSH
• Staphylococcus Phages physiology   ultrastructure   MeSH
• Staphylococcus virology   MeSH
• Transduction, Genetic * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• auR protein, Staphylococcus aureus MeSH Browser
• Phospholipases MeSH
• Metalloendopeptidases MeSH

Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated ϕB166 and ϕB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

• MeSH
• DNA, Viral chemistry   genetics   MeSH
• DNA Viruses genetics   isolation & purification   MeSH
• Disease Outbreaks MeSH
• Exfoliatins genetics   MeSH
• Phylogeny MeSH
• Genome, Viral * MeSH
• Impetigo epidemiology   microbiology   MeSH
• Cross Infection epidemiology   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Infant, Newborn MeSH
• Open Reading Frames MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Gene Order MeSH
• Hospitals, Maternity MeSH
• Gene Transfer, Horizontal MeSH
• Prophages classification   genetics   isolation & purification   MeSH
• Sequence Analysis, DNA MeSH
• Sequence Homology MeSH
• Cluster Analysis MeSH
• Staphylococcus Phages classification   genetics   isolation & purification   MeSH
• Staphylococcal Infections epidemiology   microbiology   MeSH
• Staphylococcus aureus isolation & purification   virology   MeSH
• Synteny MeSH
• Transduction, Genetic MeSH
• Check Tag
• Humans MeSH
• Infant, Newborn MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• DNA, Viral MeSH
• Exfoliatins MeSH

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.

• MeSH
• Genotype MeSH
• Humans MeSH
• Polymerase Chain Reaction MeSH
• Methicillin Resistance MeSH
• Sequence Analysis, DNA MeSH
• Staphylococcus classification   genetics   isolation & purification   MeSH
• Bacterial Typing Techniques MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH