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.

• Klíčová slova
• Kayvirus, Staphylococcus aureus, abortive infection, bacteriophage evolution, bacteriophage therapy, bacteriophages, cell death, lysogeny, phage resistance, phage therapy,
• MeSH
• lidé MeSH
• lyzogenie MeSH
• membránové proteiny genetika   MeSH
• profágy * genetika   MeSH
• stafylokokové bakteriofágy genetika   MeSH
• stafylokokové infekce * mikrobiologie   MeSH
• Staphylococcus aureus genetika   MeSH
• Staphylococcus MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH

The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10 μL of the prepared sample at the optimized flow rate of 6.5 μL min-1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 = 0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (±) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract.

• Klíčová slova
• Capillary electrophoresis, MALDI-TOF mass spectrometry, Nano-etched fused-silica capillary, Phage propagation, Staphylococcal bacteriophages, Supercritical water,
• MeSH
• bakteriofágy patogenita   MeSH
• lidé MeSH
• odběr vzorku krve přístrojové vybavení   MeSH
• oxid křemičitý chemie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oxid křemičitý MeSH

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.

• MeSH
• bakteriální léková rezistence MeSH
• bakteriofágy genetika   MeSH
• délka genomu MeSH
• genom virový MeSH
• genomika MeSH
• jednonukleotidový polymorfismus MeSH
• methicilin farmakologie   MeSH
• mutace * MeSH
• Staphylococcus aureus růst a vývoj   virologie   MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methicilin MeSH

Staphylococcus aureus may be a highly virulent human pathogen, especially when it is able to form a biofilm, and it is resistant to antibiotic. Infections caused by these bacteria significantly affect morbidity and mortality, primarily in hospitalized patients. Treatment becomes more expensive, more toxic, and prolonged. This is the reason why research on alternative therapies should be one of the main priorities of medicine and biotechnology. A promising alternative treatment approach is bacteriophage therapy. The effect of the anti-staphylococcal bacteriophage preparation Stafal® on biofilm reduction was assessed on nine S. aureus strains using both sonication with subsequent quantification of surviving cells on the catheter surface and evaluation of biofilm reduction in microtiter plates. It was demonstrated that the bacteriophages destroy planktonic cells very effectively. However, to destroy cells embedded in the biofilm effectively requires a concentration at least ten times higher than that provided by the commercial preparation. The catheter disc method (CDM) allowed easier comparison of the effect on planktonic cells and cells in a biofilm than the microtiter plate (MTP) method.

• MeSH
• antiinfekční látky * MeSH
• bakteriologické techniky MeSH
• biofilmy * MeSH
• lidé MeSH
• methicilin rezistentní Staphylococcus aureus růst a vývoj   izolace a purifikace   virologie   MeSH
• mikrobiální viabilita MeSH
• počet mikrobiálních kolonií MeSH
• stafylokokové bakteriofágy fyziologie   MeSH
• stafylokokové infekce mikrobiologie   MeSH
• Staphylococcus aureus růst a vývoj   izolace a purifikace   virologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiinfekční látky * 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.

• Klíčová slova
• Kayvirus, MALDI-MS, Staphylococcus, Viral proteins, bacteriophages, phage therapy,
• MeSH
• biologické přípravky izolace a purifikace   MeSH
• chemická frakcionace metody   MeSH
• lidé MeSH
• replikace viru MeSH
• shluková analýza MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody   MeSH
• stafylokokové bakteriofágy klasifikace   metabolismus   MeSH
• Staphylococcus aureus virologie   MeSH
• virové proteiny analýza   chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické přípravky MeSH
• viron MeSH Prohlížeč
• virové proteiny 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.

• Klíčová slova
• Endolysin, Endopeptidases, Enzybiotics, Src homology domains, Staphylococcal infections, Staphylococcus bacteriophage,
• MeSH
• endopeptidasy genetika   izolace a purifikace   metabolismus   MeSH
• hostitelská specificita * MeSH
• mutantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• Myoviridae enzymologie   genetika   fyziologie   MeSH
• peptidoglykan metabolismus   MeSH
• proteinové domény MeSH
• sekvenční delece * MeSH
• Staphylococcus virologie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endolysin MeSH Prohlížeč
• endopeptidasy MeSH
• mutantní proteiny MeSH
• peptidoglykan MeSH

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.

• Klíčová slova
• Staphylococcus, bacteriophage, contraction, genome release, structure,
• MeSH
• elektronová kryomikroskopie MeSH
• genom virový * MeSH
• Myoviridae genetika   fyziologie   ultrastruktura   MeSH
• Staphylococcus aureus virologie   MeSH
• virové plášťové proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny MeSH

Mutations extended the host range of the polyvalent bacteriophage 812 of the family Myoviridae in up to 95 % of Staphylococcus aureus strains and 43 % of strains of different coagulase-positive and -negative Staphylococcus 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 gene orf8 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 phi131. Another intron was discovered in the recA812 gene in these four mutants. An insertion was found in a non-coding region of the restriction fragment PstI-O of three mutants (812b, 812F3, 812g) and phages U16 and phi131. The above results contribute to the explanation of genetic factors affecting the host range of polyvalent staphylococcal bacteriophages.

• MeSH
• bakteriofágy genetika   MeSH
• endopeptidasy chemie   genetika   MeSH
• genom virový * MeSH
• molekulární sekvence - údaje MeSH
• mutace * MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• proteiny virových bičíků chemie   genetika   MeSH
• RNA virová chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• Staphylococcus aureus virologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endolysin MeSH Prohlížeč
• endopeptidasy MeSH
• proteiny virových bičíků MeSH
• RNA virová MeSH