Recent advances in genome editing techniques based on CRISPR-Cas have opened up new possibilities in bacteriophage engineering and, thus, enabled key developments in medicine, nanotechnology, and synthetic biology. Although staphylococcal phage genomes have already been edited, the modification of their structural proteins has not yet been reported. Here, the structure of Staphylococcus phage 812h1 of the Kayvirus genus was modified by inserting a poly histidine tag into an exposed loop of the tail sheath protein. A two-strain editing strategy was applied, utilizing homologous recombination followed by CRISPR-Cas10-assisted counter-selection of the recombinant phages. The His-tagged phage particles can be recognized by specific antibodies, enabling the modified bacteriophages to be employed in numerous techniques. The attachment of the engineered phage to bacteria was visualized by fluorescence microscopy, and its functionality was confirmed using biolayer interferometry biosensing, enzyme-linked immunosorbent assay, and flow cytometry, demonstrating that the genetic modification did not impair its biological activity.

• Keywords
• Bacteriophage, Biosensing Techniques, CRISPR-Cas10, Herelleviridae, Poly histidine Tag, Staphylococcus aureus,
• Publication type
• Journal Article MeSH

Antibiotic-resistant strains of Staphylococcus aureus pose a significant threat in healthcare, demanding urgent therapeutic solutions. Combining bacteriophages with conventional antibiotics, an innovative approach termed phage-antibiotic synergy, presents a promising treatment avenue. However, to enable new treatment strategies, there is a pressing need for methods to assess their efficacy reliably and rapidly. Here, we introduce a novel approach for real-time monitoring of pathogen lysis dynamics employing the piezoelectric quartz crystal microbalance (QCM) with dissipation (QCM-D) technique. The sensor, a QCM chip modified with the bacterium S. aureus RN4220 ΔtarM, was utilized to monitor the activity of the enzyme lysostaphin and the phage P68 as model lytic agents. Unlike conventional QCM solely measuring resonance frequency changes, our study demonstrates that dissipation monitoring enables differentiation of bacterial growth and lysis caused by cell-attached lytic agents. Compared to reference turbidimetry measurements, our results reveal distinct alterations in the growth curve of the bacteria adhered to the sensor, characterized by a delayed lag phase. Furthermore, the dissipation signal analysis facilitated the precise real-time monitoring of phage-mediated lysis. Finally, the QCM-D biosensor was employed to evaluate the synergistic effect of subinhibitory concentrations of the antibiotic amoxicillin with the bacteriophage P68, enabling monitoring of the lysis of P68-resistant wild-type strain S. aureus RN4220. Our findings suggest that this synergy also impedes the formation of bacterial aggregates, the precursors of biofilm formation. Overall, this method brings new insights into phage-antibiotic synergy, underpinning it as a promising strategy against antibiotic-resistant bacterial strains with broad implications for treatment and prevention.

• Keywords
• Staphylococcus aureus, Antimicrobial treatment, Multidrug-resistant bacteria, Phage therapy, Phage-antibiotic synergy, Piezoelectric biosensor,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacteriophages physiology   MeSH
• Bacteriolysis * drug effects   MeSH
• Biosensing Techniques * methods   instrumentation   MeSH
• Lysostaphin pharmacology   MeSH
• Quartz Crystal Microbalance Techniques * methods   MeSH
• Staphylococcus aureus * drug effects   virology   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Lysostaphin MeSH

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

Kayviruses are polyvalent broad host range staphylococcal phages with a potential to combat staphylococcal infections. However, the implementation of rational phage therapy in medicine requires a thorough understanding of the interactions between bacteriophages and pathogens at omics level. To evaluate the effect of a phage used in therapy on its host bacterium, we performed differential transcriptomic analysis by RNA-Seq from bacteriophage K of genus Kayvirus infecting two Staphylococcus aureus strains, prophage-less strain SH1000 and quadruple lysogenic strain Newman. The temporal transcriptional profile of phage K was comparable in both strains except for a few loci encoding hypothetical proteins. Stranded sequencing revealed transcription of phage noncoding RNAs that may play a role in the regulation of phage and host gene expression. The transcriptional response of S. aureus to phage K infection resembles a general stress response with differential expression of genes involved in a DNA damage response. The host transcriptional changes involved upregulation of nucleotide, amino acid and energy synthesis and transporter genes and downregulation of host transcription factors. The interaction of phage K with variable genetic elements of the host showed slight upregulation of gene expression of prophage integrases and antirepressors. The virulence genes involved in adhesion and immune evasion were only marginally affected, making phage K suitable for therapy. IMPORTANCE Bacterium Staphylococcus aureus is a common human and veterinary pathogen that causes mild to life-threatening infections. As strains of S. aureus are becoming increasingly resistant to multiple antibiotics, the need to search for new therapeutics is urgent. A promising alternative to antibiotic treatment of staphylococcal infections is a phage therapy using lytic phages from the genus Kayvirus. Here, we present a comprehensive view on the phage-bacterium interactions on transcriptomic level that improves the knowledge of molecular mechanisms underlying the Kayvirus lytic action. The results will ensure safer usage of the phage therapeutics and may also serve as a basis for the development of new antibacterial strategies.

• Keywords
• Kayvirus, RNA-Seq, Staphylococcus aureus, Staphylococcus phages, bacteriophage therapy, noncoding RNA, phage-host interactions, prophages, transcriptome, viral transcription,
• MeSH
• Humans MeSH
• Prophages genetics   MeSH
• Staphylococcus Phages genetics   MeSH
• Staphylococcal Infections * microbiology   therapy   MeSH
• Staphylococcus aureus * MeSH
• Transcriptome MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Phages are viruses which can specifically infect bacteria, resulting in their destruction. Bacterial infections are a common complication of wound healing, and experimental evidence from animal models demonstrates promising potential for phage-dependent eradication of wound-associated infections. The studies discussed suggest that phage therapy may be an effective treatment, with important advantages over some current antibacterial treatments. Phage cocktails, as well as co-administration of phages and antibiotics, have been reported to minimise bacterial resistance. Further, phage-antibiotic synergism has been reported in some studies. The ideal dose of phages is still subject to debate, with evidence for both high and low doses to yield therapeutic effects. Novel delivery methods, such as hydrogels, are being explored for their advantages in topical wound healing. There are more and more Good Manufacturing Practice facilities dedicated to manufacturing phage products and phage therapy units across the world, showing the changing perception of phages which is occurring. However, further research is needed to secure the place of phages in modern medicine, with some scientists calling upon the World Health Organisation to help promote phage therapy.

• MeSH
• Anti-Bacterial Agents therapeutic use   MeSH
• Bacteria MeSH
• Bacterial Infections * therapy   MeSH
• Bacteriophages * MeSH
• Phage Therapy * methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH