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Background. Over the last three decades, coagulase-negative staphylococci (CoNS) have been recognised as opportunistic pathogens, especially in immunocompromised patients. CoNS serve as a reservoir of accessory genes, including virulence and antimicrobial resistance factors for the genus Staphylococcus. Recent diagnostic techniques allow recognising of novel CoNS species in human clinical specimens. Methods. Here, we characterised coagulase-negative staphylococcal strains from the Staphylococcus haemolyticus phylogenetic clade obtained from human ear swabs, wounds and bile. A polyphasic taxonomic approach based on whole genomic sequencing with expert annotation, extensive biotyping, DNA fingerprinting by rep-PCR, MALDI-TOF MS, and chemotaxonomy analyses was applied to establish their phylogenetic position and characterize virulence and drug-resistance genes. Results. Five isolates of Staphylococcus sp. were obtained from various human-related clinical specimens both from mixed culture and monoculture. Based on the polyphasic taxonomic approach, the novel species named Staphylococcus brunensis sp. nov. is proposed, with the closest relative Staphylococcus petrasii. The whole genome sequence analysis revealed the presence of several variable genomic elements including staphylococcal chromosomal cassette (SCC), prophages, genomic islands, various full and partial IS elements, and plasmids conferring beta-lactam and macrolide resistance. Three strains possess an 18.8 kb-long genomic island designated SbCIccr with a ccr gene complex designated ccrDE that had a conserved structure like a ccr gene complex from known SCC types. However, the SbCIccr was integrated into the serine acetyltransferase gene rimL, not into the canonical integration site for SCCs, methyltransferase gene rlmH (orfX). The genomic island harbours genes for the restriction-modification system commonly found on SCC elements, a putative transposon with IS6 family element similar to IS431mec, a putative transcriptional regulator and accessory genes, which have been found frequently on plasmids. By a PCR assay we showed that the SbCIccr could be mobilized from the bacterial chromosome. Conclusions. Study of SbCIccr has a crucial role in the understanding of the origin, evolution and transfer of SCC-like elements. It may also represent a primordial element able to accumulate virulence and drug resistance factors, whose spread into established pathogens such as S. aureus would be a threat to the healthcare system.

The treatment of infections caused by human and veterinary pathogen Staphylococcus aureus is becoming worldwide healthcare concern due to the increasing resistance of the bacterium to antibiotics. A promising alternative to currently used drugs is represented by lytic phages from genus Kayvirus. However, the implementation of rational phage therapy into medicine requires to understand the interactions between bacteriophages and pathogens. To address this issue, we analysed RNA seqeuncing data of two Staphylococcus aureus strains infected by type phage K of the Kayvirus genus K with potential in phage therapy. The temporal transcriptional profile of phage K was similar in both analysed strains except for a few genes. Analysis of the RNA-Seq data also revealed antisense transcription and transcription from non-coding DNA with potential role in the regulation of phage and host gene expression. The transcription response of S. aureus to phage K infection firstly corresponded to a general response to stress and DNA damage1. Bacterial differentially expressed genes were involved in nucleotide biosynthesis, amino acid and energy metabolism and cell wall synthesis genes. Furthermore, we detected during the late phase of phage K infection slightly increased transcription of staphylococcal virulence genes functioning in adhesion and immune system evasion. Our results clarify the global transcriptional interaction between phage and bacterial host, which will ensure safer usage of phage therapeutics and may also serve as a basis for development of new antibacterial strategies.

Bacterial viruses (bacteriophages) have significant impact on the evolution of bacteria and regulation of bacterial populations, including the microflora of the human body. Due to the global antibiotic crisis, interest in the use of bacterial viruses, phage-derived enzybiotics and chemical compopunds inspired by structures and functions of phage proteins to combat bacterial infections has increased worldwide. Historical clinical data on the effective use of bacteriophages in patients exists from Eastern Europe, but no evidence of it has been validated under recent European Regulatory standards. Discussions in professional societies such as ESCMID Study Group for Non-traditional Antibacterial Therapy –ESGNTA are currently underway on setting the rules for production and safety of the use of bacteriophage preparations. The regulatory authorities also communicate an increased interest in the therapeutic use of bacteriophages. In July 2021, the European Pharmacopoeia Commission launched work on new general chapter on phage therapy active substances and medicinal products for human and veterinary use, establishing harmonised quality standards for phage therapy products and providing a framework for their safe use in European countries.

Bacteriophages are crucial in shaping population of pathogens, such as Staphylococcus aureus. Prophages play an important role in the virulence, pathogenesis or host preference, as well as in horizontal gene transfer. On the other hand, broad host range staphylococcal bacteriophages of the genus Kayvirus are promising agents for therapeutic applications. The lysogens become immune to infection by closely related phages, but the interactions between temperate and lytic staphylococcal phages are not understood. We describe a resistance mechanism towards lytic phages of the genus Kayvirus, mediated by S. aureus prophage accessory gene. The responsible membrane-anchored protein shows the presence of a putative membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C-terminus. We demonstrated that the mechanism of action does not prevent the infecting Kayvirus to adsorb onto the host cell, deliver its genome into the cell but, phage replication is halted. Changes in the cell membrane polarity and permeability, which lead to prophage-activated cell death were observed from 10 min after the infection. Furthermore, we describe a mechanism of overcoming resistance in a spontaneous host-range Kayvirus mutant in which a gene fusion has emerged, and which was selected on S. aureus strain harbouring a prophage encoding the immunity protein.