Strain CCM 2573 is a Gram-positive bacterium that has been intensively studied in the past due to its distinct chemotaxonomic properties, but its reliable taxonomic classification has not been satisfactorily clarified. Whole-genome sequencing and comparative genomic analyses performed in this study revealed that the strain belongs to the Macrococcus caseolyticus phylogenetic clade. Genome-to-genome comparisons confirmed the closest relationship to the type strains of M. caseolyticus subsp. hominis CCM 7927T and M. caseolyticus subsp. caseolyticus DSM 20597T. However, the strain harbored unique genomic elements distinguishing it from its nearest phylogenetic neighbors. Its accessory genome contains dozens of insertion sequences, a 92-kbp composite transposon with unique palindromic repeat loci associated with a CRISPR-Cas adaptive immune system, a pseudo-staphylococcal chromosome cassette, and several additional genomic islets. Unlike other macrococci, strain CCM 2573 exhibits a specific peptidoglycan composition (L-Lys-Gly₂-Ser₂-Gly) and shows a higher phylogenetic divergence of aminoacyltransferases (FemABX) involved in interpeptide bridge synthesis. In addition, it reveals distinct biochemical characteristics from both subspecies of M. caseolyticus, particularly in its ability to produce acid from galactose, cellobiose, melezitose, and turanose, as well as in its susceptibility to novobiocin. The MALDI-TOF mass spectra enable differentiation of the strain from other type strains of the genus Macrococcus. The results of polyphasic taxonomy obtained in this study showed that strain CCM 2573 belongs to the species M. caseolyticus, but it is distinct from both validly named M. caseolyticus subspecies. We propose to assign the analyzed strain as a new subspecies, Macrococcus caseolyticus subsp. lactis subsp. nov. The type strain is CCM 2573T (= DSM 20227T).

ABSTRACT Macrococci are usually found as commensals on the skin and mucosa of animals and have been isolated from mammal-derived fermented foods; however, they can also act as opportunistic pathogens. Here, we used whole-genome sequencing, comparative genomics, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize Macrococcus sp. strains isolated from livestock and human-related specimens. Based on the results of polyphasic taxonomy, we propose the species Macrococcus psychrotolerans sp. nov. (type strain NRL/St 95/376 T = CCM 8659 T = DSM 111350 T ) belonging to the Macrococcus caseolyticus phylogenetic clade. It grows at 4°C, and the core genome of the isolates contains suspected genes contributing to low-temperature tolerance. Variable genetic elements include prophages, chromosomal islands, a composite staphylococcal cassette chromosome island, and many plasmids that affect the overall genome expansion and adaptation to specific ecological settings of the studied isolates. Large plasmids carrying the methicillin resistance gene mecB were identified in M. psychrotolerans sp. nov. strains and confirmed as self-transmissible to Staphylococcus aureus in vitro . In addition to plasmids with circular topology, a 150-kb-long linear plasmid with 14.1-kb-long inverted terminal repeats, harboring many IS elements and putative genes for a type IV secretion system was revealed. The described strains were isolated from human clinical material, food-producing animals, meat, and a wooden cheese board and have the potential to proliferate at refrigerator temperatures. Their presence in the food chain and human infections indicates that attention needs to be paid to this potential novel opportunistic pathogen. IMPORTANCE The study offers insights into the phenotypic and genomic features of a novel species of the genus Macrococcus that occurs in livestock, food, and humans. The large number of diverse mobile genetic elements contributes to the adaptation of macrococci to various environments. The ability of the described microorganisms to grow at refrigerator temperatures, enabled by genes that are predicted to contribute to low-temperature tolerance, raises food safety concerns. Confirmed in vitro conjugative transfer of plasmid-borne mecB gene to S. aureus poses a significant risk of spread of broad β-lactam resistance. In addition, the intergeneric plasmid transfer to S. aureus is indicative of horizontal gene transfer events that may be more frequent than generally accepted. Determining a complete sequence and gene content of linear megaplasmid with exceptional topology for the Staphylococcaceae family suggests its possible role in shuttling adaptive traits through an exchange of genetic information.

Novel species of coagulase-negative staphylococci, which could serve as reservoirs of virulence and antimicrobial resistance factors for opportunistic pathogens from the genus Staphylococcus, are recognized in human and animal specimens due to advances in diagnostic techniques. Here, we used whole-genome sequencing, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize five coagulase-negative strains from the Staphylococcus haemolyticus phylogenetic clade obtained from human ear swabs, wounds, and bile. Based on the results of polyphasic taxonomy, we propose the species Staphylococcus brunensis sp. nov. (type strain NRL/St 16/872T = CCM 9024T = LMG 31872T = DSM 111349T). The genomic analysis revealed numerous variable genomic elements, including staphylococcal cassette chromosome (SCC), prophages, plasmids, and a unique 18.8 kb-long genomic island SbCIccrDE integrated into the ribosomal protein L7 serine acetyltransferase gene rimL. SbCIccrDE has a cassette chromosome recombinase (ccr) gene complex with a typical structure found in SCCs. Based on nucleotide and amino acid identity to other known ccr genes and the distinct integration site that differs from the canonical methyltransferase gene rlmH exploited by SCCs, we classified the ccr genes as novel variants, ccrDE. The comparative genomic analysis of SbCIccrDE with related islands shows that they can accumulate virulence and antimicrobial resistance factors creating novel resistance elements, which reflects the evolution of SCC. The spread of these resistance islands into established pathogens such as Staphylococcus aureus would pose a great threat to the healthcare system. IMPORTANCE The coagulase-negative staphylococci are important opportunistic human pathogens, which cause bloodstream and foreign body infections, mainly in immunocompromised patients. The mobile elements, primarily the staphylococcal cassette chromosome mec, which confers resistance to methicillin, are the key to the successful dissemination of staphylococci into healthcare and community settings. Here, we present a novel species of the Staphylococcus genus isolated from human clinical material. The detailed analysis of its genome revealed a previously undescribed genomic island, which is closely related to the staphylococcal cassette chromosome and has the potential to accumulate and spread virulence and resistance determinants. The island harbors a set of conserved genes required for its mobilization, which we recognized as novel cassette chromosome recombinase genes ccrDE. Similar islands were revealed not only in the genomes of coagulase-negative staphylococci but also in S. aureus. The comparative genomic study contributes substantially to the understanding of the evolution and pathogenesis of staphylococci.

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.

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.

Staphylococci from the Staphylococcus intermedius-Staphylococcus hyicus species group include numerous animal pathogens and are an important reservoir of virulence and antimicrobial resistance determinants. Due to their pathogenic potential, they are possible causative agents of zoonoses in humans; therefore, it is important to address the properties of these strains. Here we used a polyphasic taxonomic approach to characterize the coagulase-negative staphylococcal strain NRL/St 03/464T, isolated from the nostrils of a healthy laboratory rat during a microbiological screening of laboratory animals. The 16S rRNA sequence, MALDI-TOF mass spectrometry and positive urea hydrolysis and beta-glucuronidase tests clearly distinguished it from closely related Staphylococcus spp. All analyses have consistently shown that the closest relative is Staphylococcus chromogenes; however, values of digital DNA-DNA hybridization <35.3% and an average nucleotide identity <81.4% confirmed that the analyzed strain is a distinct Staphylococcus species. Whole-genome sequencing and expert annotation of the genome revealed the presence of novel variable genetic elements, including two plasmids named pSR9025A and pSR9025B, prophages, genomic islands and a composite transposon that may confer selective advantages to other bacteria and enhance their survival. Based on phenotypic, phylogenetic and genomic data obtained in this study, the strain NRL/St 03/464T (= CCM 9025T = LMG 31873T = DSM 111348T) represents a novel species with the suggested name Staphylococcus ratti sp. nov.

Staphylococcus epidermidis is a leading opportunistic pathogen causing nosocomial infections that is notable for its ability to form a biofilm and for its high rates of antibiotic resistance. It serves as a reservoir of multiple antimicrobial resistance genes that spread among the staphylococcal population by horizontal gene transfer such as transduction. While phage-mediated transduction is well studied in Staphylococcus aureus, S. epidermidis transducing phages have not been described in detail yet. Here, we report the characteristics of four phages, 27, 48, 456, and 459, previously used for S. epidermidis phage typing, and the newly isolated phage E72, from a clinical S. epidermidis strain. The phages, classified in the family Siphoviridae and genus Phietavirus, exhibited an S. epidermidis-specific host range, and together they infected 49% of the 35 strains tested. A whole-genome comparison revealed evolutionary relatedness to transducing S. aureus phietaviruses. In accordance with this, all the tested phages were capable of transduction with high frequencies up to 1024 among S. epidermidis strains from different clonal complexes. Plasmids with sizes from 4 to 19 kb encoding resistance to streptomycin, tetracycline, and chloramphenicol were transferred. We provide here the first evidence of a phage-inducible chromosomal island transfer in S. epidermidis. Similarly to S. aureus pathogenicity islands, the transfer was accompanied by phage capsid remodeling; however, the interfering protein encoded by the island was distinct. Our findings underline the role of S. epidermidis temperate phages in the evolution of S. epidermidis strains by horizontal gene transfer, which can also be utilized for S. epidermidis genetic studies. IMPORTANCE Multidrug-resistant strains of S. epidermidis emerge in both nosocomial and livestock environments as the most important pathogens among coagulase-negative staphylococcal species. The study of transduction by phages is essential to understanding how virulence and antimicrobial resistance genes spread in originally commensal bacterial populations. In this work, we provide a detailed description of transducing S. epidermidis phages. The high transduction frequencies of antimicrobial resistance plasmids and the first evidence of chromosomal island transfer emphasize the decisive role of S. epidermidis phages in attaining a higher pathogenic potential of host strains. To date, such importance has been attributed only to S. aureus phages, not to those of coagulase-negative staphylococci. This study also proved that the described transducing bacteriophages represent valuable genetic modification tools in S. epidermidis strains where other methods for gene transfer fail.

Members of the genus Staphylococcus are widespread in nature and occupy a variety of niches, however, staphylococcal colonization of animals in the Antarctic environment has not been adequately studied. Here, we describe the first isolation and characterization of two Staphylococcus intermedius group (SIG) members, Staphylococcus delphini and Staphylococcus pseudintermedius, in Antarctic wildlife. Staphylococcus delphini were found exclusively in Adélie penguins. The report of S. pseudintermedius from Weddell seals confirmed its occurrence in all families of the suborder Caniformia. Partial RNA polymerase beta-subunit (rpoB) gene sequencing, repetitive PCR fingerprinting with the (GTG)5 primer, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry gave consistent identification results and proved to be suitable for identifying SIG members. Comparative genomics of S. delphini isolates revealed variable genomic elements, including new prophages, a novel phage-inducible chromosomal island, and numerous putative virulence factors. Surface and extracellular protein distribution were compared between genomes and showed strain-specific profiles. The pathogenic potential of S. delphini was enhanced by a novel type of exfoliative toxin, trypsin-like serine protease cluster, and enterotoxin C. Detailed analysis of phenotypic characteristics performed on six Antarctic isolates of S. delphini and eight reference strains from different animal sources enabled us to emend the species description of S. delphini.

Staphylococcus epidermidis mobile genetic elements (MGE), including temperate phages and phage-inducible chromosomal islands (PICI), are still not well described. However, through the horizontal gene transfer, they determine properties such as antibiotic resistance and/or virulence to their hosts. Our work characterizes new bacteriophages able to transduce S. epidermidis PICIs and plasmids. Genomes of four temperate siphoviruses and their propagation strains were obtained using Oxford Nanopore and Illumina sequencing technology. Phage genomes were described and compared in detail. Differences found in otherwise conservative modules of morphogenesis and DNA packaging possibly played a role in frequency of PICI and plasmid transduction. Different PICIs localized in host genomes contain known and/or putative resistance genes as well as genes for helper phage virion assembly alteration. In portion of helper phage particles, noticeable changes in capsid morphology were observed. Our findings confirm the important role of transducing phages in the evolution of S. epidermidis strains and outline host mechanisms of phage infection disruption. This work was supported by Grant of Czech Science Foundation (GA18-13064S) and Development Fund of Masaryk University MUNI/FR/1181/2018.

Staphylococcus epidermidis is a bacterium capable of causing diseases and a potential source of antimicrobial resistance. This resistance can be transferred horizontally among bacteria, mostly by bacteriophages. Only few phages have been characterized in detail so far, despite their ability to provide bacteria with these interesting properties. Our work characterizes S. epidermidis phages that were not described previously and demonstrates their ability to transfer mobile genetic elements including antimicrobial resistance genes carrying plasmids. We selected four S. epidermidis phages ?27, ?48, ?456 and ?459 previously used for phage-typing and characterized them in detail. All phages have a narrow host range and according to electron microscopy, they belong to the family Siphoviridaeand have similar morphology. We performed transduction experiments with plasmids and confirmed the transduction potential of phage ?27, which transferred a streptomycin-resistance plasmid with high efficiency. We also detected transduction potential in phage ?48 which is naturally capable of transferring chromosomal island of its propagation strain. We confirmed the ability of this phage to package the island by PCR, detected small transducing particles by electron microscopy, and transferred this island to recipient strains. The phage was also demonstrated to transfer other chromosomal islands. Our findings support the role of S. epidermidis phages in the spread of both antimicrobial resistance and virulence genes. This work was supported by Grant of Czech Science Foundation (GA18-13064S) and Development Fund of Masaryk University MUNI/FR/1181/2018.

Bacteriophages of the significant veterinary pathogen Staphylococcus pseudintermedius are rarely described morphologically and genomically in detail, and mostly include phages of the Siphoviridae family. There is currently no taxonomical classification for phages of this bacterial species. Here we describe a new phage designated vB_SpsS_QT1, which is related to phage 2638A originally described as a Staphylococcus aureus phage. Propagating strain S. aureus 2854 of the latter was reclassified by rpoB gene sequencing as S. pseudintermedius 2854 in this work. Both phages have a narrow but different host range determined on 54 strains. Morphologically, both of them belong to the family Siphoviridae, share the B1 morphotype, and differ from other staphylococcal phage genera by a single long fibre at the terminus of the tail. The complete genome of phage vB_SpsS_QT1 was sequenced with the IonTorrent platform and expertly annotated. Its linear genome with cohesive ends is 43,029 bp long and encodes 60 predicted genes with the typical modular structure of staphylococcal siphophages. A global alignment found the genomes of vB_SpsS_QT1 and 2638A to share 84% nucleotide identity, but they have no significant similarity of nucleotide sequences with other phage genomes available in public databases. Based on the morphological, phylogenetic, and genomic analyses, a novel genus Fibralongavirus in the family Siphoviridae is described with phage species vB_SpsS_QT1 and 2638A.

Staphylococcus petrasii is recently described coagulase negative staphylococcal species and an opportunistic human pathogen, still often misidentified in clinical specimens. Four subspecies are distinguished in S. petrasii by polyphasic taxonomical analyses, however a comparative study has still not been done on the majority of isolates and their genome properties have not yet been thoroughly analysed. Here, we describe the phenotypic and genotypic characteristics of 65 isolates and the results of de novo sequencing, whole genome assembly and annotation of draft genomes of five strains. The strains were identified by MALDI-TOF mass spectrometry to the species level and the majority of the strains were identified to the subspecies level by fingerprinting methods, (GTG)5 repetitive PCR and ribotyping. Macrorestriction profiling by pulsed-field gel electrophoresis was confirmed to be a suitable strain typing method. Comparative genomics revealed the presence of new mobile genetic elements carrying antimicrobial resistance factors such as staphylococcal cassette chromosome (SCC) mec, transposones, phage-inducible genomic islands, and plasmids. Their mosaic structure and similarity across coagulase-negative staphylococci and Staphylococcus aureus suggest the possible exchange of these elements. Numerous putative virulence factors such as adhesins, autolysins, exoenzymes, capsule formation genes, immunomodulators, the phage-associated sasX gene, and SCC-associated spermidine N-acetyltransferase gene, pseudouridine and sorbitol utilization operons might explain clinical manifestations of S. petrasii isolates. The increasing recovery of S. petrasii isolates from human clinical material, the multi-drug resistance including methicillin resistance of S. petrasii subsp. jettensis strains, and virulence factors homologous to other pathogenic staphylococci demonstrate the importance of the species in human disease.

Lytic bacteriophages are used as alternative therapeutics against bacterial infections. To overcome phage resistance of some pathogens, new phages are isolated from the environment or using phage training. Another approach, based on isolation of rare single plaques of spontaneous mutants grown on phage resistant strains of S. aureus, was used to obtain five kayvirus 812 mutants. The host range of this set of mutants was 42% higher than the wild type, determined on 186 MRSA strains. Genomes of the phage mutants were investigated by deep sequencing. It revealed that some polymorphisms from the parental phage population were fixed in next-step mutants, mostly in genes for tail and baseplate components associated with host range. Genome of the most effective multi-step mutant 812h1 was highly variable due to polymorphisms associated with rearrangements between repeated sequences resulted in alterations in host-takeover and terminal genomic regions. Some regions of 812h1 genome were distinct from the wild-type phage but the corresponding sequences were found in its sequencing data with very low coverage. These results suggest that a large pool of phage sequences is maintained in minor genome variants persisting in Kayvirus populations. The high prevalence of polymorphisms in phage genomes indicates a high mutation rate that results in rapid diversification of the phage populations and enables us to isolate phage mutants effective against broad range of staphylococcal strains. This work was supported by grants from the Czech Science Foundation (18-13064S).

Panton-Valentine leucocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) strains cause life-threatening diseases. We present a draft genome sequence of PVL-positive MRSA sequence type 154 (ST154) strain NRL 08/001, isolated from a fatal case of necrotizing pneumonia. The genome consists of 2.9 Mb over 39 contigs and harbors novel composite island staphylococcal cassette chromosome mec element (SCCmec)-mercury composite type 2B&5.

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.

The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T), Macrococcus goetzii sp. nov. (type strain CCM 4927T = DSM 103683T), Macrococcus epidermidis sp. nov. (type strain CCM 7099T = DSM 103681T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100T = DSM 103680T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.

Coagulase-negative staphylococci (CoNS) are opportunistic pathogens and an important cause of both human and animal diseases. Treatment of these diseases is complicated by their common antimicrobial resistance which is caused by overuse of antibiotics mainly in the veterinary environment. Livestock strains are therefore assumed to serve as a reservoir of resistance genes often located on mobile genetic elements such as plasmids. In this study, we have characterised resistance plasmids in 62 strains belonging to 10 CoNS species of human and veterinary origin. Altogether we analyzed 12 S. petrasii strains, 6 S. sciuri, 8 S. haemolyticus, 11 S. epidermidis, 5 S. warneri, 6 S. chromogenes, 6 S. xylosus, 5 S. equorum, 2 S. simulans and 1 S. caprae and compared their plasmid content and resistance determinants. In 48 analysed strains (77 %), 107 different plasmids ranging in size between 1.3 and 38.5 kb were detected and their number in each strain varied between 1 and 6. In total 7 different antimicrobial resistance genes carried by plasmids were detected by PCR. In addition, identical plasmids found in human and veterinary strains indicate the possibility of their natural horizontal transfer and emphasize importance of CoNS as a source of resistance determinants. Acknowledgements: This study was supported by a grant of Ministry of Agriculture of the Czech Republic No. QJ1510216 and Masaryk University specific research grant No. MUNI/A/0824/2017.

Background: Exfoliative toxins A (ETA) and B (ETB), the major cause of staphylococcal epidermolytic diseases, are encoded by tempered bacteriophages and plasmids playing a crucial role in pathogenic strains origin. Objectives: Characterization and comparative genomic analysis of previously undescribed eta-positive bacteriophages (ETA-phages) and etb-positive plasmids (ETB-plasmids) isolated from clinical strains. Methods: Bioinformatics, lysogenization, PCR, PFGE, SDS-PAGE, LC-MS/MS. Results: The phages studied represent novel types of phages belonging to Siphoviridae family. Comparison with already described phages revealed, that their genomes contain sequences gained by recombination, and show numerous differences that manifested in virion structural proteins. Using the phages, we accomplished positive lysogenic conversion of non-toxigenic S. aureus strain to highly effective producer of ETA. ETB-plasmids isolated from strains of clonal complex CC121 share a large part of DNA sequence including typical virulence genes. On the other hand, they contain variable regions influencing bacterial pathogenicity. A novel ETB-plasmid showing minimal sequence homology with others was described. It represents a novel lineage of ETB-plasmids not associated with CC121 strains, and carrying conjugation genes as well as new variants of virulence genes. A multiplex PCR assay has been designed to distinguish all the types of ETB-plasmids described. Conclusions. This study presents a complex analysis of novel exfoliative toxin-encoding elements of Staphylococcus aureus with a crucial impact on pathogenicity of clinical strains. This work was supported by Grant from Ministry of Agriculture of the Czech Republic (QJ1510216), AdmireVet CZ.1.05/2.1.00/01.0006–ED0006/01/01, and from Masaryk University MUNI/A/0824/2017.

The phage therapy, an alternative to fight against methicillin-resistant Staphylococcus aureus (MRSA) strains, requires precise genome evaluation of used phages to ensure its efficiency and safety. Bacteriophages of the genus Kayvirus are commonly used in phage preparations. However, it is necessary to adapt the phage composition to be effective against emerging phage-resistant strains. The evolutionary “training” under laboratory conditions was used to obtain phages with extended host range. We focused on phage 812, characterized by a high lytic activity and a broad host range towards staphylococcal strains. Five phage 812-derived mutants were selected on strains insusceptible to the wild type. The lytic ability of the phages was tested on the set of 94 human and 92 veterinary MRSA strains. The mutant 812h1 showed the highest lytic activity when lysing 82 % of human and 97 % of veterinary strains. The causes of the phage host-range changes were investigated by comparative genomic analysis of the phage mutants. We found interspersed and tandem direct repeats of various motifs differing in copy number and genome location that served as hot-spots for rearrangements creating multiple genomic variants. Mutations specific for each host-range mutant were characterized. Genes for nucleic acid metabolism, tail tube protein and endolysin were affected. Genome of 812h1 contains regions gained from other strains of Kayvirus genus suggesting possible recombination events. Although the selection strains carried prophages and plasmids, no studied mutant contained genes of such origin as well as genes for antibiotic resistance or bacterial virulence. The results of our study demonstrate, that genomic changes observed in spontaneous host-range mutants do not possess any risk for the therapeutic use. This work was supported by Grant of Czech Ministry of Agriculture (QJ1510216), and Czech Science Foundation (GA18-13064S).

This perspective paper follows up on earlier communications on bacteriophage therapy that we wrote as a multidisciplinary and intercontinental expert-panel when we first met at a bacteriophage conference hosted by the Eliava Institute in Tbilisi, Georgia in 2015. In the context of a society that is confronted with an ever-increasing number of antibiotic-resistant bacteria, we build on the previously made recommendations and specifically address how the Nagoya Protocol might impact the further development of bacteriophage therapy. By reviewing a number of recently conducted case studies with bacteriophages involving patients with bacterial infections that could no longer be successfully treated by regular antibiotic therapy, we again stress the urgency and significance of the development of international guidelines and frameworks that might facilitate the legal and effective application of bacteriophage therapy by physicians and the receiving patients. Additionally, we list and comment on several recently started and ongoing clinical studies, including highly desired double-blind placebo-controlled randomized clinical trials. We conclude with an outlook on how recently developed DNA editing technologies are expected to further control and enhance the efficient application of bacteriophages.