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 95/376T = CCM 8659T = DSM 111350T) 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.IMPORTANCEThe 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.
- Klíčová slova
- Gram-positive cocci, cephalosporin resistance, cold temperature tolerance, conjugation, food safety, linear plasmid, methicillin resistance,
- MeSH
- antibakteriální látky * farmakologie MeSH
- bakteriální proteiny * genetika metabolismus MeSH
- beta-laktamová rezistence * genetika MeSH
- dobytek mikrobiologie MeSH
- Enterococcaceae * genetika izolace a purifikace účinky léků MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- lidé MeSH
- plazmidy MeSH
- přenos genů horizontální MeSH
- sekvenování celého genomu MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus * genetika účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky * MeSH
- bakteriální proteiny * MeSH
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.
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
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.
- Klíčová slova
- Kayvirus, RNA-Seq, Staphylococcus aureus, Staphylococcus phages, bacteriophage therapy, noncoding RNA, phage-host interactions, prophages, transcriptome, viral transcription,
- MeSH
- lidé MeSH
- profágy genetika MeSH
- stafylokokové bakteriofágy genetika MeSH
- stafylokokové infekce * mikrobiologie terapie MeSH
- Staphylococcus aureus * MeSH
- transkriptom MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Klíčová slova
- Hyicus-Intermedius species group, Staphylococcus, genomic island, laboratory rat, taxonomy, variable genetic element, whole genome sequencing,
- Publikační typ
- časopisecké články MeSH
The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.
- MeSH
- bakteriofágy * MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- povrchová plasmonová rezonance MeSH
- stafylokokové infekce * MeSH
- Staphylococcus aureus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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 10-4 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.
- Klíčová slova
- Staphylococcus epidermidis, antibiotic resistance, bacteriophages, horizontal gene transfer, pathogenicity islands, transduction,
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- genomové ostrovy genetika MeSH
- lidé MeSH
- plazmidy genetika MeSH
- stafylokokové bakteriofágy klasifikace účinky léků genetika MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus epidermidis účinky léků virologie MeSH
- transdukce genetická * MeSH
- virulence MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
Bacteria of the genus Massilia often colonize extreme ecosystems, however, a detailed study of the massilias from the Antarctic environment has not yet been performed. Here, sixty-four Gram-stain-negative, aerobic, motile rods isolated from different environmental samples on James Ross Island (Antarctica) were subjected to a polyphasic taxonomic study. The psychrophilic isolates exhibited slowly growing, moderately slimy colonies revealing bold pink-red pigmentation on R2A agar. The set of strains exhibited the highest 16S rRNA gene sequence similarities (99.5-99.9%) to Massilia violaceinigra B2T and Massilia atriviolacea SODT and formed several phylogenetic groups based on the analysis of gyrB and lepA genes. Phenotypic characteristics allowed four of them to be distinguished from each other and from their closest relatives. Compared to the nearest phylogenetic neighbours the set of six genome-sequenced representatives exhibited considerable phylogenetic distance at the whole-genome level. Bioinformatic analysis of the genomic sequences revealed a high number of putative genes involved in oxidative stress response, heavy-metal resistance, bacteriocin production, the presence of putative genes involved in nitrogen metabolism and auxin biosynthesis. The identification of putative genes encoding aromatic dioxygenases suggests the biotechnology potential of the strains. Based on these results four novel species and one genomospecies of the genus Massilia are described and named Massilia rubra sp. nov. (P3094T=CCM 8692T=LMG 31213T), Massilia aquatica sp. nov. (P3165T=CCM 8693T=LMG 31211T), Massilia mucilaginosa sp. nov. (P5902T=CCM 8733T=LMG 31210T), and Massilia frigida sp. nov. (P5534T=CCM 8695T=LMG 31212T).
- Klíčová slova
- Antarctica, Massilia, Oxalobacteraceae, Polyphasic taxonomy, Psychrophilic bacteria, Whole-genome sequence,
- MeSH
- bakteriální geny MeSH
- DNA bakterií genetika MeSH
- fenotyp MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- geny rRNA MeSH
- geologické sedimenty mikrobiologie MeSH
- jezera mikrobiologie MeSH
- Oxalobacteraceae klasifikace genetika izolace a purifikace fyziologie MeSH
- řeky mikrobiologie MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Antarktida MeSH
- Názvy látek
- DNA bakterií MeSH
- RNA ribozomální 16S MeSH
A taxonomic study was carried out on four Gram-stain-negative strains P5773T, P6169, P4708 and P6245, isolated from anus or mouth samples of Weddell seals at James Ross Island, Antarctica. The results of initial 16S rRNA gene sequence analysis showed that all four strains formed a group placed in the genus Pseudomonas and found Pseudomonas guineae and Pseudomonas peli to be their closest neighbours with 99.9 and 99.2 % sequence similarity, respectively. Sequence analysis of rpoD, rpoB and gyrB housekeeping genes confirmed the highest similarity of isolates to P. peli (rpoD) and to P. guineae (rpoB and gyrB). The average nucleotide identity value below 86 %, as calculated from the whole-genome sequence data, showed the low genomic relatedness of P5773T to its phylogenetic neighbours. The complete genome of strain P5773T was 4.4 Mb long and contained genes encoding proteins with biotechnological potential. The major fatty acids of the seal isolates were summed feature 8 (C18 : 1 ω7c), summed feature 3 (C16 : 1 ω 7 c/C16 : 1 ω6c) and C16:0. The major respiratory quinone was Q9. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Putrescine and spermidine are predominant in the polyamine pattern. Further characterization performed using repetitive sequence-based PCR fingerprinting and MALDI-TOF MS analysis showed that the studied isolates formed a coherent cluster separated from the remaining Pseudomonas species and confirmed that they represent a novel species within the genus Pseudomonas, for which the name Pseudomonas leptonychotis sp. nov. is suggested. The type strain is P5773T (=CCM 8849T=LMG 30618T).
- Klíčová slova
- Antarctica, Pseudomonas leptonychotis, Weddell seal, polyphasic taxonomy,
- MeSH
- bakteriální geny MeSH
- DNA bakterií genetika MeSH
- fosfolipidy chemie MeSH
- fylogeneze * MeSH
- mastné kyseliny chemie MeSH
- Pseudomonas klasifikace MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- tuleňovití mikrobiologie MeSH
- ubichinon chemie MeSH
- zastoupení bazí MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Antarktida MeSH
- Názvy látek
- DNA bakterií MeSH
- fosfolipidy MeSH
- mastné kyseliny MeSH
- RNA ribozomální 16S MeSH
- ubichinon MeSH
- ubiquinone 9 MeSH Prohlížeč
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.
- Klíčová slova
- Coagulase-negative staphylococci, MALDI-TOF MS, Methicillin resistance, Mobile genetic elements, Molecular subtyping, Virulence factors,
- MeSH
- faktory virulence genetika MeSH
- fenotyp MeSH
- genom bakteriální * MeSH
- genomika MeSH
- genotyp MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- pulzní gelová elektroforéza MeSH
- ribotypizace MeSH
- rozptýlené repetitivní sekvence * MeSH
- Staphylococcus klasifikace genetika patogenita MeSH
- techniky typizace bakterií MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- faktory virulence MeSH
