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.

• Klíčová slova
• cassette chromosome recombinase, coagulase-negative staphylococci, comparative genomics, genomic islands, gram-positive pathogens, mobile genetic elements, phylogenetic analysis, polyphasic taxonomy,
• Publikační typ
• časopisecké články 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 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.

• Klíčová slova
• CRISPR-Cas, Gram-positive pathogens, Macrococcus, Staphylococcal Cassette Chromosome (SCC), bacteriophage, methicillin resistance, plasmids, prokaryotic transformation,
• Publikační typ
• časopisecké články MeSH

Two Gram-stain-positive, coagulase-negative staphylococcal strains were isolated from abiotic sources comprising stone fragments and sandy soil in James Ross Island, Antarctica. Here, we describe properties of a novel species of the genus Staphylococcus that has a 16S rRNA gene sequence nearly identical to that of Staphylococcus saprophyticus However, compared to S. saprophyticus and the next closest relatives, the new species demonstrates considerable phylogenetic distance at the whole-genome level, with an average nucleotide identity of <85% and inferred DNA-DNA hybridization of <30%. It forms a separate branch in the S. saprophyticus phylogenetic clade as confirmed by multilocus sequence analysis of six housekeeping genes, rpoB, hsp60, tuf, dnaJ, gap, and sod Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and key biochemical characteristics allowed these bacteria to be distinguished from their nearest phylogenetic neighbors. In contrast to S. saprophyticus subsp. saprophyticus, the novel strains are pyrrolidonyl arylamidase and β-glucuronidase positive and β-galactosidase negative, nitrate is reduced, and acid produced aerobically from d-mannose. Whole-genome sequencing of the 2.69-Mb large chromosome revealed the presence of a number of mobile genetic elements, including the 27-kb pseudo-staphylococcus cassette chromosome mec of strain P5085T (ψSCCmecP5085), harboring the mecC gene, two composite phage-inducible chromosomal islands probably essential to adaptation to extreme environments, and one complete and one defective prophage. Both strains are resistant to penicillin G, ampicillin, ceftazidime, methicillin, cefoxitin, and fosfomycin. We hypothesize that antibiotic resistance might represent an evolutionary advantage against beta-lactam producers, which are common in a polar environment. Based on these results, a novel species of the genus Staphylococcus is described and named Staphylococcus edaphicus sp. nov. The type strain is P5085T (= CCM 8730T = DSM 104441T).IMPORTANCE The description of Staphylococcus edaphicus sp. nov. enables the comparison of multidrug-resistant staphylococci from human and veterinary sources evolved in the globalized world to their geographically distant relative from the extreme Antarctic environment. Although this new species was not exposed to the pressure of antibiotic treatment in human or veterinary practice, mobile genetic elements carrying antimicrobial resistance genes were found in the genome. The genomic characteristics presented here elucidate the evolutionary relationships in the Staphylococcus genus with a special focus on antimicrobial resistance, pathogenicity, and survival traits. Genes encoded on mobile genetic elements were arranged in unique combinations but retained conserved locations for the integration of mobile genetic elements. These findings point to enormous plasticity of the staphylococcal pangenome, shaped by horizontal gene transfer. Thus, S. edaphicus can act not only as a reservoir of antibiotic resistance in a natural environment but also as a mediator for the spread and evolution of resistance genes.

• Klíčová slova
• beta-lactams, chromosomal islands, coagulase-negative staphylococci, drug resistance evolution, genomics, methicillin resistance, mobile genetic elements, pathogenicity islands, penicillin-binding proteins, phylogenetic analysis, polyphasic taxonomy,
• MeSH
• bakteriální geny fyziologie   MeSH
• biologická adaptace genetika   MeSH
• extrémně chladné počasí * MeSH
• extrémní prostředí * MeSH
• genomové ostrovy fyziologie   MeSH
• Staphylococcus klasifikace   genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Antarktida MeSH

Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.

• MeSH
• agar MeSH
• analýza hlavních komponent MeSH
• časové faktory MeSH
• diagnostické testy rutinní MeSH
• fluorescence MeSH
• odběr biologického vzorku MeSH
• Ramanova spektroskopie metody   MeSH
• Staphylococcus izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• agar MeSH