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.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Experimental Biology Czech Collection of Microorganisms Faculty of Science Masaryk University Brno Czech Republic

Department of Experimental Biology Division of Genetics and Molecular Biology Faculty of Science Masaryk University Brno Czech Republic

Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures Braunschweig Germany

Reference Laboratory for Staphylococci National Institute of Public Health Praha Czech Republic

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Heilmann C, Ziebuhr W, Becker K. 2019. Are coagulase-negative staphylococci virulent? Clin Microbiol Infect 25:1071–1080. doi:10.1016/j.cmi.2018.11.012 PubMed DOI

Marincola G, Liong O, Schoen C, Abouelfetouh A, Hamdy A, Wencker FDR, Marciniak T, Becker K, Köck R, Ziebuhr W. 2021. Antimicrobial resistance profiles of coagulase-negative staphylococci in community-based healthy individuals in Germany. Front Public Health 9:684456. doi:10.3389/fpubh.2021.684456 PubMed DOI PMC

Takeuchi F, Watanabe S, Baba T, Yuzawa H, Ito T, Morimoto Y, Kuroda M, Cui L, Takahashi M, Ankai A, Baba S, Fukui S, Lee JC, Hiramatsu K. 2005. Whole-genome sequencing of Staphylococcus haemolyticus uncovers the extreme plasticity of its genome and the evolution of human-colonizing staphylococcal species. J Bacteriol 187:7292–7308. doi:10.1128/JB.187.21.7292-7308.2005 PubMed DOI PMC

Chaudhry V, Patil PB. 2020. Evolutionary insights into adaptation of Staphylococcus haemolyticus to human and non-human niches. Genomics 112:2052–2062. doi:10.1016/j.ygeno.2019.11.018 PubMed DOI

Pain M, Wolden R, Jaén-Luchoro D, Salvà-Serra F, Iglesias BP, Karlsson R, Klingenberg C, Cavanagh JP. 2020. Staphylococcus borealis sp. nov., isolated from human skin and blood. Int J Syst Evol Microbiol 70:6067–6078. doi:10.1099/ijsem.0.004499 PubMed DOI

Lin Y-T, Hung W-C, Wan T-W, Li H, Lee T-F, Hsueh P-R, Teng L-J. 2022. Staphylococcus taiwanensis sp. nov., isolated from human blood. Int J Syst Evol Microbiol 72. doi:10.1099/ijsem.0.005262 PubMed DOI

Pantůček R, Švec P, Dajcs JJ, Machová I, Černohlávková J, Šedo O, Gelbíčová T, Mašlaňová I, Doškař J, Zdráhal Z, Růžičková V, Sedláček I. 2013. Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections. Syst Appl Microbiol 36:90–95. doi:10.1016/j.syapm.2012.11.004 PubMed DOI

Švec P, De Bel A, Sedláček I, Petráš P, Gelbíčová T, Černohlávková J, Mašlaňová I, Cnockaert M, Varbanovová I, Echahidi F, Vandamme P, Pantůček R. 2015. Staphylococcus petrasii subsp. pragensis subsp. nov., occurring in human clinical material. Int J Syst Evol Microbiol 65:2071–2077. doi:10.1099/ijs.0.000220 PubMed DOI

Madhaiyan M, Wirth JS, Saravanan VS. 2020. Phylogenomic analyses of the Staphylococcaceae family suggest the reclassification of five species within the genus Staphylococcus as heterotypic synonyms, the promotion of five subspecies to novel species, the taxonomic reassignment of five Staphylococcus species to Mammaliicoccus gen. nov., and the formal assignment of Nosocomiicoccus to the family Staphylococcaceae. Int J Syst Evol Microbiol 70:5926–5936. doi:10.1099/ijsem.0.004498 PubMed DOI

Vrbovská V, Kovařovic V, Mašlaňová I, Indráková A, Petráš P, Šedo O, Švec P, Fišarová L, Šiborová M, Mikulášek K, Sedláček I, Doškař J, Pantůček R. 2019. Staphylococcus petrasii diagnostics and its pathogenic potential enhanced by mobile genetic elements. Int J Med Microbiol 309:151355. doi:10.1016/j.ijmm.2019.151355 PubMed DOI

Uehara Y. 2022. current status of staphylococcal cassette chromosome mec (SCCmec). Antibiotics 11:86. doi:10.3390/antibiotics11010086 PubMed DOI PMC

Qin L, McCausland JW, Cheung GYC, Otto M. 2016. PSM-Mec-A virulence determinant that connects transcriptional regulation, virulence, and antibiotic resistance in staphylococci. Front Microbiol 7:1293. doi:10.3389/fmicb.2016.01293 PubMed DOI PMC

Bleiziffer I, Eikmeier J, Pohlentz G, McAulay K, Xia G, Hussain M, Peschel A, Foster S, Peters G, Heilmann C. 2017. The plasmin-sensitive protein pls in methicillin-resistant Staphylococcus aureus (MRSA) is a glycoprotein. PLoS Pathog 13:e1006110. doi:10.1371/journal.ppat.1006110 PubMed DOI PMC

Chen H-J, Lin Y-T, Hung W-C, Tsai J-C, Hsueh P-R, Teng L-J. 2016. Distribution of staphylococcal cassette chromosome (SCC) mec element types in fusidic acid-resistant Staphylococcus epidermidis and identification of a novel SCC7684 element. Antimicrob Agents Chemother 60:5006–5009. doi:10.1128/AAC.00231-16 PubMed DOI PMC

Tsubakishita S, Kuwahara-Arai K, Sasaki T, Hiramatsu K. 2010. Origin and molecular evolution of the determinant of methicillin resistance in staphylococci. Antimicrob Agents Chemother 54:4352–4359. doi:10.1128/AAC.00356-10 PubMed DOI PMC

Scharn CR, Tenover FC, Goering RV. 2013. Transduction of staphylococcal cassette chromosome mec elements between strains of Staphylococcus aureus. Antimicrob Agents Chemother 57:5233–5238. doi:10.1128/AAC.01058-13 PubMed DOI PMC

Ray MD, Boundy S, Archer GL. 2016. Transfer of the methicillin resistance genomic island among staphylococci by conjugation. Mol Microbiol 100:675–685. doi:10.1111/mmi.13340 PubMed DOI PMC

Maree M, Thi Nguyen LT, Ohniwa RL, Higashide M, Msadek T, Morikawa K. 2022. Natural transformation allows transfer of SCCmec-mediated methicillin resistance in Staphylococcus aureus biofilms. Nat Commun 13:2477. doi:10.1038/s41467-022-29877-2 PubMed DOI PMC

Boundy S, Safo MK, Wang L, Musayev FN, O’Farrell HC, Rife JP, Archer GL. 2013. Characterization of the Staphylococcus aureus rRNA methyltransferase encoded by orfX, the gene containing the staphylococcal chromosome cassette mec (SCCmec) insertion site. J Biol Chem 288:132–140. doi:10.1074/jbc.M112.385138 PubMed DOI PMC

Misiura A, Pigli YZ, Boyle-Vavra S, Daum RS, Boocock MR, Rice PA. 2013. Roles of two large serine recombinases in mobilizing the methicillin-resistance cassette SCCmec. Mol Microbiol 88:1218–1229. doi:10.1111/mmi.12253 PubMed DOI PMC

Ito T, Ma XX, Takeuchi F, Okuma K, Yuzawa H, Hiramatsu K. 2004. Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob Agents Chemother 48:2637–2651. doi:10.1128/AAC.48.7.2637-2651.2004 PubMed DOI PMC

Takahashi T, Satoh I, Kikuchi N. 1999. Phylogenetic relationships of 38 taxa of the genus Staphylococcus based on 16S rRNA gene sequence analysis. Int J Syst Bacteriol 49:725–728. doi:10.1099/00207713-49-2-725 PubMed DOI

Pantůček R, Sedláček I, Indráková A, Vrbovská V, Mašlaňová I, Kovařovic V, Švec P, Králová S, Krištofová L, Kekláková J, Petráš P, Doškař J. 2018. Staphylococcus edaphicus sp. nov., isolated in Antarctica, harbors the mecC gene and genomic islands with a suspected role in adaptation to extreme environments. Appl Environ Microbiol 84:e01746-17. doi:10.1128/AEM.01746-17 PubMed DOI PMC

Kim M, Oh HS, Park SC, Chun J. 2014. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64:346–351. doi:10.1099/ijs.0.059774-0 PubMed DOI

Vrbovská V, Sedláček I, Zeman M, Švec P, Kovařovic V, Šedo O, Laichmanová M, Doškař J, Pantůček R. 2020. Characterization of Staphylococcus intermedius group isolates associated with animals from Antarctica and emended description of Staphylococcus delphini. Microorganisms 8:204. doi:10.3390/microorganisms8020204 PubMed DOI PMC

Sabat AJ, Hermelijn SM, Akkerboom V, Juliana A, Degener JE, Grundmann H, Friedrich AW. 2017. Complete-genome sequencing elucidates outbreak dynamics of CA-MRSA USA300 (ST8-spa t008) in an academic hospital of Paramaribo, Republic of Suriname. Sci Rep 7:41050. doi:10.1038/srep41050 PubMed DOI PMC

Pumirat P, Boonyuen U, Vanaporn M, Pinweha P, Tandhavanant S, Korbsrisate S, Chantratita N. 2014. The role of short-chain dehydrogenase/oxidoreductase, induced by salt stress, on host interaction of B. pseudomallei. BMC Microbiol 14:1. doi:10.1186/1471-2180-14-1 PubMed DOI PMC

North RA, Wahlgren WY, Remus DM, Scalise M, Kessans SA, Dunevall E, Claesson E, Soares da Costa TP, Perugini MA, Ramaswamy S, Allison JR, Indiveri C, Friemann R, Dobson RCJ. 2018. The sodium sialic acid symporter from Staphylococcus aureus has altered substrate specificity. Front Chem 6:233. doi:10.3389/fchem.2018.00233 PubMed DOI PMC

International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) . 2009. Classification of staphylococcal cassette chromosome mec (SCCmec): guidelines for reporting novel SCCmec elements. Antimicrob Agents Chemother 53:4961–4967. doi:10.1128/AAC.00579-09 PubMed DOI PMC

McClure JA, Conly JM, Zhang K. 2021. Characterizing a novel staphylococcal cassette chromosome mec with a composite structure from a clinical strain of Staphylococcus hominis, C34847. Antimicrob Agents Chemother 65:e00777-21. doi:10.1128/AAC.00777-21 PubMed DOI PMC

Lin Y-T, Tsai J-C, Chen H-J, Hung W-C, Hsueh P-R, Teng L-J. 2014. A novel staphylococcal cassette chromosomal element, SCCfusC, carrying fusC and speG in fusidic acid-resistant methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 58:1224–1227. doi:10.1128/AAC.01772-13 PubMed DOI PMC

Wilson LK, Coombs GW, Christiansen K, Grubb WB, O’Brien FG. 2016. Characterization of a novel staphylococcal cassette chromosome composite island from community-associated MRSA isolated in aged care facilities in Western Australia. J Antimicrob Chemother 71:3372–3375. doi:10.1093/jac/dkw317 PubMed DOI

Xiao J, Huang J, Xue X, Wang C, Sun Y, Zheng L, Zhao X, Wang X, Zhao X, Xue H. 2023. Novel cassette chromosome recombinases CcrA8B9 catalyse the excision and integration of the staphylococcal cassette chromosome mec element. J Antimicrob Chemother 78:440–444. doi:10.1093/jac/dkac410 PubMed DOI

Vasu K, Nagaraja V. 2013. Diverse functions of restriction-modification systems in addition to cellular defense. Microbiol Mol Biol Rev 77:53–72. doi:10.1128/MMBR.00044-12 PubMed DOI PMC

Chen L, Hu JX, Liu C, Liu J, Ma ZB, Tang ZY, Li YF, Zeng ZL. 2021. Identification of the multiresistance gene poxtA in oxazolidinone-susceptible Staphylococcus haemolyticus and Staphylococcus saprophyticus of pig and feed origins. Pathogens 10:601. doi:10.3390/pathogens10050601 PubMed DOI PMC

Berbel Caban A, Pak TR, Obla A, Dupper AC, Chacko KI, Fox L, Mills A, Ciferri B, Oussenko I, Beckford C, Chung M, Sebra R, Smith M, Conolly S, Patel G, Kasarskis A, Sullivan MJ, Altman DR, van Bakel H. 2020. PathoSPOT genomic epidemiology reveals under-the-radar nosocomial outbreaks. Genome Med 12:96. doi:10.1186/s13073-020-00798-3 PubMed DOI PMC

Ziebuhr W, Hennig S, Eckart M, Kränzler H, Batzilla C, Kozitskaya S. 2006. Nosocomial infections by Staphylococcus epidermidis: how a commensal bacterium turns into a pathogen. Int J Antimicrob Agents 28 Suppl 1:14–20. doi:10.1016/j.ijantimicag.2006.05.012 PubMed DOI

Foster TJ. 2019. The MSCRAMM family of cell-wall-anchored surface proteins of Gram-positive cocci. Trends Microbiol 27:927–941. doi:10.1016/j.tim.2019.06.007 PubMed DOI

Lindsay JA. 2019. Staphylococci: evolving genomes. Microbiol Spectr 7:GPP3-0071. doi:10.1128/microbiolspec.GPP3-0071-2019 PubMed DOI PMC

Everitt RG, Didelot X, Batty EM, Miller RR, Knox K, Young BC, Bowden R, Auton A, Votintseva A, Larner-Svensson H, Charlesworth J, Golubchik T, Ip CLC, Godwin H, Fung R, Peto TEA, Walker AS, Crook DW, Wilson DJ. 2014. Mobile elements drive recombination hotspots in the core genome of Staphylococcus aureus. Nat Commun 5:3956. doi:10.1038/ncomms4956 PubMed DOI PMC

Firth N, Jensen SO, Kwong SM, Skurray RA, Ramsay JP, Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, Braunstein M, Rood JI. 2018. Staphylococcal Plasmids, Transposable and integrative elements. Microbiol Spectr 6:GPP3-0030. doi:10.1128/microbiolspec.GPP3-0030-2018 PubMed DOI PMC

Shearer JES, Wireman J, Hostetler J, Forberger H, Borman J, Gill J, Sanchez S, Mankin A, Lamarre J, Lindsay JA, Bayles K, Nicholson A, O’Brien F, Jensen SO, Firth N, Skurray RA, Summers AO. 2011. Major families of multiresistant plasmids from geographically and epidemiologically diverse staphylococci. G3 (Bethesda) 1:581–591. doi:10.1534/g3.111.000760 PubMed DOI PMC

Caryl JA, Smith MCA, Thomas CD. 2004. Reconstitution of a staphylococcal plasmid-protein relaxation complex in vitro. J Bacteriol 186:3374–3383. doi:10.1128/JB.186.11.3374-3383.2004 PubMed DOI PMC

Kwong SM, Ramsay JP, Jensen SO, Firth N. 2017. Replication of staphylococcal resistance plasmids. Front Microbiol 8:2279. doi:10.3389/fmicb.2017.02279 PubMed DOI PMC

Bender J, Strommenger B, Steglich M, Zimmermann O, Fenner I, Lensing C, Dagwadordsch U, Kekulé AS, Werner G, Layer F. 2015. Linezolid resistance in clinical isolates of Staphylococcus epidermidis from German hospitals and characterization of two cfr-carrying plasmids. J Antimicrob Chemother 70:1630–1638. doi:10.1093/jac/dkv025 PubMed DOI

Pain M, Hjerde E, Klingenberg C, Cavanagh JP. 2019. Comparative genomic analysis of Staphylococcus haemolyticus reveals key to hospital adaptation and pathogenicity. Front Microbiol 10:2096. doi:10.3389/fmicb.2019.02096 PubMed DOI PMC

Saroha T, Chaudhry V, Patil PB. 2022. Novel insights into the role of the mobilome in ecological diversification and success of Staphylococcus haemolyticus as an opportunistic pathogen. Microb Genom 8:000755. doi:10.1099/mgen.0.000755 PubMed DOI PMC

Hiramatsu K, Ito T, Tsubakishita S, Sasaki T, Takeuchi F, Morimoto Y, Katayama Y, Matsuo M, Kuwahara-Arai K, Hishinuma T, Baba T. 2013. Genomic basis for methicillin resistance in Staphylococcus aureus. Infect Chemother 45:117–136. doi:10.3947/ic.2013.45.2.117 PubMed DOI PMC

Mir-Sanchis I, Roman CA, Misiura A, Pigli YZ, Boyle-Vavra S, Rice PA. 2016. Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative. Nat Struct Mol Biol 23:891–898. doi:10.1038/nsmb.3286 PubMed DOI PMC

Sivertsen A, Janice J, Pedersen T, Wagner TM, Hegstad J, Hegstad K, Ellermeier CD. 2018. The Enterococcus cassette chromosome, a genomic variation enabler in enterococci. mSphere 3:e00402-18. doi:10.1128/mSphere.00402-18 PubMed DOI PMC

Bjørkeng EK, Tessema GT, Lundblad EW, Butaye P, Willems R, Sollid JE, Sundsfjord A, Hegstad K. 2010. ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium. Microbiol 156:3624–3634. doi:10.1099/mic.0.041491-0 PubMed DOI PMC

Schwendener S, Perreten V. 2022. The bla and mec families of beta-lactam resistance genes in the genera Macrococcus, Mammaliicoccus and Staphylococcus: an in-depth analysis with emphasis on Macrococcus. J Antimicrob Chemother 77:1796–1827. doi:10.1093/jac/dkac107 PubMed DOI

Bebel A, Walsh MA, Mir-Sanchis I, Rice PA. 2020. A novel DNA primase-helicase pair encoded by SCCmec elements. Elife 9:e55478. doi:10.7554/eLife.55478 PubMed DOI PMC

Zeman M, Mašlaňová I, Indráková A, Šiborová M, Mikulášek K, Bendíčková K, Plevka P, Vrbovská V, Zdráhal Z, Doškař J, Pantůček R. 2017. Staphylococcus sciuri bacteriophages double-convert for staphylokinase and phospholipase, mediate interspecies plasmid transduction, and package mecA gene. Sci Rep 7:46319. doi:10.1038/srep46319 PubMed DOI PMC

Chlebowicz MA, Mašlaňová I, Kuntová L, Grundmann H, Pantůček R, Doškař J, van Dijl JM, Buist G. 2014. The staphylococcal cassette chromosome mec type V from Staphylococcus aureus ST398 is packaged into bacteriophage capsids. Int J Med Microbiol 304:764–774. doi:10.1016/j.ijmm.2014.05.010 PubMed DOI

Rolo J, Worning P, Nielsen JB, Bowden R, Bouchami O, Damborg P, Guardabassi L, Perreten V, Tomasz A, Westh H, de Lencastre H, Miragaia M. 2017. Evolutionary origin of the Staphylococcal cassette Chromosome MEC (Sccmec). Antimicrob Agents Chemother 61:e02302–16. doi:10.1128/AAC.02302-16 PubMed DOI PMC

Novick RP, Ram G. 2017. Staphylococcal pathogenicity islands-movers and shakers in the genomic firmament. Curr Opin Microbiol 38:197–204. doi:10.1016/j.mib.2017.08.001 PubMed DOI PMC

Jiang N, Li J, Feßler AT, Wang Y, Schwarz S, Wu C. 2019. Novel pseudo-staphylococcal cassette chromosome mec element (phiSCCmecT55) in MRSA ST9. J Antimicrob Chemother 74:819–820. doi:10.1093/jac/dky457 PubMed DOI

Wendlandt S, Li B, Ma Z, Schwarz S. 2013. Complete sequence of the multi-resistance plasmid pV7037 from a porcine methicillin-resistant Staphylococcus aureus. Vet Microbiol 166:650–654. doi:10.1016/j.vetmic.2013.07.017 PubMed DOI

Kovařovic V, Sedláček I, Petráš P, Králová S, Mašlaňová I, Švec P, Neumann-Schaal M, Botka T, Gelbíčová T, Staňková E, Doškař J, Pantůček R. 2022. Staphylococcus ratti sp. nov. isolated from a lab rat. Pathogens 11:51. doi:10.3390/pathogens11010051 PubMed DOI PMC

Mašlaňová I, Wertheimer Z, Sedláček I, Švec P, Indráková A, Kovařovic V, Schumann P, Spröer C, Králová S, Šedo O, Krištofová L, Vrbovská V, Füzik T, Petráš P, Zdráhal Z, Ružičková V, Doškař J, Pantuček R. 2018. Description and comparative genomics of Macrococcus caseolyticus subsp. hominis subsp. nov., Macrococcus goetzii sp. nov., Macrococcus epidermidis sp. nov., and Macrococcus bohemicus sp. nov., novel macrococci from human clinical material with virulence potential and suspected uptake of foreign DNA by natural transformation. Front Microbiol 9:1178. doi:10.3389/fmicb.2018.01178 PubMed DOI PMC

Freeman DJ, Falkiner FR, Keane CT. 1989. New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol 42:872–874. doi:10.1136/jcp.42.8.872 PubMed DOI PMC

EUCAST . 2022. Breakpoint tables for interpretation of MICs and zone diameters, in press. The European Committee on Antimicrobial Susceptibility Testing, Version 12. Available from: http://www.eucast.org

Fišarová L, Botka T, Du X, Mašlaňová I, Bárdy P, Pantůček R, Benešík M, Roudnický P, Winstel V, Larsen J, Rosenstein R, Peschel A, Doškař J. 2021. Staphylococcus epidermidis phages transduce antimicrobial resistance plasmids and mobilize chromosomal islands. mSphere 6:e00223-21. doi:10.1128/mSphere.00223-21 PubMed DOI PMC

De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34:2666–2669. doi:10.1093/bioinformatics/bty149 PubMed DOI PMC

Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. doi:10.1371/journal.pcbi.1005595 PubMed DOI PMC

Wick RR, Judd LM, Cerdeira LT, Hawkey J, Méric G, Vezina B, Wyres KL, Holt KE. 2021. Trycycler: consensus long-read assemblies for bacterial genomes. Genome Biol 22:266. doi:10.1186/s13059-021-02483-z PubMed DOI PMC

Wick RR, Holt KE. 2022. Polypolish: short-read polishing of long-read bacterial genome assemblies. PLoS Comput Biol 18:e1009802. doi:10.1371/journal.pcbi.1009802 PubMed DOI PMC

Okonechnikov K, Golosova O, Fursov M, UGENE team . 2012. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics 28:1166–1167. doi:10.1093/bioinformatics/bts091 PubMed DOI

Li W, O’Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, Coulouris G, Chitsaz F, Derbyshire MK, Durkin AS, Gonzales NR, Gwadz M, Lanczycki CJ, Song JS, Thanki N, Wang J, Yamashita RA, Yang M, Zheng C, Marchler-Bauer A, Thibaud-Nissen F. 2021. RefSeq: expanding the prokaryotic genome annotation pipeline reach with protein family model curation. Nucleic Acids Res 49:D1020–D1028. doi:10.1093/nar/gkaa1105 PubMed DOI PMC

Madeira F, Pearce M, Tivey ARN, Basutkar P, Lee J, Edbali O, Madhusoodanan N, Kolesnikov A, Lopez R. 2022. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res 50:W276–W279. doi:10.1093/nar/gkac240 PubMed DOI PMC

Sullivan MJ, Petty NK, Beatson SA. 2011. Easyfig: a genome comparison visualizer. Bioinformatics 27:1009–1010. doi:10.1093/bioinformatics/btr039 PubMed DOI PMC

Bertelli C, Gray KL, Woods N, Lim AC, Tilley KE, Winsor GL, Hoad GR, Roudgar A, Spencer A, Peltier J, Warren D, Raphenya AR, McArthur AG, Brinkman FSL. 2022. Enabling genomic island prediction and comparison in multiple genomes to investigate bacterial evolution and outbreaks. Microb Genom 8:mgen000818. doi:10.1099/mgen.0.000818 PubMed DOI PMC

Edwards R, Decewicz P, Katelyn D, Laurasisk S. 2021. Linsalrob/Phispy: Dropped Prophages (v.4.2.19). Zenodo. doi:10.5281/zenodo.5070204 DOI

Ou H-Y, He X, Harrison EM, Kulasekara BR, Thani AB, Kadioglu A, Lory S, Hinton JCD, Barer MR, Deng Z, Rajakumar K. 2007. MobilomeFINDER: web-based tools for in silico and experimental discovery of bacterial genomic islands. Nucleic Acids Res 35:W97–W104. doi:10.1093/nar/gkm380 PubMed DOI PMC

Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O, Villa L, Møller Aarestrup F, Hasman H. 2014. In silico detection and typing of plasmids using plasmidfinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 58:3895–3903. doi:10.1128/AAC.02412-14 PubMed DOI PMC

Bertelli C, Laird MR, Williams KP, Simon Fraser University Research Computing Group, Lau BY, Hoad G, Winsor GL, Brinkman FSL. 2017. IslandViewer 4: expanded prediction of genomic islands for larger-scale datasets. Nucleic Acids Res 45:W30–W35. doi:10.1093/nar/gkx343 PubMed DOI PMC

Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. 2006. Isfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res 34:D32–6. doi:10.1093/nar/gkj014 PubMed DOI PMC

Kaya H, Hasman H, Larsen J, Stegger M, Johannesen TB, Allesøe RL, Lemvigh CK, Aarestrup FM, Lund O, Larsen AR, Limbago BM. 2018. Sccmecfinder, a web-based tool for typing of Staphylococcal cassette Chromosome MEC in Staphylococcus aureus using whole-genome sequence data. mSphere 3:e00612–17. doi:10.1128/mSphere.00612-17 PubMed DOI PMC

Russel J, Pinilla-Redondo R, Mayo-Muñoz D, Shah SA, Sørensen SJ. 2020. CRISPRCasTyper: automated identification, annotation, and classification of CRISPR-Cas loci. CRISPR J 3:462–469. doi:10.1089/crispr.2020.0059 PubMed DOI

Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM, Pereira S, Sharma AN, Doshi S, Courtot M, Lo R, Williams LE, Frye JG, Elsayegh T, Sardar D, Westman EL, Pawlowski AC, Johnson TA, Brinkman FSL, Wright GD, McArthur AG. 2017. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res 45:D566–D573. doi:10.1093/nar/gkw1004 PubMed DOI PMC

Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640–2644. doi:10.1093/jac/dks261 PubMed DOI PMC

Chen L, Zheng D, Liu B, Yang J, Jin Q. 2016. VFDB 2016: hierarchical and refined dataset for big data analysis-10 years on. Nucleic Acids Res 44:D694–D697. doi:10.1093/nar/gkv1239 PubMed DOI PMC

Pantůček R, Sedláček I, Petráš P, Koukalová D, Švec P, Štětina V, Vancanneyt M, Chrastinová L, Vokurková J, Růžičková V, Doškař J, Swings J, Hájek V. 2005. Staphylococcus simiae sp. nov., isolated from South American squirrel monkeys. Int J Syst Evol Microbiol 55:1953–1958. doi:10.1099/ijs.0.63590-0 PubMed DOI

Tamura K, Stecher G, Kumar S, Battistuzzi FU. 2021. Mega11: molecular evolutionary genetics analysis version 11. Mol Biol Evol 38:3022–3027. doi:10.1093/molbev/msab120 PubMed DOI PMC

Na SI, Kim YO, Yoon SH, Ha SM, Baek I, Chun J. 2018. UBCG: up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction. J Microbiol 56:280–285. doi:10.1007/s12275-018-8014-6 PubMed DOI

Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:5114. doi:10.1038/s41467-018-07641-9 PubMed DOI PMC

Meier-Kolthoff JP, Klenk H-P, Göker M. 2014. Taxonomic use of DNA G+C content and DNA-DNA hybridization in the genomic age. Int J Syst Evol Microbiol 64:352–356. doi:10.1099/ijs.0.056994-0 PubMed DOI

Xu L, Dong Z, Fang L, Luo Y, Wei Z, Guo H, Zhang G, Gu YQ, Coleman-Derr D, Xia Q, Wang Y. 2019. OrthoVenn2: a web server for whole-genome comparison and annotation of orthologous clusters across multiple species. Nucleic Acids Res 47:W52–W58. doi:10.1093/nar/gkz333 PubMed DOI PMC

Versalovic J, Schneider M, de Bruijn FJ, Lupski JR. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5:25–40.

Švec P, Pantůček R, Petráš P, Sedláček I, Nováková D. 2010. Identification of Staphylococcus spp. using (GTG)5-PCR fingerprinting. Syst Appl Microbiol 33:451–456. doi:10.1016/j.syapm.2010.09.004 PubMed DOI

Freiwald A, Sauer S. 2009. Phylogenetic classification and identification of bacteria by mass spectrometry. Nat Protoc 4:732–742. doi:10.1038/nprot.2009.37 PubMed DOI

Vieira S, Huber KJ, Neumann-Schaal M, Geppert A, Luckner M, Wanner G, Overmann J. 2021. Usitatibacter rugosus gen. nov., sp. nov. and Usitatibacter palustris sp. nov., novel members of Usitatibacteraceae fam. nov. within the order Nitrosomonadales isolated from soil. Int J Syst Evol Microbiol 71:004631. doi:10.1099/ijsem.0.004631 PubMed DOI

Schumann P, Kalensee F, Cao J, Criscuolo A, Clermont D, Köhler JM, Meier-Kolthoff JP, Neumann-Schaal M, Tindall BJ, Pukall R. 2021. Reclassification of Haloactinobacterium glacieicola as Occultella glacieicola gen. nov., comb. nov., of Haloactinobacterium album as Ruania alba comb. nov, with an emended description of the genus Ruania, recognition that the genus names Haloactinobacterium and Ruania are heterotypic synonyms and description of Occultella aeris sp. nov., a halotolerant isolate from surface soil sampled at an ancient copper smelter. Int J Syst Evol Microbiol 71:004769. doi:10.1099/ijsem.0.004769 PubMed DOI

Sasser M. 1990. MIDI Technical Note 101, Revision July 2006 ed, in press. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Inc, Newark, DE. http://midi-inc.com/pdf/MIS_Technote_101.pdf.

Schumann P. 2011. Peptidoglycan structure. Methods Microbiol 38:101–129. doi:10.1016/b978-0-12-387730-7.00005-x DOI

Kämpfer P, McInroy JA, Clermont D, Neumann-Schaal M, Criscuolo A, Busse H-J, Glaeser SP. 2021. Leucobacter soli sp. nov., from soil amended with humic acid. Int J Syst Evol Microbiol 71:005156. doi:10.1099/ijsem.0.005156 PubMed DOI