- Open access
- Published:
Non-contiguous finished genome sequence of Staphylococcus capitis CR01 (pulsetype NRCS-A)
Standards in Genomic Sciences volume 9, pages 1118–1127 (2014)
Abstract
Staphylococcus capitis is a coagulase-negative staphylococcus (CoNS) commonly found in the human microflora. Recently, a clonal population of Staphylococcus capitis (denominated NRCS-A) was found to be a major cause of late-onset sepsis (LOS) in several neonatal intensive care units in France. Here, we report the complete genome sequence and annotation of the prototype Staphylococcus capitis NCRS-A strain CR01. The 2,504,472 bp long genome (1 chromosome and no plasmids) exhibits a G+C content of 32.81%, and contains 2,468 protein-coding and 59 tRNA genes and 4 rRNA genes.
Introduction
A frequent cause of low-weight newborns mortality and morbidity in Neonatal Intensive Care Units (NICUs) are late-onset sepsis (LOS), that are defined as sepsis occurring after 3 days of age. The most frequently encountered pathogens are coagulase-negative staphylococci (CoNS) and within those Staphylococcus epidermidis has been shown to be the most prevalent [1,2]. However, a few studies have reported the emergence of Staphylococcus capitis as a main CoNS- and LOS-causative pathogen in NICU settings [2–4]. A study in French NICUs [2] has demonstrated the spread of a single clonal population of methicillin-resistant S. capitis (pulsotype NRCS-A) associated to reduced susceptibility to vancomycin, the first line of antibiotics used in cases of LOS. Moreover, this clone has also been recently identified in NICUs in Belgium, United Kingdom and Australia, which suggests a worldwide distribution. In contrast, in adult bacteremia, S. capitis are rarely found and when detected, it presents a bigger diversity in terms of genotypes as well as antimicrobial susceptibility profiles than neonates bacteremia.
In order to elucidate the molecular mechanisms behind the wide spreading of the S. capitis NRCS-A clone in NICUs throughout the world, we sequenced a prototype strain (CR01).
Classification and information
A strain belonging to the clonal population of Staphylococcus capitis NCRS-A pulsetype (Table 1) was isolated from the blood culture of a preterm infant with LOS, hospitalized in the NICU of the Northern Hospital Group Center (Hospices Civils de Lyon, Lyon, France) and suffering of LOS.
Species identification of the bacterial isolates and antimicrobial susceptibility testing (AST) were performed, respectively, using Vitek MS (bioMérieux, Marcy l’Etoile), 16S rDNA sequencing, the automated BD Phoenix system (Becton Dickinson, Sparks, MD) and with Shimadzu-MALDI-TOF MS system (Shimadzu Corporation), as implemented on [21].
The strain was identified as being a Staphylococcus capitis by VITEK MS with 99.9% and at 93.7% by the MALDI-TOF MS, using the Shimadzu Launchpad software program and the SARAMIS database application (AnagnosTec GmbH) for automatic measurement and identification (Figure 1). Based on the information provided by the manufacture, when the score is ≥70%, identification is considered of high confidence.
The antimicrobial susceptibility test (AST) results were analyzed according to the recommendations of the French Microbiology Society [22]. The S. capitis bacteremia was considered positive based on a single positive blood culture [2,23]. The S. capitis NCRS-A isolate CR01, as all isolates from this clone, is resistant to penicillin, methicillin, gentamicin, rifampicin, hetero-resistant to vancomycin and sensitive to fusidic acid and fluoroquinolones.
Table 1, Figure 2 and Figure 3 show detailed information concerning general features of Staphylococcus capitis strain (CR01) and position within the genus Staphylococcus.
The 16S rRNA sequences were aligned using the MUSCLE software, with the default parameters as implemented on Seaview version 4 [24], and a tree was inferred based on 1285 sites using the distance model of observed divergence, as implemented in the BioNJ algorithm, and a bootstrapping process repeated 500 times.
The final tree was rooted using the 16S rRNA sequence of Macrococcus equipercicus Type strain that belongs to a closely-related sister genus.
Genome sequencing information
The genome sequence of S. capitis strain CR01 was determined by high-throughput sequencing performed on a Genome Sequencer FLX + system (454 Life Sciences/Roche) using FLX Titanium reagents according to the manufacturer’s protocols and instructions, with approximately 47-fold coverage of the genome. This platform provides longer read lengths than other sequencing platforms to obtain raw sequences. De novo assemblies were performed using the Roche Newbler (v 2.7) software package.
Genome project history
Table 2 presents the project information and its association with MIGS version 2.0 compliance [5].
Growth conditions and DNA isolation
The sample was prepared for sequencing by growing S. capitis CR01, aerobically at 37°C in Blood Agar for 24–48 hours. Genomic DNA was extracted using the PureLinkTM genomic DNA kit (InvitrogenTM) according to the manufacturer’s recommended protocol. The quantity of DNA obtained was determined using a NanoVue TM Plus (HVD Life Sciences), and 1 µg of DNA was used for sequencing of whole-genome of this strain.
Genome sequencing and assembly
The isolated DNA of S. capitis CR01, was used to create 454-shotgun libraries following the GS Rapid library protocol (Roche 454, Roche). The resulting 454 DNA libraries were sequenced using a whole-genome shotgun strategy by GS FLX Titanium sequencing kit XL+ [25] (202,108 reads totaling 2.5 Mb, X48 fold coverage of the genome). Genome sequences were processed by Roche’s sequencing software according to the manufacturer’s instructions (454 Life Science). The resulting shotgun reads were assembled de novo using the Roche Newbler assembly software 2.7 (454 Life Science) and 26 large contigs (Contig00001 to Contig00026) were obtained. The N50 was 176239 bp.
Genome annotation
An automatic syntactic and functional annotation of the draft genome was performed using the MicroScope platform pipeline [26,27]. The syntactic analysis combines a set of programs including AMIGene [28], tRNAscan-SE [29], RNAmmer [30], Rfam scan [31] and Prodigal software [32] to predict genomic objects that are mainly CDSs and RNA genes. More than 20 bioinformatics methods are then used for functional and relational analyses: homology search in the generalist databank UniProt [33] and in more specialized databases as COG [34], InterPro [35], PRIAM profiles for enzymatic classification [36], prediction of protein localization using TMHMM [37], SignalP [38] and PsortB [39] tools.
Genome properties
The genome includes one circular chromosome of 2,504,472 bp (32.81% GC content). A total of 2,565 genes were predicted with 2,453 being protein-coding genes, 59 tRNA-enconding genes, 4 rRNA-encoding genes (including 2 copies of 5S rRNA, 1 copy of both the large and the small-subunits, respectively, 23S and 16S rRNA) and 34 other RNA related ORFs. No plasmid was detected.
Of the 2,453 protein-coding genes, 1,892 genes (76.7%) were assigned to a putative function with the remaining annotated as hypothetical proteins. The predicted coding density in S. capitis strain CR01 was 86%.
Table 3 and 4 and Figure 4 detailed description of the properties and the statistics of Staphylococcus capitis strain CR01 genome. The distribution of the genes into COGs functional categories is presented in Table 4.
Conclusion
Here, we described a new genome sequence of Staphylococcus capitis (strain CR01 belonging to NRCS-A clone) as a first step toward comparing its content with other sequenced Staphylococcus capitis genomes as well as CoNS genomes of species associated with late-onset sepsis. Detailed analyses are in progress to identify virulence factors and mobile genetic elements (MBE), such as the staphylococcal chromosome cassette (SCCmec) [18], potentially related to the high specificity of the NRCS-A clone to the NICU environment.
Abbreviations
- EMBL:
-
European Molecular Biology Laboratory
- NCBI:
-
National Center for Biotechnology Information (Bethesda, MD, USA)
- RDP:
-
Ribosomal Database Project (East Lansing, MI, USA)
References
Klingenberg C, Rønnestad A, Anderson AS, Abrahamsen TG, Zorman J, Villaruz A, Flægstad T, Otto M. Sollid, Ericson J. Persistent strains of coagulase-negative staphylococci in a neonatal intensive care unit: virulence factors and invasiveness. Clin Microbiol Infect 2007; 13:1100–1111. PubMed http://dx.doi.org/10.1111/j.1469-0691.2007.01818.x
Rasigade JP, Raulin O, Picaud JC, Tellini C, Bes M, Grando J, Ben Saïd M, Claris O, Etienne J, Tigaud S, Laurent F. Methicillin-ResistantStaphylococcus capitis with Reduced Vancomycin Susceptibility Causes Late-Onset Sepsis in Intensive Care Neonates. PLoS ONE 2012; 7:e31548. PubMed http://dx.doi.org/10.1371/journal.pone.0031548
Ng PC, Chow VC, Lee CH, Ling JM, Wong HL, Chang RCY. Persistent Staphylococcus capitis septicemia in a preterm infant. Pediatr Infect Dis J 2006; 25:652–654. PubMed http://dx.doi.org/10.1097/01.inf.0000225785.32137.d3
de Silva GDI, Kantzanou M, Justice A, Massey RC, Wilkinson AR, Day NPJ, Peacock SJ. The ica Operon and Biofilm Production in Coagulase-Negative Staphylococci Associated with Carriage and Disease in a Neonatal Intensive Care Unit. J Clin Microbiol 2002; 40:382–388. PubMed http://dx.doi.org/10.1128/JCM.40.02.382-388.2002
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed http://dx.doi.org/10.1038/nbt1360
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eukarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576
Gibbons NE, Murray RGE. Proposals Concerning the Higher Taxa of Bacteria. Int J Syst Bacteriol 1978; 28:1–6; http://dx.doi.org/10.1099/00207713-28-1-1.
Murray RGE. The Higher Taxa, or, a Place for Everything…? In: Holt JG (ed), Bergey’s Manual of Systematic Bacteriology, First Edition, Volume 1, The Williams and Wilkins Co., Baltimore, 1984, p. 31–34.
List of new names and new combinations previously effectively, but not validly, published. List no. 132. Int J Syst Evol Microbiol 2010; 60:469–472. http://dx.doi.org/10.1099/ijs.0.022855-0
Ludwig W, Schleifer KH, Whitman WB. Class I. Bacilli class nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 3, Springer-Verlag, New York, 2009, p. 19–20.
Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. http://dx.doi.org/10.1099/00207713-30-1-225
Prévot AR. In: Hauderoy P, Ehringer G, Guillot G, Magrou. J., Prévot AR, Rosset D, Urbain A (eds), Dictionnaire des Bactéries Pathogènes, Second Edition, Masson et Cie, Paris, 1953, p. 1–692.
List Editor. List of new names and new combinations previously effectively, but not validly, published. List no. 132. Int J Syst Evol Microbiol 2010; 60:469–472. http://dx.doi.org/10.1099/ijs.0.022855-0
Schleifer KH, Bell JA. Family VIII. Staphylococcaceae fam. nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 3, Springer-Verlag, New York, 2009, p. 392.
Rosenbach FJ. In: Bergmann JF (ed), Microorganismen bei den Wund-Infections-Krankheiten des Menschen., Wiesbaden, 1884, p. 1–122.
Judicial Commission. Opinion 17. Conservation of the Generic name Staphylococcus Rosenbach, Designation of Staphylococcus aureus Rosenbach as the Nomenclatural Type of the Genus Staphylococcus Rosenbach, and Designation of the Neotype culture of Staphylococcus aureus Rosenbach. Int Bull Bacteriol Nomencl Taxon 1958; 8:153–154.
Kloos WE, Musselwhite MS. Distribution and Persistence of Staphylococcus and Micrococcus Species and Other Aerobic Bacteria on Human Skin. Appl Microbiol 1975; 30:381–385. PubMed
Martins Simões P, Rasigade JP, Lemriss H, Butin M, Ginevra C, Lemriss S, Goering RV, Ibrahimi A, Picaud JC, Vandenesch FEL, et al. Characterization of a novel staphylococcal chromosome cassette (SCCmec) within a composite SCC island in neonatal sepsis-associated Staphylococcus capitis pulsotype NRCS-A. Antimicrob Agents Chemother 2013; 57:6354. PubMed http://dx.doi.org/10.1128/AAC.01576-13
Schleifer KH, Bell JA. Family VIII. Staphylococcaceae fam. nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 3, Springer-Verlag, New York, 2009, p. 392.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556
Cherkaoui A, Hibbs J, Emonet S, Tangomo M, Girard M, Francois P, Schrenzel J. Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. J Clin Microbiol 2010; 48:1169–1175. PubMed http://dx.doi.org/10.1128/JCM.01881-09
French Society for Microbiology. Recommandations du Comite de l’Antibiogramme de la Société Française de Microbiologie. 2009. Available at: http://www.sfmasofr/doc/downloadphp?doc=DiU8C&fic=casfm_2009pdf. Accessed 22 February 2009.
Hall KK, Lyman JA. Updated review of blood culture contamination. Clin Microbiol Rev 2006; 19:788–802. PubMed http://dx.doi.org/10.1128/CMR.00062-05
Gouy M, Guindon S, Gascuel O. SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building. Mol Biol Evol 2010; 27:221–224. PubMed http://dx.doi.org/10.1093/molbev/msp259
Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM, et al. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 1995; 269:496–512. PubMed http://dx.doi.org/10.1126/science.7542800
Vallenet D, Belda E, Calteau A, Cruveiller S, Engelen S, Lajus A, Le Fèvre F, Longin C, Mornico D, Roche D, et al. MicroScope—an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data. Nucleic Acids Res 2013; 41:D636–D647. PubMed http://dx.doi.org/10.1093/nar/gks1194
Vallenet D, Labarre L, Rouy Z, Barbe V, Bocs S, Cruveiller S, Lajus A, Pascal G, Scarpelli C, Médigue C. MaGe: a microbial genome annotation system supported by synteny results. Nucleic Acids Res 2006; 34:53–65. PubMed http://dx.doi.org/10.1093/nar/gkj406
Bocs S, Cruveiller S, Vallenet D, Nuel G, Médigue C. AMIGene: Annotation of MIcrobial Genes. Nucleic Acids Res 2003; 31:3723–3726. PubMed http://dx.doi.org/10.1093/nar/gkg590
Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997; 25:955–964. PubMed http://dx.doi.org/10.1093/nar/25.5.0955
Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 2007; 35:3100–3108. PubMed http://dx.doi.org/10.1093/nar/gkm160
Gardner PP, Daub J, Tate JG, Nawrocki EP, Kolbe DL, Lindgreen S, Wilkinson AC, Finn RD, Griffiths-Jones S, Eddy SR, Bateman A. Rfam: updates to the RNA families database. Nucleic Acids Res 2009; 37:D136–D140. PubMed http://dx.doi.org/10.1093/nar/gkn766
Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119. PubMed http://dx.doi.org/10.1186/1471-2105-11-119
UnitProt Consortium. The Universal Protein Resource (UniProt) 2009. Nucleic Acids Res 2009; 37:D169–D174. PubMed http://dx.doi.org/10.1093/nar/gkn664
Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN, et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics 2003; 4:41. PubMed http://dx.doi.org/10.1186/1471-2105-4-41
Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L, et al. InterPro: the integrative protein signature database. Nucleic Acids Res 2009; 37:D211–D215. PubMed http://dx.doi.org/10.1093/nar/gkn785
Claudel-Renard C, Chevalet C, Faraut T, Kahn D. Enzyme-specific profiles for genome annotation: PRIAM. Nucleic Acids Res 2003; 31:6633–6639. PubMed http://dx.doi.org/10.1093/nar/gkg847
Sonnhammer EL, von Heijne G, Krogh A. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 1998; 6:175–182. PubMed
Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004; 340:783–795. PubMed http://dx.doi.org/10.1016/j.jmb.2004.05.028
Gardy JL, Laird MR, Chen F, Rey S, Walsh CJ, Ester M, Brinkman FS. PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 2005; 21:617–623. PubMed http://dx.doi.org/10.1093/bioinformatics/bti057
Moller S, Croning MDR, Apweiler R. Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 2001; 17:646–653. PubMed http://dx.doi.org/10.1093/bioinformatics/17.7.646
Acknowledgements
This work was supported by a grant from the Fondation pour la Recherche Médicale (FRM, grant ING20111223510) and by the Institut National de la Recherche Médicale (INSERM) and the French Ministry of Health.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
Lemriss, H., Simões, P.M., Lemriss, S. et al. Non-contiguous finished genome sequence of Staphylococcus capitis CR01 (pulsetype NRCS-A). Stand in Genomic Sci 9, 1118–1127 (2014). https://doi.org/10.4056/sigs.5491045
Published:
Issue Date:
DOI: https://doi.org/10.4056/sigs.5491045