- Short genome report
- Open Access
Complete genome sequence of an agr-dysfunctional variant of the ST239 lineage of the methicillin-resistant Staphylococcus aureus strain GV69 from Brazil
Standards in Genomic Sciences volume 11, Article number: 34 (2016)
Staphylococcus aureus is a versatile Gram-positive coccus frequently found colonizing the skin and nasal membranes of humans. The acquisition of the staphylococcal cassette chromosome mec was a major milestone in the evolutionary path of methicillin-resistant S. aureus. This genetic element carries the mecA gene, the main determinant of methicillin resistance. MRSA is involved in a plethora of opportunistic infectious diseases. The accessory gene regulator is the major S. aureus quorum sensing system, playing an important role in staphylococcal virulence, including the development of biofilms. We report the complete genome sequence (NCBI BioProject ID: PRJNA264181) of the methicillin-resistant S. aureus strain GV69 (= CMVRS P4521), a variant of the ST239 lineage that presents with a natural attenuation of agr-RNAIII transcription and a moderate accumulation of biofilm.
Staphylococcus aureus is an adaptable pathogen capable of infecting nearly all tissues and organs of the human body. Methicillin-resistant S. aureus is a major bacterial pathogen in terms of its incidence and the severity of associated illnesses. MRSA infections can affect either hospitalized patients or healthy individuals within the community . Hospital-associated MRSA show a highly clonal population, and clonality have usually been characterized based on pulsed-field gel electrophoresis analysis, SCCmec typing and multilocus sequence typing. One of the most globally disseminated HA-MRSA lineages is the ST239-SCCmecIII .
We previously reported the complete genome sequence of the ST239 strain BMB9393 from Brazil that expresses high levels of agr-RNAIII transcripts, and has a superior ability to accumulate ica-independent biofilm . The accessory gene regulator operon is the main quorum-sensing system of S. aureus . It is well-known that agr regulates a plethora of virulence factors and key mechanisms associated with the pathogenesis of S. aureus infections, including the development of biofilm . The agr-RNAIII transcripts and the AgrA protein are the regulatory molecules (effectors) of the agr operon .
We report here the complete genome sequence of an ST239 variant, strain GV69, which has a natural attenuation of the agr-rnaIII gene expression and forms a thinner biofilm layer in comparison to BMB9393.
Classification and features
We sequenced the complete genome of a variant of the ST239 MRSA lineage called GV69. This strain was isolated in 1996 from a skin wound infection in a patient admitted at a burn unit in a general public hospital in Teresina city, located at the northeast region of Brazil . In Brazil, ST239 isolates are only associated with hospital infections, and they are broadly disseminated, multiresistant, and frequently grouped in the Brazilian epidemic clone, based on PFGE analysis, MLST, and mec typing [6–8]. Strain GV69 has a natural agr dysfunction and a moderate biofilm phenotype. The ability of many bacteria to develop biofilm is considered an important mechanism of colonization, primarily in infections associated with the use of indwelling medical devices . GV69 strain is a non-motile, non-spore forming, non-hemolytic Gram-positive cocci in the family Staphylococcaceae , order Bacillales , and class Bacilli . Figure 1 shows the phylogenetic position of the GV69 in relation to other Staphylococcus spp. The GV69 strain is a facultatively anaerobic, mesophilic bacterium that can grow at temperatures of 30–37 °C. S. aureus isolates exhibit a preference for glycolytic carbon sources. Acid is produced aerobically and anaerobically from glucose, lactose, maltose and mannitol, and aerobically from fructose, galactose, mannose, ribose, sucrose, trehalose, turanose and glycerol . Figure 2 shows a photomicrograph of the S. aureus GV69 strain using Gram stain technique.
GV69 cultures were grown at 37 °C with aeration (250 rpm) in rich media (tripticase soy broth) for 18 h, and the strain was initially identified by routine diagnostics based on Gram stain, mannitol fermentation, catalase testing and tube coagulase testing. A summary of the general information gathered for the GV69 is listed in the Table 1. Data from antimicrobial disc susceptibility test demonstrated that, in addition to methicillin and other β-lactam drugs, this strain is resistant to several different groups of antimicrobial drugs, although vancomycin and the more recent commercially available antibiotics are exceptions. In addition, GV69 strain shows an average biofilm unit of 0.86 (moderate biofilm phenotype), whereas BMB9393 has an average BU of 3.7 (strong biofilm phenotype) . This strain was deposited at the public collection “Coleção de Micro-organismos de Referência em Vigilância Sanitária” of the Fundação Oswaldo Cruz with the reference name P4521 .
Genome sequencing information
Genome project history
A collaboration between the Laboratório Nacional de Computação Científica, operated by the Ministério de Ciência e Tecnologia e Inovação of the Brazilian government, and the Universidade Federal do Rio de Janeiro sequenced, assembled, and annotated the complete GV69 genome as part of the ST239 Genome Program. This organism was selected for sequencing as a representative of the approximately 30 % of Brazilian ST239 isolates that display an agr dysfunction. The raw sequence data was deposited in NCBI’s Sequence Read Archive (experiment accession number SRX1322312 and GV69 run accession number SRR2601051). The complete genome sequence of the GV69 strain was deposited in GenBank (accession number CP009681). Table 2 presents the project information and its association with MIGS version 2.0 compliance .
Growth conditions and genomic DNA preparation
A volume of 0.5 mL of a GV69 culture (37 °C/18 h) was inoculated into a 250 mL-Erlenmeyer flask containing 50 mL of pre-sterilized TSB. The culture was grown at 37 °C for 18 h under normal atmospheric conditions and shaking at 250 RPM. The bacteria were harvested by centrifugation (1500 × g at 4 °C), washed twice in cold sterile water and the whole pellet used for DNA preparation. Cells were lysed with 20U/mL lysostaphin and DNA obtained by phenol extraction and ethanol precipitation . The concentration and purity of the resulting DNA was assessed using a Qubit® 2.0 fluorometer (Invitrogen; Eugene, Oregon, USA). This genomic DNA (5 μg) was used to prepare a paired-end library.
Genome sequencing and assembly
The genome sequencing was performed using a 454 GS FLX Titanium (3-kb paired-end library) approach (Roche Diagnostics Corporation, Indianapolis, IN, USA). The assembly, based on 362,284 reads that corresponded to 62,981,906 bp (23-fold coverage), was performed using Newbler v2.6 (Roche) and Celera Assembler v6.1 . Gaps within scaffolds resulting from repetitive sequences were resolved by in silico gap filling. For determining the small insertions and deletions occurring into homopolymer regions (at least three consecutive equal base pairs), the complete genomic sequence of the GV69 isolate was compared to that of the ST239 isolate, TW20, from United Kingdom, whose complete sequence is deposited in the GenBank (accession number: FN433596). For this comparison we applied Crossmatch (version 0.990329) with more stringent default parameter (mismatch = 14). The result of the alignment showed 541 inserts (of which 174 occurring into homopolymeric regions) and 575 deletes (of which 244 occurring into homopolymeric regions). In summary, the complete genome sequence of the GV69 isolate harbors 418 InDels occurring into homopolymer regions in relation to the genome sequence of the TW20 (Additional file 1: Table S1).
The genome annotation was performed using the System for Automated Bacterial Integrated Annotation . This software uses an automated annotation pipeline, where each open reading frame is submitted to comparison with several databases (NCBI-nr, KEGG, InterPro and UniProtKB/Swiss-Prot), and the results are made available on the screen for the assessment of expert users. All possible ORFs are predicted by Glimmer  and GeneMark  and tRNAs are detected by tRNAscan-SE . The identification of bona fide ORFs and their probable functions takes in account the results of similarity searches using both nucleotide and amino acid sequences by BLAST against KEGG, NCBI-nr and UniProtKB/Swiss-Prot databases, and also the prediction of protein domains and important sites using InterPro . ORFs with a good BLAST coverage in the NCBI-nr database, with a minimum of 60 % positive identity, 80 % query coverage, 80 % subject coverage, and 10−5 e-value cutoff were assigned as “valid”, with known function or hypothetical. On the other hand, when identified truncated version of a gene, because of nonsense or frameshift mutations in the coding sequence, the corresponding ORF was annotated as pseudogene. In addition, other analyses using SABIA pipeline comprised the classification of gene products according with biological processes, cellular components and molecular functions based on Gene Ontology [19, 20]. The functional classification according with biological systems was based on KEGG and COG databases. The identification and classification of membrane transport proteins was based on Transporter Classification system available in TCDB database, and subcellular localization of proteins was predicted using PSORT tool . CRISPRFinder was used for identifying clustered regularly interspaced short palindromic repeats .
The GV69 genome consists of one circular chromosome of 3,046,210 bp with a G + C content of 32.94 % (Fig. 3). Using the SABIA pipeline , we functionally annotated 2,758 protein-coding sequences of which 2,285 were assigned to known functions, with the remaining 473 assigned to unknown categories. Seventy-six were assigned as putative pseudogenes. The genome harbors 5 rRNA operons (5 copies of 16S rRNA, 5 of 23S rRNA, and 6 of 5S rRNA) and 60 tRNA genes, which were identified with RNAmmer  and tRNAscan-SE , respectively. This information is summarized in Table 3. A total of 2,098 genes were assigned to COG; a breakdown of their functional assignments is shown in Table 4.
Comparative analyses were performed using the SABIA pipeline . The bidirectional best hit (90 % amino acid identity and 90 % alignment coverage) comparison with six other published ST239 S. aureus genomes revealed that GV69 shares 2,415 CDS with BMB9393, another Brazilian ST239 variant; 2,328 CDS with strain JKD6008; 2,357 with strain TW20; 2,342 with strain T0131; 2,380 with Z172; and 2,290 with XN108. Despite that, GV69 has 170 unique CDS relative to the other six genomes, including an extra copy of a gene encoding a putative N-acetylmuramoyl-L-alanine amidase, an enzyme related to the bacterial cell autolytic function. This gene is located in a phage-associated mobile genetic element (phage-associated) inserted in the chromosome.
Although belonging to the same lineage and clonal type, strains GV69 and BMB9393 have differences in their flexible genomes. In addition to 343 CDS (150 of unknown function, including several related to MGEs) found exclusively in GV69, this strain lacks a small 2,908 bp plasmid found in BMB9393 that carries the cat gene, a determinant for chloramphenicol resistance.
accessory gene regulator
bidirectional best hit
Brazilian epidemic clone
hospital-acquired methicillin-resistant Staphylococcus aureus
mobile genetic element
multilocus sequence typing
system for automated bacterial integrated annotation
- SCCmec :
staphylococcal cassette chromosome mec
ST239 genome program
Figueiredo AM, Ferreira FA. The multifaceted resources and microevolution of the successful human and animal pathogen methicillin-resistant Staphylococcus aureus. Mem Inst Oswaldo Cruz. 2014;109:265–78.
Costa MO, Beltrame C, Ferreira FA, Botelho AMN, Lima NB, Souza RC, et al. Complete genome sequence of a variant of the methicillin-resistant Staphylococcus aureus ST239 lineage, strain BMB9393, displaying superior ability to accumulate ica-independent biofilm. Genome A. 2013;1:1–2.
Coelho LR, Souza RR, Ferreira FA, Guimarães MA, Ferreira-Carvalho BT, Sá Figueiredo AM. agr RNAIII divergently regulates glucose-induced biofilm formation in clinical isolates of Staphylococcus aureus. Microbiology. 2008;154:3480–90.
Reynolds J, Wigneshweraraj S. Molecular insights into the control of transcription initiation at the Staphylococcus aureus agr operon. J Mol Biol. 2011;412:862–81.
Dos Santos Soares MJ, da Silva-Carvalho MC, Ferreira-Carvalho BT, Figueiredo AM. Spread of methicillin-resistant Staphylococcus aureus belonging to the Brazilian epidemic clone in a general hospital and emergence of heterogenous resistance to glycopeptide antibiotics among these isolates. J Hosp Infect. 2000;44:301–8.
Amaral MM, Coelho LR, Flores RP, Souza RR, Silva-Carvalho MC, Teixeira LA, et al. The predominant variant of the Brazilian epidemic clonal complex of methicillin-resistant Staphylococcus aureus has an enhanced ability to produce biofilm and to adhere to and invade airway epithelial cells. J Infect Dis. 2005;192:801–10.
de Sousa-Junior FC, Silva-Carvalho MC, Fernandes MJBC, Vieira MFP, Pellegrino FLP, Figueiredo AMS, et al. Genotyping of methicillin-resistant Staphylococcus aureus isolates obtained in the Northeast region of Brazil. Braz J Med Biol Res. 2009;42:877–81.
Vieira MA, Minamisava R, Pessoa-Júnior V, Lamaro-Cardoso J, Ternes YM, Andre MCP, et al. Methicillin-resistant Staphylococcus aureus nasal carriage in neonates and children attending a pediatric outpatient clinics in Brazil. Braz J Infect Dis. 2013;18:42–7.
Garrity G, Holt J. The road map to the manual. In: Castenholz R, editor. Bergey’s manual of systematic bacteriology. 2nd ed. New York: Springer; 2001. p. 119–69.
Coleção de Micro-organismos de Referência em Vigilância Sanitária. Available at: http://cmrvs.fiocruz.br.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.
Sambrook J, Fritch E, Maniatis T. Commonly used techniques in molecular cloning. In: Sambrook J, Fritch E, Maniatis T, editors. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989. p. E3–4.
Myers EW, Sutton GG, Delcher AL, Dew IM, Fasulo DP, Flanigan MJ, et al. A whole-genome assembly of drosophila. Science. 2000;287:2196–204.
Almeida LGP, Paixão R, Souza RC, da Costa GC, Barrientos FJ, Trindade dos Santos M, et al. A system for automated bacterial (genome) integrated annotation - SABIA. Bioinformatics. 2004;20:2832–3.
Delcher AL, Bratke KA, Powers EC, Salzberg SL. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007;23:673–9.
Besemer J, Borodovsky M. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res. 2005;33:W451–4.
Schattner P, Brooks AN, Lowe TM. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005;33:W686–9.
Mitchell A, Chang H, Daugherty L, Fraser M, Hunter S, Lopez R, et al. The InterPro protein families database : the classification resource after 15 years. Nucleic Acids Res. 2015;43:D213–21.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.
The Gene Ontology Group. Gene ontology consortium: going forward. Nucleic Acids Res. 2014;43:1049–56.
Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, et al. PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics. 2010;26:1608–15.
Grissa I, Vergnaud G, Pourcel C, Bland C, Ramsey TL, Sabree F, et al. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007;35:W52–7.
Lagesen K, Hallin P, Rødland EA, Stærfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35:3100–8.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.
Murray R. The higher taxa, or, a place for everything…? In: Holt J, editor. Bergey’s manual of systematic bacteriology. 1st ed. Baltimore: The Williams and Wilkins Co; 1984. p. 31–4.
Skerman V, McGowan V, Sneath P. Approved lists of bacterial names (Amended). (Skerman V, McGowan V, Sneath P, eds.). Washinton (DC): ASM Press; 1989.
Gibbons N, Murray R. Proposals concerning the higher taxa of bacteria. Int J Syst Bacteriol. 1978;28:1–6.
Ludwig W, Schleifer K, Whitman W. Class I. Bacilli. In: Whitman W, editor. Bergey’s manual of systematic bacteriology. 2nd ed. New York: Springer; 2009. p. 19–20.
Oren A, Garrity GM. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2014;64:2184–7.
Schleifer K, Bell J. Family VIII. Staphylococcaceae fam. nov. In: Whitman W, editor. Bergey’s manual of systematic bacteriology. 2nd ed. New York: Springer; 2009. p. 392.
Bruno WJ, Socci ND, Halpern AL. Weighted neighbor joining: a likelihood-based approach to distance-based phylogeny reconstruction. Mol Biol Evol. 2000;17:189–97.
Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohideen AS, McGarrell DM, et al. The ribosomal database project (RDP-II): Introducing myRDP space and quality controlled public data. Nucleic Acids Res. 2007;35:D169–72.
Stothard P, Wishart DS. Circular genome visualization and exploration using CGView. Bioinformatics. 2005;21:537–9.
This work was supported in part by grants from the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Grant no.: E26/102.901/2011, E26/110.625/2011 and E26/111.663/2013), Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant no. 472034/2012-0). PTB has a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.
The authors declare that they have no competing interests.
LGPA and RCS participated in the genome assembly. AMNB, AMSF, MFN and ATRV carried out the molecular studies and acquisition of data. AMNB, FAF, MOCC, COB, MFC, PTB, NCBL, RCS, LGPA, ATRV, MFN and AMSF participated in the sequence alignment and manually curated annotations. AMNB, AMSF, MFN and LGPA participated in the drafted the manuscript and in the design of the study. AMSF conceived the study. All authors read and approved the final manuscript.
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Botelho, A.M.N., Costa, M.O.C., Beltrame, C.O. et al. Complete genome sequence of an agr-dysfunctional variant of the ST239 lineage of the methicillin-resistant Staphylococcus aureus strain GV69 from Brazil. Stand in Genomic Sci 11, 34 (2016). https://doi.org/10.1186/s40793-016-0154-x
- Complete genome sequence
- Methicillin-resistant Staphylococcus aureus
- Skin hospital infection
- agr dysfunction