Complete genomes of two clinical Staphylococcus aureus strains: Evidence for the rapid evolution of virulence and drug resistance Matthew T. G. Holden*, Edward J. Feil†, Jodi A. Lindsay‡, Sharon J. Peacock§¶, Nicholas P. J. Day§¶, Mark C. Enright†, Tim J. Fosterʈ, Catrin E. Moore§, Laurence Hurst†, Rebecca Atkin*, Andrew Barron*, Nathalie Bason*, Stephen D. Bentley*, Carol Chillingworth*, Tracey Chillingworth*, Carol Churcher*, Louise Clark*, Craig Corton*, Ann Cronin*, Jon Doggett*, Linda Dowd*, Theresa Feltwell*, Zahra Hance*, Barbara Harris*, Heidi Hauser*, Simon Holroyd*, Kay Jagels*, Keith D. James*, Nicola Lennard*, Alexandra Line*, Rebecca Mayes*, Sharon Moule*, Karen Mungall*, Douglas Ormond*, Michael A. Quail*, Ester Rabbinowitsch*, Kim Rutherford*, Mandy Sanders*, Sarah Sharp*, Mark Simmonds*, Kim Stevens*, Sally Whitehead*, Bart G. Barrell*, Brian G. Spratt**, and Julian Parkhill*†† *The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom; †Department of Biology and Biochemistry, University of Bath, South Building, Claverton Down, Bath BA2 7AY, United Kingdom; ‡Department of Cellular and Molecular Medicine, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, United Kingdom; §Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; ʈDepartment of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland; and **Department of Infectious Disease Epidemiology, Faculty of Medicine, Imperial College, St. Mary’s Hospital, Norfolk Place, London W2 1PG, United Kingdom Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved May 13, 2004 (received for review April 8, 2004) Staphylococcus aureus is an important nosocomial and community- pounded by the recent emergence of methicillin-resistant S. acquired pathogen. Its genetic plasticity has facilitated the evolu- aureus (MRSA) carriage and disease in the community (3, 4). tion of many virulent and drug-resistant strains, presenting a major The genomes of two recently isolated clinical S. aureus strains and constantly changing clinical challenge. We sequenced the were sequenced: a hospital-acquired MRSA strain (MRSA252), Ϸ2.8-Mbp genomes of two disease-causing S. aureus strains iso- representative of the highly successful epidemic EMRSA-16 lated from distinct clinical settings: a recent hospital-acquired clone, and a community-acquired methicillin-susceptible S. au- representative of the epidemic methicillin-resistant S. aureus reus (MSSA) strain (MSSA476). More complete genomes are EMRSA-16 clone (MRSA252), a clinically important and globally now available for S. aureus than for any other bacterial species, prevalent lineage; and a representative of an invasive community- thus providing a detailed insight into the evolutionary processes acquired methicillin-susceptible S. aureus clone (MSSA476). A com- leading to strains of differing virulence and drug-resistance parative-genomics approach was used to explore the mechanisms potential. Our comparative analyses used the previously pub- lished genomes of closely related hospital-acquired MRSA and of evolution of clinically important S. aureus genomes and to vancomycin intermediately susceptible S. aureus strains (N315 identify regions affecting virulence and drug resistance. The ge- and Mu50, respectively) (5), and a community-acquired MRSA nome sequences of MRSA252 and MSSA476 have a well conserved strain (MW2) (6). Furthermore, extensive multilocus sequence core region but differ markedly in their accessory genetic elements. typing (MLST) data have been generated for this species, MRSA252 is the most genetically diverse S. aureus strain sequenced allowing the comparison of the sequenced strains within the to date: Ϸ6% of the genome is novel compared with other context of the whole S. aureus population (7). MRSA252 belongs published genomes, and it contains several unique genetic ele- to the clinically important EMRSA-16 clone that is responsible ments. MSSA476 is methicillin-susceptible, but it contains a novel for half of all MRSA infections in the U.K. (8) and is one of the Staphylococcal chromosomal cassette (SCC) mec-like element (des- major MRSA clones found in the U.S. (USA200) (9). Its ignated SCC476), which is integrated at the same site on the emergence in the last 10 years has been associated with a chromosome as SCCmec elements in MRSA strains but encodes a significant increase in the numbers of S. aureus infections in U.K. putative fusidic acid resistance protein. The crucial role that acces- hospitals, and it is regarded as endemic in the majority of U.K. sory elements play in the rapid evolution of S. aureus is clearly hospitals (8). MSSA476 was chosen because it caused severe illustrated by comparing the MSSA476 genome with that of an invasive disease in an immunocompetent child in the community extremely closely related MRSA community-acquired strain; the and belonged to a major clone associated with community- differential distribution of large mobile elements carrying viru- acquired disease that also contains the community-acquired lence and drug-resistance determinants may be responsible for the MRSA strain MW2 (10). clinically important phenotypic differences in these strains. In this study, we describe general features of the two new genomes and highlight the significance of mobile genetic ele- he impact on human health of Staphylococcus aureus infec- Ttions in community and hospital settings has lead to intensive This paper was submitted directly (Track II) to the PNAS office. investigation of this organism over recent years. It is the caus- Abbreviations: MLST, multilocus sequence typing; MRSA, methicillin-resistant Staphylo- ative agent of a wide range of diseases, from carbuncles and food coccus aureus; MSSA, methicillin-sensitive Staphylococcus aureus; SCC, Staphylococcal chromosomal cassette; ST, sequence type; SaPI, Staphylococcus aureus pathogenicity poisoning, through more serious device and wound-related islands. infections, to life threatening conditions, such as bacteremia, Data deposition: The sequences and annotation of the MRSA252 and MSSA476 genomes, necrotizing pneumonia, and endocarditis. S. aureus produces a including the pSAS1 plasmid from MSSA476, have been deposited in the EMBL database plethora of virulence factors that facilitate attachment, coloni- (accession nos. BX571856–BX571858). zation, cell–cell interactions, immune evasion, and tissue dam- ¶Present address: Faculty of Tropical Medicine, Mahidol University, 420͞6 Rajvithi Road, age. The number of effective antibiotics has been reduced by the Bangkok 10400, Thailand. emergence of resistance to penicillin, methicillin, and, more ††To whom correspondence should be addressed. E-mail: [email protected]. recently, vancomycin (1, 2), a problem that has been com- © 2004 by The National Academy of Sciences of the USA 9786–9791 ͉ PNAS ͉ June 29, 2004 ͉ vol. 101 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402521101 Downloaded by guest on September 25, 2021 ments for the acquisition of traits of clinical importance. We also acquired DNA, often at loci that have been previously identified present comparisons of very similar and very diverged genomes as mobile genetic elements or ‘‘genomic islands’’ (Fig. 1) (5, 6). that reveal the patterns of genomic diversification over time and Many of the genes contained in these regions are associated with in particular illustrate the speed with which resistance genes and virulence or drug resistance. A summary of the genomic islands putative virulence genes move between strains by lateral gene of MRSA252 and MSSA476 and the important determinants transfer. they carry is given in Table 1. Methods Genetic Elements Carrying Resistance Genes in MRSA252 and Bacterial Strains. Hospital-acquired, epidemic strain MRSA252. MSSA476. MSSA476 is resistant to penicillin and fusidic acid but MRSA252 was isolated in 1997 from a 64-year-old female who susceptible to methicillin. Surprisingly MSSA476 contains a acquired MRSA postoperatively. The organism was part of a novel Staphylococcal chromosomal cassette (SCC)mec-like ele- miniature outbreak at an intensive treatment unit where all three ment (designated SCC476) integrated at the same site on the of the patients who became infected died as a direct consequence chromosome as SCCmec elements in MRSA strains (Fig. 1). The of MRSA septicemia (septic shock or multiorgan failure subse- 22.8-kb SCC476 contains 18 CDSs and shares the same left and quent to septic shock). MRSA252 is resistant to penicillin, right boundaries (attL and attR, respectively) and similar in- ciprofloxacin, erythromycin, and methicillin and sensitive to verted repeat sequences as SCCmec elements (Fig. 2) (14). fusidic acid, rifampicin, tetracycline, trimethoprim, gentamicin, However, unlike SCCmec elements, SCC476 does not carry the and amikacin. The strain has been typed as belonging to the mecA gene (encoding penicillin-binding protein 2a) that confers EMRSA-16 clone by pulsed-field gel electrophoresis and to resistance to methicillin, but it carries a CDS (SAS0043) encod- sequence type (ST)36 by MLST (8, 10). ing a protein similar (43.7% amino acid identity) to the plasmid- Community-acquired, invasive strain MSSA476. MSSA476 was isolated borne fusidic acid-resistance determinant Far1 (15). The only in 1998 from a 9-year-old boy with community-acquired primary other apparent resistance protein