MECHANISMS OF DISEASE Mechanisms of disease Genome and virulence determinants of high virulence community-acquired MRSA Tadashi Baba, Fumihiko Takeuchi, Makoto Kuroda, Harumi Yuzawa, Ken-ichi Aoki, Akio Oguchi, Yoshimi Nagai, Natsuko Iwama, Kazuyuki Asano, Timothy Naimi, Hiroko Kuroda, Longzhu Cui, Kenji Yamamoto, Keiichi Hiramatsu Summary Introduction Meticillin-resistant Staphylococcus aureus (MRSA), which Background A new type of meticillin-resistant Staphylococcus arose in the 1960s, typically causes infections in inpatients, aureus (MRSA), designated community-acquired MRSA, is who have risk factors associated with health care. In the becoming increasingly noticeable in the community, some past 5 years however, MRSA infections have been strains of which cause fatal infections in otherwise healthy described in the general population. These infections arise individuals. By contrast with hospital-acquired MRSA, in the community, and thus affected people lack traditional community-acquired MRSA is more susceptible to non risk factors such as recent admittance, surgery, or long- ␤-lactam antibiotics. We investigated the high virulence term residence in care facilities. Community-acquired potential of certain strains of this bacterium. MRSA infections can cause serious and even fatal infections in otherwise healthy hosts.1,2 Methods We ascertained the whole genome sequence of Community-acquired MRSA typically affects children MW2, a strain of community-acquired MRSA, by shotgun and young adults, and it causes a range of infections cloning and sequencing. MW2 caused fatal septicaemia and similar to those caused by community-acquired meticillin- septic arthritis in a 16-month-old girl in North Dakota, USA, in susceptible S aureus (MSSA).3 Furthermore, these 1998. The genome of this strain was compared with those of infections are more likely to be susceptible to many hospital-acquired MRSA strains, including N315 and Mu50. antimicrobial classes and to have different pulsed-field gel electrophoresis subtypes than isolates that are associated Findings Meticillin resistance gene (mecA) in MW2 was carried with health care. There is an ominous sign that MRSA by a novel allelic form (type IVa) of staphylococcal cassette strains circulating beyond nosocomial settings are chromosome mec (SCCmec), by contrast with type II in N315 replacing community-acquired MSSA as the flora of and Mu50. Type IVa SCCmec did not carry any of the multiple healthy human beings in some communities. antibiotic resistance genes reported in type II SCCmec. By Recently, we established the whole genome sequence of contrast, 19 additional virulence genes were recorded in the two MRSA strains, N315 and Mu50, both of which are MW2 genome. All but two of these virulence genes were noted strains associated with health care.4 Two further hospital- in four of the seven genomic islands of MW2. acquired MRSA strains have been sequenced by others (COL, E-MRSA-16 [strain 252]; see Methods). However, Interpretation MW2 carried a range of virulence and resistance the genome of any community-acquired strain with high genes that was distinct from those displayed on the virulence has not been analysed. In this study, we chromosomes of extant S aureus strains. Most genes were sequenced the whole genome of the community-acquired carried by specific allelic forms of genomic islands in the MW2 MRSA strain MW2, and aimed to investigate the genetic chromosome. The combination of allelic forms of genomic basis for this strain’s high virulence by comparison of its islands is the genetic basis that determines the pathogenicity genome with those of MRSA strains that are associated of medically important phenotypes of S aureus, including those with health care. of community-acquired MRSA strains. Methods Lancet 2002; 359: 1819–27 MW2 is a typical community-acquired strain of MRSA, See Commentary page 1791 which was isolated in 1998 in North Dakota, USA.1 MW2 is so-called because strains of similar pulsed-field gel electrophoresis genotypes are reported widely in mid- western USA. MW2 caused fatal septicaemia and septic arthritis in a 16-month-old American-Indian girl who had no risk factors associated with health care. She died within 2 h of arrival at hospital. The MW2 strain is susceptible to Department of Bacteriology, Juntendo University, 2-1-1 Hongo, many antibiotic classes, apart from ␤-lactam antibiotics, to Bunkyo-ku, Tokyo 113-8421, Japan (T Baba PhD, M Kuroda PhD, which the strain shows a typical heterogeneous-type H Yuzawa PhD, H Kuroda MS, L Cui MD, Prof K Hiramatsu MD); National resistance. Institute of Technology and Evaluation, Tokyo (A Oguchi ASc, We established the whole genome sequence as described 4 Y Nagai ASc, K Aoki BSc, K Asano MSc); New Energy and Industrial previously, with the exception that we used genomic DNA Technology Development Organisation, Tokyo (N Iwama MSc); fragments of 1·5–3·0 kb instead of 1·0–2·2 kb to make the Research Institute, International Medical Centre of Japan, Tokyo plasmid library. We based the final genome sequence (F Takeuchi PhD, K Yamamoto MD); Organisation for Pharmaceutical of MW2 on about 64 000 sequences. We have entered Safety and Research, Tokyo (F Takeuchi); and Minnesota the whole genome sequence of MW2 primarily in the Department of Health, Minneapolis, MN, USA (T Naimi MD) DNA Database of Japan, with accession numbers Correspondence to: Prof Keiichi Hiramatsu AP004822–AP004831 (chromosome), AP004832 (plas- (e-mail: [email protected]) mid), and BA000033 (11 entries and contig). THE LANCET • Vol 359 • May 25, 2002 • www.thelancet.com 1819 For personal use. Only reproduce with permission from The Lancet Publishing Group. MECHANISMS OF DISEASE Kawasaki, Japan), and mutually overlapping orfs were GLOSSARY identified and removed in some cases. We searched a non- redundant protein database with ORFs—peptides BLA OPERON 7 ␤ encoded by orfs—with BLAST software. We identified Usually present on a plasmid in S aureus; its function is -lactam-induced 8 9 production of penicillinase. The operon is composed of three genes: blaZ, transfer RNA and tmRNA genes by tRNAscan-SE and 10 encoding a penicillin-hydrolysing enzyme (penicillinase), and its with a procedure available online. transcription regulator genes, blaR1 and blaI. When penicillin is in the The best-hit entries identified by BLASTP7 searches environment, membrane-bound signal transducer protein BlaR1 recognises with e-values of 1·0ϫ10–5 or less were classified into it and transmits the signal to the cytoplasm. Then, the repressor protein taxonomic groups in accordance with the NCBI (National BlaI, which binds near to the promoter of blaZ preventing its transcription, is cleaved off, leaving blaZ to be transcribed efficiently to produce Center for Biotechnology Information) taxonomy 11 penicillinase. database. We excluded hits against entries for Staphylococcus genus from this analysis. GENOMIC ISLAND ALLOTYPING We calculated bias in SYNONYMOUS CODON USAGE against Clinically important properties, drug resistance, and the pathogenic averages of all orfs longer than 150 bp (B ) and of potential of S aureus strains are mostly established by the set of genomic all islands that strain possesses. Each island is found in several alternative ribosomal proteins (Brp) and GC content at the third forms with different sets of virulence or resistance genes among S aureus codon position (GC3), as described previously.4 With strains—ie, it exists in several allelic forms. Since different allelic forms of these values, we defined putative highly expressed genes islands encode different properties, identifying the allelic set of islands and alien genes as orfs with Brp<0·35 and Ball>0·30, and that the strain carries gives information on the overall properties of the B >0·52 and B >0·345, respectively. We also regarded orfs strain, including its pathogenic potential and the pattern of antibiotic rp all susceptibilities. as possible alien genes when their GC3 values differed more than 1·5 SD from the average of all orfs longer than MEC OPERON 150 bp, which corresponds to GC3<0·16 or GC3>0·29. Composed of mecA, mecR1, and mecI. The latter two are gene To compare the MW2 genome with those of other homologues of blaR1 and blaI, respectively. MecR1 is the membrane- bound signal transducer recognising ␤-lactam antibiotics, and BlaI is a S aureus strains, we first processed homologous regions of repressor protein. Therefore, the same sequence of events arises as with the two chromosomes with bl2seq (blast for two bla operon. The only difference is that mecA encodes cell-wall synthesis sequences),12 and we visualised them for gross enzyme PBP2Ј instead of penicillinase encoded by blaZ: ␤-lactams induce comparison. To do this, we divided a 2·8-Mb genome meticillin resistance in this case. into three 1-Mb fragments, and blast homology was PANTON-VALENTINE LEUKOCIDIN calculated for the nine pairs of fragments. Then, we Member of the leukocidin group of bacterial toxins that kill leucocytes by processed blast results by a script written in Perl creation of pores in the cell membrane. Apart from S aureus Panton- (http://www.perl.com) and visualised with Mathematica Valentine leukocidin, the group includes Pasteurella haemolytica (Wolfram Research, Champaign, IL, USA). Similarity leukotoxin, Actinobacillus actimycetemcomitans leukotoxin, Listeria monocytogenes listeriolysin, Escherichia coli haemolysin, and between the two virtual chromosomes—which are Fusobacterium necrophorum leukotoxin. obtained