Evolution and pathogenesis of Staphylococcus aureus : lessons learned from genotyping and...

R E V I E W A R T I C L E

Evolutionand pathogenesis ofStaphylococcus aureus :lessons learned fromgenotypingand comparative genomicsYe Feng1,2, Chih-Jung Chen3, Lin-Hui Su3, Songnian Hu1,2, Jun Yu1,2 & Cheng-Hsun Chiu3

1James D. Watson Institute of Genome Sciences, Zhejiang University, Hangzhou, China; 2Beijing Institute of Genomics, Chinese Academy of Sciences,

Beijing, China; and 3Division of Pediatric Infectious Diseases, Department of Pediatrics, Chang Gung Children’s Hospital, Chang Gung University College

of Medicine, Taoyuan, Taiwan

Correspondence: Cheng-Hsun Chiu,

Division of Pediatric Infectious Diseases,

Department of Pediatrics, Chang Gung

Children’s Hospital, Chang Gung University

College of Medicine, 5 Fu-Hsin Street,

Kweishan 333, Taoyuan, Taiwan.

Tel.: 1886 3 3281200; fax: 1886 3 3288957;

e-mail: [email protected]

Received 12 April 2007; revised 26 July 2007;

accepted 27 August 2007.

First published online 5 November 2007.

DOI:10.1111/j.1574-6976.2007.00086.x

Editor: Ramon Diaz Orejas

Keywords

methicillin-resistant Staphylococcus aureus ;

comparative genomics; clonal complex;

genotype.

Abstract

Staphylococcus aureus is an opportunistic pathogen and the major causative agent

of numerous hospital- and community-acquired infections. Multilocus sequence

typing reveals a highly clonal structure for S. aureus. Although infrequently

occurring across clonal complexes, homologous recombination still contributed

to the evolution of this species over the long term. agr-mediated bacterial

interference has divided S. aureus into four groups, which are independent of

clonality and provide another view on S. aureus evolution. Genome sequencing of

nine S. aureus strains has helped identify a number of virulence factors, but the key

determinants for infection are still unknown. Comparison of commensal and

pathogenic strains shows no difference in diversity or clonal assignments. Thus,

phage dynamics and global transcriptome shifts are considered to be responsible

for the pathogenicity. Community-acquired methicillin-resistant S. aureus (C-

MRSA) is characterized by a short SCCmec and the presence of a Panton–Valentine

leukocidin locus, but no studies have proven their exact biologic roles in C-MRSA

infection, indicating the existence of other mechanisms for the genesis of C-MRSA.

Introduction

Staphylococcus aureus is an extraordinarily versatile patho-

gen that can survive in hostile environmental conditions,

colonize mucous membranes and skin, and can cause severe,

nonpurulent, toxin-mediated disease or invasive pyogenic

infections in humans. In the 1940s, penicillin G was the

treatment of choice for infections caused by S. aureus.

However, since the 1960s, S. aureus strains resistant to the

penicillinase-resistant penicillins, as represented by the

original member of the class, methicillin, have gradually

emerged worldwide (Ayliffe, 1997; Chambers, 2001). These

strains have been historically referred to as methicillin-

resistant S. aureus (MRSA) and are resistant to all b-lactam

agents. Recently, these strains have become multi-resistant,

exhibiting resistance to macrolides and lincosamides, and

often to tetracyclines and gentamicin as well. Resistance to

trimethoprim and sulfonamides is also prevalent in some

countries. This type of MRSA is now a common cause of

nosocomial infections in both developing and developed

countries.

Different types of MRSA have been described with origins

in the communities of different countries worldwide

(Chambers, 2001; Vandenesch et al., 2003; Zetola et al.,

2005). Resistance to penicillin and methicillin, but not to

most or all other drug classes, characterizes these types

of MRSA. For the most part, it appears to be an organism

occurring in the community setting (Riley et al., 1995;

Chambers, 2001), but hospital outbreaks have also been

described (O’Brien et al., 1999).

Comparative genomics, including comparison at the

sequence, transcriptome, and proteome levels, has been an

increasingly important approach for scientists to improve

knowledge on the pathogenesis and drug resistance of

S. aureus. For example, vancomycin, as the last resort against

multi-resistant MRSA, has gradually lost its potency due to

the appearance of vancomycin-resistant strains. Whereas

high-level vancomycin resistance in S. aureus has been

FEMS Microbiol Rev 32 (2008) 23–37 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

shown to rely on horizontal transfer of vanA from Enter-

ococcus faecalis (Chang et al., 2003; Weigel et al., 2003), the

mechanisms underlying vancomycin-intermediate-resistant

remain poorly understood. The first two sequenced

S. aureus strains, Mu50 and N315, are a pair of sister strains

whose genome sequences are nearly identical, making it

difficult to target vancomycin-related genes. Cui et al.

(2005) identified c. 100 genes that show differential tran-

scription by use of microarray expression analysis. These

genes are thought to increase vancomycin resistance by

involving the cell wall metabolic pathway.

The more the details regarding the evolution and patho-

genesis of S. aureus are elucidated, the more the questions

generated, awaiting further laboratory, epidemiologic, and

clinical studies. Herein, the progress made on S. aureus

during recent years, as well as the major challenges con-

fronting researchers in this field is reviewed.

Evolution of the core genome

Clonal structure

The population of S. aureus presents a highly clonal struc-

ture. The clonality of S. aureus was initially discovered by

multilocus enzyme electrophoresis and pulsed field gel

electrophoresis, and later gained support from multilocus

sequence typing (MLST). MLST is currently the most

popular typing method through the sequencing of seven

housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and

yqiL). For each gene, the different sequences are assigned as

alleles and the alleles at the seven loci provide an allelic

profile, which unambiguously defines the sequence type

(ST) of each isolate. Furthermore, isolates with at least six

of seven matching genes are thought to belong to the same

clonal complex (CC). It has been shown that most MRSA

strains can be grouped into five lineages: CC8, CC5, CC30,

CC45, and CC22 (Enright et al., 2002), and 87% of S. aureus

isolates, including both carriage and clinical isolates, are

grouped into the 11 most frequent clonal complexes (Feil

et al., 2004).

The clear clonal structure has inferred few genetic ex-

changes between lineages; in contrast, in a sexual species,

frequent recombination disrupts linkage associations be-

tween alleles and the relationships between clonal complexes

are more accurately represented as a network, rather than

the usual bifurcating phylogenic tree. Examination of the

sequence changes at MLST loci has proven that point

mutations give rise to new alleles at least 15-fold more

frequently than recombination (Feil et al., 2003). Most

prokaryocytes exhibit a clonal structure to some extent.

The clonality may result from geographic subdivision

that can block genetic exchanges, a rapid propagation of

certain clones that can overwhelm other sporadic clones, or

some cryptic mechanism that can produce true clonality

(i.e. long-term clonal evolution).

It seems that S. aureus belongs to the true clonality type,

as the arbitrary mobility of mobile genetic elements (MGEs)

is not allowed in S. aureus. In the laboratory, S. aureus is

notoriously difficult to manipulate genetically, as evidenced

by the rejection of exogenous plasmids. In addition, each

lineage of S. aureus has its own phage range. As one of the

earliest typing methods used for S. aureus, phage typing is

based on the selective phage sensitivity of this species.

Differences in the phage pattern between lineages are caused

by the restriction–modification (RM) system, which has

been observed in many taxonomically unrelated bacteria.

Waldron & Lindsay (2006) showed that in S. aureus, the RM

systems not only serve to protect the bacterial cell from

phage lysis, but stringently control all types of foreign DNA

acquisition, namely, transduction, conjugation, and trans-

formation. Here, the RM systems specifically refer to two

type I RM systems located in the genomic islands, nSaa and

nSab, respectively, the only RM systems in S. aureus chro-

mosome. The two islands have been found in all S. aureus

strains, and the gene hsdS in the RM systems, which is

responsible for sequence specificity, varies substantially

between lineages. Therefore, it is tempting to speculate that

the RM system plays a major role in forming the clonal

structure in S. aureus.

Compared with S. aureus, Staphylococcus epidermidis does

not have nSaa and nSab in its chromosome. The ratio of the

recombination-to-point mutation in S. epidermidis is ap-

proximately twofold, far higher than that in S. aureus

(Miragaia et al., 2007). Therefore, S. epidermidis has a

putative population with an epidemic structure, in which

its nine clones have emerged upon a recombining back-

ground and evolved quickly through lateral genetic ex-

changes. In staphylococci, it is thought that recombination

often occurs in a phage-mediated fashion. Therefore, it is

very likely that the absence of the type I RM systems results

in the free transfer of phages between lineages, which can be

regarded as additional evidence that the RM system has an

effect on limiting recombination and the evolution of the

population structure.

Recombination

Although recombination occurs in S. aureus at a low

frequency, its significance in the pathogenesis should not be

overlooked. By calculating nucleotide substitution rates

among orthologous genes of different strains, 45 genes have

been identified to demonstrate anomalously high divergence

at synonymous sites (Hughes & Friedman, 2005). Apart

from those with hypothetical functions, most of the genes

involved in recombination are related to pathogenesis,

such as genes encoding staphylocoagulase, exotoxins,

FEMS Microbiol Rev 32 (2008) 23–37c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

24 Y. Feng et al.

enterotoxins, and fibrinogen-binding proteins. Some of

these genes have been verified by independent studies. For

example, staphylocoagulase is an extracellular protein that

causes coagulation of plasma and is regarded as the hallmark

protein for the classification of S. aureus infections. Phylo-

genetic relations among coa do not seem to correlate with

those among the flanking regions or the housekeeping genes

used for MLST, indicating that coa can be laterally trans-

ferred among different lineages (Watanabe et al., 2005).

Sometimes, the recombination can even change the clonal

structure. The relationships between STs are not always

consistent, even between the seven housekeeping loci (Feil

et al., 2003). More than half of these incongruent compar-

isons involve the arcC locus; this is often accounted for as a

‘hitchhiking effect.’ arcC is in close proximity to three

putative virulence genes, namely clfB, aur, and isaB. Because

these genes encode proteins that are exposed to the host

immune response, these loci are more likely to become

recombination hot spots in order to introduce genetic

diversity for adaptation to selection pressure. Such recom-

bination will frequently extend into the arcC locus and may

influence its sequence evolution.

Large chromosomal replacements have been identified

in S. aureus, although rarely occurring naturally. The ST239

mosaic chromosome has �557 kb spanning oriC from its

ST30 parent and �2220 kb spanning terC from its ST8

parent (Robinson & Enright, 2004). ST239 has thrived to

become a pandemic lineage of MRSA represented by

numerous clones, including epidemic EMRSA-1, -4, -7, -9,

-11, Brazilian, Portuguese, and Vienna clones (Aires de

Sousa et al., 1998; Witte, 1999), suggesting that a successful

recombination event can breed a new pandemic clone.

Difference of gene content between lineagesand between species

To date, nine S. aureus strains have been sequenced, includ-

ing one laboratory strain (NCTC8325), one bovine strain

(RF122), and seven human strains (COL, USA300, MW2,

MSSA476, MRSA252, Mu50, and N315). The overall struc-

tures of all sequenced S. aureus chromosomes exhibit good

synteny between each other. Approximately 78% of the

genes are conserved among strains and constitute the ‘core

genome.’ The remaining 22% of the genes comprise an

‘accessory genome,’ including genomic islands, pathogeni-

city islands (SaPIs), prophages, integrated plasmids, and

transposons.

The entire ‘core genome’ is not as stable as the term

suggests. Some regions in the core genome are exceptionally

variable between lineages; therefore, the core genome can be

further divided into stable core and core variable genomes,

which can be easily discriminated by microarray analysis

(Lindsay et al., 2006). Specifically, many of the ‘core variable’

genes encode virulence factors involved in pathogenesis, e.g.,

toxins, superantigens, exoenzymes, and regulatory elements.

Apart from a higher nucleotide substitution rate, core

variable genes often contain variable number tandem re-

peats (VNTRs). The best-studied VNTR loci in S. aureus are

genes encoding microbial surface components recognizing

adhesive matrix molecules (MSCRAMMs). Attachment to

tissue, a key step during the infection process, is primarily

mediated by the binding of MSCRAMMs to fibrinogen,

fibronectin, collagen, and other components of the host

extracellular matrix (Foster & Hook, 1998). A number of

MSCRAMMs (e.g. ClfA and B; SdrC, D, and E; and FnbA

and B) are characteristics of peptide repeats, which are

prone to allow slippage error in replication or to induce

recombination in these loci. It is well understood that

hypervariation of virulence genes is due to competition with

the host immune system and/or the fact that they are not

critical for basic metabolism.

Homologue analysis has shown that MRSA252 and

RF122 are more divergent than the other seven S. aureus

strains (Fig. 1). Some other small details also demonstrate,

and thus support, this notion. For example, SarT and U, two

regulators that are believed to have evolved from SarA, are

Fig. 1. Protein homology between nine sequenced Staphylococcus

aureus genomes. In each box is the number of orthologues shared by

the corresponding strains and median nucleotide divergence that reflects

divergence between the two strains. The orthologue was constructed by

the ORTHOMCL program (Li et al., 2003). Nucleotide divergence is defined

as the number of mismatch bases divided by the number of comparable

bases. The color intensity in each box is in inverse proportion to the

nucleotide divergence. The accession numbers of the S. aureus genomes

are: NC_002745 (N315), NC_002758 (Mu50), NC_003923 (MW2),

NC_002953 (MSSA476), NC_002951 (COL), NC_007795 (NCTC8325),

NC_007793 (USA300), NC_002952 (MRSA252), NC_007622 (RF122).


25Comparative genomics of Staphylococcus aureus

only missing from MRSA252 and RF122, and are present in

all other seven strains. When S. aureus and the four sequenced

coagulase-negative staphylococci (CoNS) strains (two

S. epidermidis strains, one Staphylococcus haemolyticus strain,

and one Staphylococcus saprophyticus strain) are combined for

comparison, a large proportion of genes are conserved in

their sequence and order on the chromosome comprising the

backbone of the staphylococci genus genome (Fig. 2). A 0.4-

Mbp region downstream staphylococcal cassette chromo-

some (SCC) has little homology among species, in which

many important S. aureus-specific genes are located, such as

spa (encoding protein A) and coa (encoding coagulase).

Takeuchi et al. (2005) designated it as an ‘oriC environ’ and

hypothesized that this region is related to chromosomal

inversion events within the staphylococci genus and has made

an important contribution to the evolution and differentia-

tion of the staphylococcal species.

Table 1 lists the known virulence factors and regulators of

S. aureus that are present or not present in the four sequenced

CoNS strains. Nearly all prophages, genomic islands, and

pathogenicity islands that harbor toxin genes are absent from

the CoNS strains, which is supposed to be the most impor-

tant reason for exceeding virulence of S. aureus. Adhesins and

exoenzymes are also different between species. In contrast, agr

and sarA, the two regulators responsible for the global

regulation of virulence factors in S. aureus, are conserved in

all staphylococcal species. Theoretically, most of the toxins

and other S. aureus-specific virulence factors emerged in

S. aureus after speciation. It would be interesting, then, to

determine how agr and sarA have developed their new

function in regulating virulence factors. It is possible that a

functional coevolution occurred between agr/sarA and viru-

lence factors, whereas those not regulated by agr/sarA, such as

enterotoxins A and K (Tremaine et al., 1993), have possibly

not completed coevolution.

agr Groups

To gain an insight into the relatedness among the S. aureus

species, including those strains not sequenced, the concate-

nated sequence of MLST alleles is often used for reconstruct-

ing a phylogenetic tree. Sometimes, SAS genes that encode

putative surface proteins are also included to provide more

informing sites. Based on MLST, SAS sequence, and agr

typing, Robinson et al. (2005a) proposed a ‘two-subspecies’

hypothesis stating that both subspecies contain agr I, II, and

III groups. The topology derived from the hypothesis is in

agreement with the conditional tree constructed by the use

of microarray analysis of core variable genes from 161

isolates (Fig. 3; Lindsay et al., 2006).

The essential part of the hypothesis is the agr locus.

Bacterial interference is a commonly observed phenomenon

in which strains of different species or lineages exclude each

other in the sites of infection or colonization. In S. aureus,

agr is responsible for this phenomenon. It encodes a two-

component signaling pathway with the activating ligand of

an auto-inducing peptide (AIP). Polymorphism in the

sequence of AIP and its corresponding receptor divide S.

aureus strains into four major groups. Within a given group,

each strain produces a peptide that can activate the agr

response in the other member strains, whereas AIPs belong-

ing to different groups are mutually inhibitory (Ji et al.,

1997; Jarraud et al., 2000).

However, the species are not subdivided into three or five

monophyletic agr groups. Strains of the same agr group are

not related to each other. For example, MRSA252 and two

CC1 strains, MW2 and MSSA476, belong to agr III, but

MRSA252 is the most divergent among the seven human

strains compared according to the proportion of strain-

specific genes and pairwise synonymous substitution rates

(Holden et al., 2004; Hughes & Friedman, 2005). Thus,

Robinson et al. (2005a) proposed that the evolution of

S. aureus includes four phases. The initial phase is the

speciation event that led to the origin of S. aureus; the

Fig. 2. Circular representation of the MW2 chromosome compared

with other Staphylococcus aureus and CoNS strains. The outmost

magenta arcs represent mobile genetic elements and a large surface-

anchored protein-encoding gene (ebh); the black curve line represents

the ‘oriC environ’ that starts from SCCmec and ends at about 0.4 Mbp

on the chromosome. The four blue circles from the outside inward

represent orthologues of MW2’s coding sequences on Staphylococcus

haemolyticus JCSC1435 (accession no. NC_007168), Staphylococcus

saprophyticus ATCC15305 (NC_007350), Staphylococcus epidermidis

RP62a (NC_002976), and Staphylococcus aureus MRSA252

(NC_002952), respectively. The green circle represents MW2’s coding

sequences. The innermost circle represents GC skew (orange, positive

value; purple, negative value).


26 Y. Feng et al.

Table 1. Major virulence factors and regulators in Staphylococcus aureus that are present or absent from the sequenced CoNS strains�

Product Gene name Locationz S. epidermidis S. saprophyticus S. haemolyticus

Exoenzymes

1-Phosphatidylinositol phosphodiesterase plc � � �Staphylocoagulase coa � � �Triacylglycerol lipase lip 1 1 1

Lipase geh 1 1 �Serine protease htrA 1 1 1

Cysteine protease sspB,C 1 � �Serine V8 protease sspA 1 1 �Thermonuclease nuc 1 1 1

Serine proteases spl(s)z nSab � � �Staphylokinase sak Prophage � � �Hyaluronidase hysA � � �Zinc metalloproteinase aureolysin aur 1 1 �Cell wall hydrolase lytN � � �proteases ClpX clpX 1 1 1

Toxins

Exotoxins/superantigen-like proteins set(s)z,w nSaa � � �a-Hemolysin hly � � �b-Hemolysin hlb 1 � �d-Hemolysin hld 1 1 1

Leukotoxins lukD,E nSab � � �Leukocidins lukF,M SaPI � � �Panton-Valentine leukocidin lukS,F-PV SaPI � � �Toxic shock syndrome toxin 1 tst SaPI � � �g-Hemolysin components hlgA,B,C � � �Enterotoxins SE(s)z nSab‰ � � �exfoliative toxins eta,etb � � �

Adhesins

Extracellular matrix binding proteins ebhA,B 1 � �Elastin-binding protein ebpS 1 1 1

Fibronectin-binding proteins fnbA,B � � �Intercellular adhesion proteins icaA,B,C,D 1 � �Collagen adhesin precursor cna � � �Clumping factors clfA,B � � �Ser-Asp rich proteins sdr 1 1 1

Others

Immunoglobulin G (IgG)-binding protein A spa � � �Capsular polysaccharide synthesis proteins capA-G � 1 1

Lipoproteins lpl(s)z nSaa � � �Ferrichrome ABC transporter fhuD � 1 1

IgG-binding protein SBI sbi � � �Iron uptake Isd isdA-G,srtB � �

Two-component regulatory systems

Accessory gene regulator agrA,B,C,D 1 1 1

S. aureus exoprotein expression regulator saeS,R 1 � �Staphylococcal respiratory response protein srrA,B 1 1 1

Autolysis-related locus arlS,R 1 1 1

– lytR,S 1 1 1

SarA protein family

Staphylococcal accessory regulator A sarA 1 1 1

Staphylococcal accessory regulator R sarR 1 1 1

Staphylococcal accessory regulator S sarS � � �Staphylococcal accessory regulator T,U sarT,U � � �Repressor of toxins rot 1 1 1

�Some products, such as sarTand U, toxins located in mobile genetic elements, are not present in all S. aureus strains. The two sequenced S. epidermidis

strains are RP62a (accession no. NC_002976) and ATCC12228 (NC_004461); the sequenced S. saprophyticus strain is ATCC15305 (NC_007350); the

sequenced S. haemolyticus strain is JCSC1435 (NC_007168).wset cluster are now re-designated as ssl (Staphylococcal Superantigen-Like proteins) cluster Lina et al. (2004).z(s) indicates it is a gene cluster rather than a single gene.‰Most enterotoxin genes are located in nSab, but some enterotoxin genes, such as sea, seg2, sek2, sel, sec3, are located in prophages and SaPIs.znSaa and nSab are two genomic islands. Except those located in genomic islands, prophages, and pathogenicity islands (SaPIs), other virulence factors

and regulators are located in the core genome.



second phase is the divergence of S. aureus into two

subspecies groups, each having agr I, II, and III; the third

phase is the divergence of agr I and IV within subspecies

group 1; and the final phase is the recombination event

between agr I and IV, resulting in agr I/IV.

It is still unclear whether the divergence of the two

subspecies groups precedes the divergence of the agr groups.

However, it can be speculated that some more important

events may have occurred during S. aureus evolution based

on the hypothesis (Fig. 4). CCs should arise, at least after the

divergence of the three agr groups, because it seems im-

possible that the ancient agr is able to evolve to the same agr

variants in different lineages convergently. Meanwhile, the

genomic islands, nSaa and b, which exist in all S. aureus

strains, must have entered the genome shortly after the

speciation of S. aureus. Given the important role of nSaaand b in lineage formation, the hypothesis must be accepted

that the divergence of RM systems within the islands did not

occur at least until the divergence of agr groups.

Some diseases are known to be related to certain agr

groups, such as the association of agr III with menstrual

toxic shock syndrome (Ji et al., 1997) and Panton–Valentine

leukocidin (PVL)-induced necrotizing pneumonitis (Gillet

et al., 2002), the association of agr IV with exfoliatin

production (Jarraud et al., 2000), and the association of agr

I and II with reduced vancomycin susceptibility (Sakoulas

et al., 2002). It is probable that the genome of a certain agr

group has specific gene combinations that give rise to a

specific phenotype.

Evolution of the accessory genome

SCC

Methicillin resistance in MRSA results from the presence of

a modified penicillin-binding protein (PBP-2a alias PBP2’

and MecA), which has a reduced affinity for methicillin and

other b-lactams, and hence retains critical functions neces-

sary for cell homeostasis (Chambers et al., 1985; Lowy, 1998;

Mallorqui-Fernandez et al., 2004). PBP-2a is encoded by the

mecA gene located in the staphylococcal chromosome with-

in a discrete region called the SCC (SCCmec; Hiramatsu

et al., 2001). Apart from the mec divergon that further

encodes a transmembrane signal-transduction system to

trigger the resistance response, SCCmec possesses another

essential genetic component, the ccr complex, which is

responsible for the mobility of SCCmec. The rest of SCCmec

is designated as the junkyard (J) region, whose presence

does not appear to be essential for bacterial cells (Ito et al.,

2003). Five types of SCCmec have been described, according

Fig. 3. Comparison of relatedness derived from two different methods.

(a) Conditional tree constructed by the use of microarray analysis of core

variable genes from 161 isolates (Lindsay et al., 2006). (b) Phylogenetic

tree based on MLST, SAS sequences, and agr typing (Robinson et al.,

2005a). The dotted line separates lineages into two putative subspecies.

Fig. 4. Illustration of the hypothetical Staphylo-

coccus aureus evolutionary history. The whole

S. aureus species can be divided into two

putative subspecies (Robinson et al., 2005a). The

circles with different colors represent different

agr groups, and the circles with numbers inside

represent the corresponding clonal complexes.

The arrows on the right side indicate the

important phases during the S. aureus evolution.


28 Y. Feng et al.

to the combination of different variants of mec and ccr

complex and subtypes of J regions.

The first MRSA clinical isolate was reported in 1961, only 1

year after the introduction of the drug into the clinic (Jevons,

1961; Hiramatsu et al., 2001). Although the origin of SCCmec

is unknown, evidence of an interspecies exchange of DNA has

been found between CoNS and S. aureus (Wielders et al.,

2001; Wisplinghoff et al., 2003; Hanssen et al., 2004). Frequent

conversion of methicillin-sensitive S. aureus (MSSA) to MRSA

by the lateral transfer of SCCmec has also been described

(Enright et al., 2000; Fitzgerald et al., 2001; Robinson &

Enright, 2003), suggesting that MSSA is the origin of MRSA

and that MRSA strains may evolve multiple times indepen-

dently, rather than from a single ancestral strain. It is

noteworthy that MRSA is restricted to five CCs, but that

S. aureus as a whole is distributed among 11 CCs. It may be

the case that the five MRSA lineages have a greater capacity to

accept SCCmec by some unknown mechanism, even though

SCCmec is routinely inserted into a region adjacent to orfX

that seems to exist in all S. aureus variants. It may also be the

case that the five lineages are more virulent and prevalent;

selection pressure from antibiotics in the hospital setting has

perhaps necessitated the five lineages to retain SCCmec.

A variety of insertion sequences (ISs), transposons, and

plasmids have been found in SCCmec, including Tn554,

IS1272, IS431, pUB110, pT181, and p1258. Perhaps the mec

complex could even be regarded as a mobile element, as its

integration into SCC probably causes the conversion of SCC

into an antibiotic determinant. Apart from increasing the

range of drug resistance to antibiotics, such as methicillin,

macrolides, aminoglycosides, tetracycline, and bleomycin,

the insertion of these mobile elements provides potential

hot spots for recombination, therefore helping remodel the

structure of SCCmec and giving rise to a greater number

of structural variants. SCCmec III appears to be composed

of two SCC elements because it contains two copies of the

ccr complex and two copies of Tn554, which may be

explained by the sequential integration of two copies of

SCC, followed by deletion of internal parts (Ito et al., 2001).

Another mobile element often integrated into SCC is

the RM system, which exists in SCC476, SCCmec V, and

SCCpbp4. The origins of these RM systems are unknown,

but differences in the nucleotide sequences show that they

originated from different places. It is interesting to ponder

why RM systems prefer insertion into SCC; however, their

roles in S. aureus evolution should not be overemphasized

because only a small proportion of S. aureus possess SCC

that contain RM systems.

Genomic islands

The two islands, nSaa and nSab, have been found in nearly

all S. aureus isolates of divergent clonal, geographic, and

disease origins (Fitzgerald et al., 2003). Both islands are

nurseries of tandem paralogous gene clusters. nSaa encodes

for a cluster of staphylococcal superantigen-like proteins,

the so-called set cluster (now redesignated as the ssl cluster;

Lina et al., 2004), and a cluster of lipoproteins (lpl cluster),

while nSab encodes for a serine protease cluster (spl cluster)

and an enterotoxin cluster. All these clusters are virulence

factors, especially the enterotoxin gene cluster. Staphylococ-

cal diseases are often the result of the intake of enterotoxin-

contaminated food (Bunning et al., 1997).

Although the two genomic islands are ancient features of

the S. aureus genome, the evolution of these clusters is still

active. Frequent recombination and deletion events lead to

a variation of the copy number of toxin genes between the

isolates. Interestingly, Thomas et al. (2006) found that

within the enterotoxin gene cluster, most isolates have a

prevalent archetype that carries two pseudo-enterotoxins,

jent1 and 2, while in a few isolates, recombination between

the two pseudogenes has led to the emergence of new toxins.

It is therefore tempting to speculate that the accumulation

of virulence genes may not always confer an optimal

selective advantage on isolates. Likewise, Fitzgerald et al.

(2003) proposed an ‘independent loss’ model for the set

cluster, such that the ancestral state of the set cluster may be

represented by a complete complement of set genes and then

the loss of the set genes has occurred several times indepen-

dently within separate lineages. These phenomena contra-

dict the traditional view that more toxin variants offer the

pathogen more choices against the host immune system,

and that amplification may be selected if the paralogues have

a weak, but slightly selected product.

SaPIs and prophages

SaPIs and prophages are both important vectors carrying

virulence factors. Identified virulence factors include sta-

phylokinase, enterotoxins, toxic shock syndrome toxin, and

PVLs. Horizontal transfer of SaPIs relies on the ‘helper’

phage. It is now known that SaPI-1 can be excised and

circularized by staphylococcal phages F13 and 80a, and then

it can be efficiently encapsidated into special small phage

heads and replicates during the latter growth, which trans-

duces it at a very high frequency (Lindsay et al., 1998; Ruzin

et al., 2001). SaPI-2 and SaPI-3 can also be excised from

chromosomes and form extrachromosomal closed circular

DNA (Baba et al., 2002).

Many of the genes contained in SaPIs are homologous to

the described phage genes, suggesting they are of bacter-

iophage origin. Yarwood et al. (2002b) proposed a recombi-

nation model for SaPI genesis, following which a mis-

recombination event could have led to the replacement of

a segment of phage DNA necessary for complete phage

function with a chromosomal segment. In this way, SaPI



would have become dependent on a wild-type helper phage

for excision, packaging, and/or mobilization.

A remarkable feature of SaPIs, prophages, and phages is

their mosaic structure. Phage genes can be classified into six

functional categories: DNA replication, integration, packa-

ging, head, tail, and lysis (Kwan et al., 2005). Accordingly,

the distribution of these phage genes maps to discretely

functional modules. One functional module found in one

phage can be replaced in another phage by a sequence-

unrelated module that frequently fulfills the same or a

related function. Based on this theory, a module, rather than

the entire phage genome, has a relatively independent

evolutionary history (Brussow et al., 2004).

The mosaic structure confuses the nomenclature of the

prophage and SaPI to some extent. Lindsay & Holden

(2004) suggested classifying MGEs on the basis of integrase

gene homology, as this enzyme usually determines the MGE

insertion site within the genome. However, due to a module

exchange, an SaPI/prophage with the same integrase may

have an entirely different gene content. For example, even

though FPVL shares an integrase and the PVL locus with

FSLT, most genes of FPVL are more like prophage FSa3,

while genes of FSLT are more similar to FSa2. SaPI-3 in

Mu50 and MW2 are clustered together according to the

integrase sequence, but with respect to gene content,

SaPI-3 in MW2 seems to be more similar to SaPI-5 in

USA300 (Fig. 5).

Pathogenesis

Phage dynamics

Staphylococcus aureus is often considered to be an opportu-

nistic pathogen. On the one hand, it can cause life-threaten-

ing diseases; on the other, healthy people also carry S. aureus

in their anterior nares. From longitudinal studies, it has

become clear that 10–35% of individuals carry S. aureus

persistently, 20–75% carry S. aureus intermittently, and

5–50% never carry S. aureus in their noses (Armstrong-

Esther, 1976).

Another staphylococci species, S. epidermidis, is also an

opportunistic pathogen. The essential pathogenesis of for-

eign-body-associated S. epidermidis infection is biofilm

formation, which is a two-step process. The first step,

bacterial attachment to a surface, is related to a cell surface

protein (an autolysin) encoded by the chromosomal atlE

gene. The second step, including cell aggregation and

biofilm accumulation, is mediated by the products of the

chromosomal intercellular adhesion (ica) operon. Phase

variation of virulence in S. epidermidis can occur by

Fig. 5. Illustration of the mosaic structure of phages and SaPI in Staphylococcus aureus. Segments having sequence identities of more than 90% are

linked by green shading. Known functions of ORFs are colored as follows: lysogeny, blue; replication and recombination, red; packaging and head

protein, yellow; tail protein, green; lysis, cyan; toxin, black. (a) Alignment of phage/prophage sequences. Structures of the four sequences are indicated

based on the following nucleotide sequences: FSa3 in NCTC8325 (accession no. NC_007795), FPVL (NC_002321), FSLT (NC_002661), and FSa2 in

MW2 (NC_003923). (b) Alignment of SaPI sequences. Structures of the three sequences are indicated based on the following nucleotide sequences:

SaPI3 in Mu50 (NC_002758), SaPI3 in MW2 (NC_003923), and SaPI5 in USA300 (NC_007793).


30 Y. Feng et al.

insertion/excision of IS256 or by a rearrangement-mediated

genetic defect, which results in inactivation of the ica operon

(Ziebuhr et al., 1999, 2000).

There has been no report on chromosomal rearrange-

ment in S. aureus to date, and the number of IS in S. aureus

is evidently less than in CoNS. IS256 has only been found to

influence teicoplanin resistance in S. aureus by insertion

inactivation of the tca gene (Maki et al., 2004). It has also

been shown that a mutation in the ica genes of a clinical

S. aureus isolate has little effect on biofilm formation (Beenken

et al., 2004). The pathogenic mechanism of S. epidermidis thus

differs from S. aureus. Because a considerable part of the toxin

genes are located in SaPIs and prophages, phage dynamics are

of apparent importance for the pathogenesis of S. aureus.

Assays of consecutive isolates have revealed that commensal

strains possess a very low transformation rate and evolved

slowly over time; in contrast, phages are remarkably active

within pathogenic strains and the genome plasticity of patho-

genic strains is evidently elevated (Goerke et al., 2004).

According to clinical sampling and animal model experi-

ments, the role of certain phages for pathogenesis has been

proven by the fact that isogenic isolates, with and without the

phage, can have a strikingly different ability to cause disease

(Moore & Lindsay, 2001; Bae et al., 2006).

Toxin genes do not accumulate within the chromosome

without limit because MGEs can often exclude each other.

For example, no clinical isolates have been found to simul-

taneously contain TSST-1 (in SaPI-1) and SEB (in SaPI-3),

perhaps because the two SaPIs share identical att sequences

and therefore compete for the same insertion site in the

chromosome (Yarwood et al., 2002b). Negative correlations

between tst and lukE-splB and between lukE-splB and seg-sei

have also been reported (Moore & Lindsay, 2001). Because

lukE-splB are located in nSab, which exists in all S. aureus

strains, it is likely that phages that have tst or seg-sei are

inhibited by the type I RM system of certain lineages.

Although free transfer of MGEs is not allowed across

lineages, it is allowed within the same sequence type.

The TW strain has accumulated all detectable MGEs

that were variably expressed by other epidemic ST239

strains in the United Kingdom, and developed an enhanced

ability to cause bloodstream infection (Edgeworth et al.,

2007). Epidemiologists should always be vigilant of such

‘superbugs.’

The actual pathogenesis of phages in S. aureus is much

more complex, as exemplified by phage FSa3, which has

been studied by Goerke et al. (2006) in detail. This phage

encodes for the immune evasion molecules (SAK, SCIN, and

CHIPS) that are widely distributed in clinical isolates.

Usually, they insert specifically within the b-hemolysin

(Hlb) gene, so that the recipient is negatively affected by

the inactivation of a virulence factor, but atypical integra-

tion (not in hlb) has also been found. Analysis of the

sequence of the integrase gene demonstrated no difference

between typical and atypical sak-encoding prophages. Thus,

the integrase allows illegal integration, which contradicts the

classical view that the integrase specifically recognizes the

chromosome attB site. In addition, FSa3 was also found to

able to be stabilized extra-chromosomally during its life

cycle, although it is not known whether the phage is able to

express toxin genes in this state.

The knowledge of the type I RM system in S. aureus is still

unsatisfactory, although it is thought to be, at least partly,

responsible for lineage segregation. The extent of how the

type II RM systems restrict gene flow is also unclear. Phage K

is a large, virulent bacteriophage that infects a broad range

of staphylococci, including multiple-drug-resistant strains

of S. aureus. A remarkable paucity of the sau3A1 restriction

site (GATC) is thought to be an efficient mechanism that

phage K developed to avoid host restriction-modification

systems (O’Flaherty et al., 2004). In contrast, vanA-encod-

ing Tn1546 and the PVL locus have an abundance of sau3A1

restriction sites, which may be the basis for the two elements

residing in only a small proportion of strains. While these

sequence properties support the role of the type II RM

system in restricting gene flow, the contradicting phenom-

enon in Helicobacter pylori forces one to abandon the

notion. More than 20 RM systems, comprising more than

4% of the total genome, have been identified in sequenced

H. pylori strains (Lin et al., 2001), but H. pylori is naturally

competent to take up DNAs.

Expression and regulation of virulence factors

A number of virulence genes have been identified in the

S. aureus genome, conferring upon S. aureus the ability to

cause various types of disease; which genes are necessary for

which infection is still unclear. Many of the previous

epidemiologic studies have focused on the presence or

absence of a given genetic determinant. Regrettably, this

type of research cannot explain the phenomenon that toxic

shock syndrome cases are rare, while c. 20% of S. aureus

strains carry the toxin gene tst (Moore & Lindsay, 2001;

Peacock et al., 2002).

A direct comparison of S. aureus isolates collected from

both disease and asymptomatic carriers revealed no differ-

ence in the diversity or clonal assignments (Feil et al., 2003).

Thus, commensal and pathogenic strains are not two

distinct types of organisms, but the same organism in

different states. When S. aureus switches from commensal

to pathogen, it has to face a completely different environ-

ment and undergo a much more severe host defense system.

Thus, a global change of expression pattern is expected.

Voyich et al. (2005) found that under in vitro growth

conditions, the highly expressed genes involved in transcrip-

tion and protein biosynthesis, maturation, and folding



typically dominate bacterial gene expression; when phago-

cytized by neutrophils, the overall functional profile of

highly expressed genes would shift to pathogenicity-related

genes, such as those involved in virulence, metabolism,

capsule synthesis, and gene regulation.

Phage dynamics also cause conversion between com-

mensalism and pathogenicity, as discussed above. It should

be noted that virulence factors in phages do not express their

pathogenic roles independently. A recent study has shown

that the expression of PVL leukotoxin induced global

changes in transcriptional levels of genes encoding secreted-

and cell wall-anchored staphylococcal proteins that are

located in the core chromosome (Labandeira-Rey et al.,

2007).

The expression of staphylococcal virulence factors and

cell surface adhesion proteins is largely regulated by two-

component regulatory systems (agr, saeRS, srrAB, arlSR, and

lytRS) and the SarA protein family (SarA, SarR, Rot, SarS,

SarT, and SarU). agr is thought to be the most important

locus governing growth-phase-dependent regulation of

virulence factors (Novick et al., 1993; Novick, 2003). By

effecting promoter P3 transcript RNAIII, agr controls the

up-regulation of genes encoding secreted proteins (a-toxin,

b-hemolysin, TSST-1, and leukotoxins) and down-regula-

tion of genes encoding surface proteins (protein A, coagu-

lase, and fibronectin binding protein). SarA activates agr via

an SarA-agr promoter interaction (Chien & Cheung, 1998;

Rechtin et al., 1999) during the postexponential phase and

therefore alters the synthesis of virulence factors. SarA also

regulates several cell wall-associated proteins and exopro-

teins directly in an agr-independent way (Chien et al., 1999).

Most of these studies on agr regulation were performed

under in vitro conditions. When it comes to in vivo condi-

tions (i.e. in animal models of infection), expression of agr

does not significantly affect the expression of virulence

factors (Goerke et al., 2000, 2001; Heyer et al., 2002;

Yarwood et al., 2002a), perhaps because the environment of

an actual infection generates many signals that are not

present in laboratory media. Furthermore, cell intensity

may be an important source that gives S. aureus different

stimuli. When in the laboratory, S. aureus are usually

cultured under planktonic conditions. Staphylococcus aureus

more often grows in biofilm form during an infection

because a biofilm can help it withstand stronger host defense

responses and antibiotic stress. By microarray analysis, it is

known that the processes involved in cell wall synthesis and

other distinct physiologic activities of the cell play a crucial

role in biofilm persistence (Resch et al., 2005).

What the exact role of agr in the actual pathogenic

process is still unknown and there are many other phenom-

ena that cannot be explained by the current knowledge. For

example, epidemic MRSA (EMRSA) is thought to retain a

high secretion of toxin, given its enhanced ability to colonize

and infect. However, all EMRSA isolates tested are poor a-

toxin producers, while the sporadic strain maintains a

relatively high level of protein A (spa) transcripts during

the exponential and postexponential growth phases (Saber-

sheikh & Saunders, 2004). Given the enormous difficulty of

deciphering the complicated network of virulence genes, a

compromising approach would be to find an association

between lineage and disease. Theoretically, strains from the

same lineage have the same core variable genes and share the

common pool of SaPIs and phages for genetic exchange.

Therefore, they would potentially infect the same popula-

tion of people and cause the same type of disease. However,

it would be impertinent to conclude that all strains are

equally virulent. Even though each lineage may have a

specific set of core variable genes, especially adhesin genes,

which could determine the power of adhering to epithelial

cells of certain populations and decide the potential group

of the host population, many other prerequisites, such as

fast growth rate and strong survival ability, work together to

determine whether a clone would develop into a successful

pathogen. Indeed, some lineages deserve careful surveillance

based on epidemiologic investigations. For example, CC1

has emerged as the leading lineage that has a strong

association with community-acquired diseases.

Community-acquired MRSA (C-MRSA)

During the last two decades, staphylococci have shown a

trend of increasing virulence. CoNS have generally been

regarded as saprophytes or organisms with no or very low

virulence. However, there has been an increase in the

documentation of human infections due to CoNS, especially

with S. epidermidis. With respect to S. aureus, C-MRSA

outbreaks have been reported worldwide. The extreme

heterogeneity of the genetic background in C-MRSA indi-

cates that any strain of S. aureus has the potential to become

a C-MRSA. The reemergence of 80/81 isolates is the best

example (Robinson et al., 2005b). This early MSSA clone

waned in the 1960s when methicillin was introduced into

clinical use. However, it did not really disappear, but

probably took refuge, in healthy people as a commensal

strain. Upon acquisition of SCCmec IV, it reemerged as a

pandemic clone. Given the giant pool of MSSA strains,

including those carried in healthy people, it is unavoidable

that a new C-MRSA outbreak will occur sooner or later.

To investigate the pathogenesis of C-MRSA, it is most

important to distinguish C-MRSA from hospital-acquired

MRSA (H-MRSA), which is seemingly easy, but is actually

difficult. On the one hand, nosocomial colonization with

MRSA usually goes undetected and may lead to infection

many months after hospital discharge (when the patient is in

the community). On the other hand, enhancement of drug

resistance in recent years has made C-MRSA strains achieve


32 Y. Feng et al.

more success in replacing other MRSA strains in some

hospitals (Seybold et al., 2006). A general definition now is

that C-MRSA strains should be isolated in an outpatient

setting or from patients within 48 h of hospital admission;

such patients must have no history of MRSA infection and

no history in the previous year of either admission to a

nursing home, hospitalization, dialysis, or surgery.

SCCmec IV and the PVL locus are now considered to be

two characteristic features of C-MRSA strains. SCCmec IV is

rarely seen in H-MRSA strains, but it is the dominant allotype

in C-MRSA. C-MRSA is now thought to arise from horizon-

tal transmission of SCCmec IV into MSSA. SCCmec IV was

highly prevalent among S. epidermidis from the 1970s and has

not been found among MRSA isolates recovered during that

time. The first S. aureus isolates carrying SCCmec IV were

recovered in the early 1980s and then spread worldwide

(Wisplinghoff et al., 2003). Robinson & Enright (2003) found

that nearly one-half of conversions from MSSA clones to

MRSA clones involved SCCmec IV. Within the past 2 years,

SCCmec V has emerged with an intimate association with

C-MRSA, especially in Australia and Taiwan (Ito et al., 2004;

Boyle-Vavra et al., 2005; O’Brien et al., 2005). Compared with

multi-resistance conferred by SCCmec II and III, both

SCCmec IVand V have a small size, which has been attributed

to less metabolic burdens of protein synthesis during replica-

tion. Previous observations have reported that C-MRSA

strains grew significantly faster than H-MRSA strains (Baba

et al., 2002; Okuma et al., 2002). This high growth rate may be

a prerequisite for C-MRSA to achieve successful colonization

in humans by outcompeting the numerous bacterial species

in the environment.

PVL is a bi-component, synergohymenotropic toxin that

exerts cytolytic pore-forming activity directed at the cell

membranes of neutrophils, monocytes, and macrophages

(Kaneko & Kamio, 2004). Clinically, PVL is associated with

skin abscesses and necrotizing pneumonitis (Lina et al.,

1999; Gillet et al., 2002). Although PVL genes are usually

found in only 2% of S. aureus clinical isolates (Holmes et al.,

2005), they have been found in most C-MRSA strains

(Vandenesch et al., 2003). Within the two sequenced

C-MRSA strains, MW2 and USA300, the PVL locus resides

in prophage FSa2. However, Lindsay et al. (2006) have found

that in a collection of lukS, F-PV-positive isolates, only one

carried FSa2 genes, suggesting that the PVL locus is able to be

horizontally transferred among a wide range of phages.

ST36:USA200 and ST30:USA1100 provide an ideal model

for investigating the role of SCCmec IV and the PVL locus in

the pathogenesis of C-MRSA infections. They both belong to

CC30 and share very similar genetic backgrounds. The

SCCmec II, PVL-negative ST36:USA200 strain was endemic in

health care facilities during 1996–2000, while the SCCmec IV,

PVL-positive ST30:USA1100 strain was epidemic in commu-

nity populations during 1998–2001 (Binswanger et al., 2000).

Although the short SCCmec and PVL locus are important

for C-MRSA based on results of epidemiologic analyses, no

studies have proven their exact biologic roles in the patho-

genesis of community-acquired infections. In fact, there are

also C-MRSA strains that possess neither the short SCCmec

nor the PVL locus. Moreover, there has been research

suggesting that the PVL locus is not the major determinant

of C-MRSA because the PVL-negative and -positive strains

performed similarly during neutrophil lysis (Said-Salim

et al., 2005) and were equally lethal in a sepsis model

(Voyich et al., 2006).

Because the mechanism of C-MRSA infection is poorly

understood, the genomics approach has been applied in

some studies. Oligoarrays were used to detect specific

chromosomal regions for C-MRSA (Koessler et al., 2006).

Proteomics on MW2 and LAC were also explored, indicat-

ing that exoproteins accounted in part for the success of

C-MRSA (Burlak et al., 2007). Transcriptomic comparison

between H-MRSA (COL and MRSA252) and C-MRSA

(MW2 and MnCop) revealed that several putative mem-

brane or exported proteins of unknown function were up-

regulated only in the community strains (Voyich et al.,

2005). The difference found between these representative

strains may be the difference between lineages, rather than

the real difference between C-MRSA and H-MRSA. Ob-

viously, a direct comparison of C-MRSA and H-MRSA of

the same sequence type would generate more persuasive

data.

Concluding remarks

Genetic exchanges inside S. aureus and between S. aureus

and other staphylococcal species are one of the most

important topics in the research of the evolution and

pathogenesis of S. aureus. A scarcity of recombination

contributes to the highly clonal structure of S. aureus; lateral

transfer of MGEs is controlled by some cryptic rules,

resulting in mutual exclusion of toxin genes and preference

of specific toxin genes within certain lineages. A better

understanding of the underlying mechanism of genetic

exchange would help one to reconstruct the history of

lineage formation and to make some interesting predictions,

for example, whether those dangerous determinants, such as

vanA and the PVL locus, would disseminate to a larger

extent or whether the barriers to gene flow would make

clonal complexes evolve to become a new biological species.

The short SCCmec and the presence of PVL locus

characterize C-MRSA, but no studies have demonstrated

their exact biologic roles in C-MRSA infection. It is often

thought that C-MRSA and H-MRSA belong to different

lineages within a geographic area, but this is probably not

the case. In Taiwan, ST59 accounts for nearly all C-MRSA

infections and c. 20% of H-MRSA infections; the proportion



is still in an increasing trend. Thus, it is hypothesized that all

lineages have the potential to develop into both C-MRSA

and H-MRSA clones, if without competition from other

lineages. Given the fact that C-MRSA and H-MRSA can be

isolated within the same lineage, it is likely that the

difference of virulence gene expression has differentiated

the two types of organisms. Likewise, commensal and

pathogenic strains are the same organism of two different

states rather than two different types of organisms. Conver-

sion from commensal to pathogen must also be achieved by

a shift of the global expression profile. agr and sarA are two

global transcription regulators based on in vitro experi-

ments, but their regulatory effects are dramatically wea-

kened under in vivo conditions. The suspicion therefore

arises as to whether their roles in pathogenesis are over-

estimated. Thus, a future challenge for researchers is to

investigate the interaction between regulators and the viru-

lence genes in the pathogenesis of S. aureus.

Acknowledgements

The study of methicillin-resistant Staphylococcus aureus

in the Department of Pediatrics, Chang Gung Memorial

Hospital, Chang Gung University College of Medicine, was

supported in part by grants 94-2321-B-182A-002 from

National Science Council, Taiwan, and CMRPG33029 from

Chang Gung Memorial Hospital, Taiwan.

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