Evolution of virulence in epidemic community-associated methicillin-resistant Staphylococcus aureus.

Abstract

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged worldwide. The United States, in particular, is experiencing a serious epidemic of CA-MRSA that is almost entirely caused by an extraordinarily infectious strain named USA300. However, the molecular determinants underlying the pathogenic success of CA-MRSA are mostly unknown. To gain insight into the evolution of the exceptional potential of USA300 to cause disease, we compared the phylogeny and virulence of USA300 with that of closely related MRSA clones. We discovered that the sublineage from which USA300 evolved is characterized by a phenotype of high virulence that is clearly distinct from other MRSA strains. Namely, USA300 and its progenitor, USA500, had high virulence in animal infection models and the capacity to evade innate host defense mechanisms. Furthermore, our results indicate that increased virulence in the USA300/USA500 sublineage is attributable to differential expression of core genome-encoded virulence determinants, such as phenol-soluble modulins and alpha-toxin. Notably, the fact that the virulence phenotype of USA300 was already established in its progenitor indicates that acquisition of mobile genetic elements has played a limited role in the evolution of USA300 virulence and points to a possibly different role of those elements. Thus, our results highlight the importance of differential gene expression in the evolution of USA300 virulence. This finding calls for a profound revision of our notion about CA-MRSA pathogenesis at the molecular level and has important implications for design of therapeutics directed against CA-MRSA.

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Evolution of virulence in epidemic
community-associated methicillin-resistant
Staphylococcus aureus
Min Lia,b,1, Binh An Diepc,1, Amer E. Villaruza, Kevin R. Braughtona, Xiaofei Jiangb, Frank R. DeLeoa, Henry F. Chambersc,
Yuan Lub,2, and Michael Ottoa,2
aNational Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th Street, Hamilton, MT 59840, and 9000 Rockville Pike,
Bethesda, MD 20892;bDepartment of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, 12 Central Urumqi Road,
Shanghai 200040, People’s Republic of China; andcDivision of Infectious Diseases, Department of Medicine, University of California, San Francisco,
1001 Potrero Avenue, Box 0811, San Francisco, CA 94110
Edited by Richard M. Krause, National Institutes of Health, Bethesda, MD, and approved February 24, 2009 (received for review January 22, 2009)
Community-associated methicillin-resistant Staphylococcus aureus
(CA-MRSA) has recently emerged worldwide. The United States, in
particular, is experiencing a serious epidemic of CA-MRSA that is
almost entirely caused by an extraordinarily infectious strain
named USA300. However, the molecular determinants underlying
the pathogenic success of CA-MRSA are mostly unknown. To gain
insight into the evolution of the exceptional potential of USA300
to cause disease, we compared the phylogeny and virulence of
USA300 with that of closely related MRSA clones. We discovered
thatthesublineagefromwhichUSA300evolvedischaracterizedby
a phenotype of high virulence that is clearly distinct from other
MRSA strains. Namely, USA300 and its progenitor, USA500, had
highvirulenceinanimalinfectionmodelsandthecapacitytoevade
innate host defense mechanisms. Furthermore, our results indicate
that increased virulence in the USA300/USA500 sublineage is
attributable to differential expression of core genome-encoded
virulence determinants, such as phenol-soluble modulins and
?-toxin. Notably, the fact that the virulence phenotype of USA300
was already established in its progenitor indicates that acquisition
of mobile genetic elements has played a limited role in the
evolution of USA300 virulence and points to a possibly different
role of those elements. Thus, our results highlight the importance
of differential gene expression in the evolution of USA300 viru-
lence. This finding calls for a profound revision of our notion about
CA-MRSA pathogenesis at the molecular level and has important
implications for design of therapeutics directed against CA-MRSA.
C
United States (1). In contrast to hospital-associated (HA)-
MRSA, CA-MRSA strains cause infections in healthy individ-
uals without predisposing risk factors and outside of the hospital
setting. Although CA-MRSA has emerged worldwide, it is
epidemic in the United States (2, 3). The vast majority of
CA-MRSA infections in the United States are caused by a
recently emerged S. aureus clone known as pulsed-field type
USA300 (USA300). USA300 infections are primarily those of
skin and soft tissue, but the pathogen can cause severe invasive
disease and spreads easily and sustainably among humans (3–5).
CA-MRSA infections in other countries have not attained
comparable levels, produce less severe disease phenotypes, and
are commonly caused by clones unrelated to USA300 (2, 6, 7).
Given the high transmissibility of USA300, it is possible that this
clone could become a problem worldwide.
The molecular determinants underlying the success of
USA300 as a pathogen are not understood. Based on genome
comparisons and epidemiological evidence, it has been specu-
lated that determinants encoded on mobile genetic elements
(MGEs), such as the Panton-Valentine leukocidin (PVL), have
a predominant impact on virulence (8–10). However, recent
reports indicate that the contribution of these unique MGEs to
ommunity-associated methicillin-resistant Staphylococcus
aureus (CA-MRSA) is a major public health problem in the
CA-MRSA virulence may be comparatively minor (11–14). This
view is supported by reports on CA-MRSA infections that are
caused by PVL-negative strains (2, 6, 7). Thus, there likely exist
alternative explanations for the basis and evolution of the
enhanced capacity of USA300 to cause widespread disease.
Increases in disease severity or frequency are often linked to
the emergence of distinct bacterial clones of high virulence (15,
16). Therefore, to understand the evolution of USA300 better,
we performed a comprehensive analysis of the major MRSA
subclones of the clonal complex (CC) 8 lineage from which
USA300 arose (5, 10). We analyzed CC8 isolates obtained from
different geographical regions around the world to determine
presence and expression of key virulence factors and evaluated
virulence in vitro using human leukocytes and in vivo using
animal infection models. We demonstrate that the S. aureus
sublineage that led to the emergence of USA300 is characterized
by enhanced virulence, clearly distinguishing that sublineage
from others within CC8. Furthermore, our analyses indicate that
enhanced virulence of USA300 is mainly based on high expres-
sion of core genome-encoded virulence determinants rather
than the acquisition of additional virulence genes via MGEs.
Thesefindingsrepresentaparadigmaticshiftofournotionabout
how the pathogenic potential of USA300 evolved and are
importantforongoingeffortsaimedtofindtherapeuticsdirected
against CA-MRSA.
Results
Major MRSA Subclones of the CC8 Lineage. To investigate the
evolution of USA300 virulence, we selected 6 S. aureus isolates
representing all major epidemic clones of CC8 (17–19) (Fig. 1
and Table S1). First, to infer the genetic relation of these clones,
we analyzed DNA sequence variations within 7 housekeeping
and 7 surface protein-encoding gene fragments (Fig. 1 and Table
S1). According to our analysis, CC8 is split into 3 distinct
sublineages. Importantly, the data indicate that USA300 evolved
from a USA500 clone. Both USA500 and USA300 are epidemic
in hospital and community settings, predominantly in the United
States. The second sublineage, comprising the Archaic MRSA
clone and its contemporary successor, the Iberian MRSA clone,
was widely disseminated in hospitals in the United Kingdom and
Author contributions: M.L., B.A.D., F.R.D., H.F.C., Y.L., and M.O. designed research; M.L.,
B.A.D., A.E.V., K.R.B., X.J., and M.O. performed research; M.L., B.A.D., and M.O. analyzed
data; and B.A.D., F.R.D., and M.O. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1M.L. and B.A.D. contributed equally to this work.
2To
yuanlu@hsh.stn.sh.cn.
whom correspondencemay beaddressed.E-mail:motto@niaid.nih.gov or
This article contains supporting information online at www.pnas.org/cgi/content/full/
0900743106/DCSupplemental.
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other parts of Europe shortly after the introduction of ?-lacta-
mase–resistant penicillin into clinical use in 1961 (20). The third
sublineage, comprising the Brazilian/Portuguese MRSA clone
and its successor, the Chinese MRSA clone, is widespread in
hospitals in South America and Europe and hospitals in Asia,
respectively (17, 18). This sublineage evolved through a rare
genetic event involving lateral transfer and homologous recom-
bination with a ?557-kb fragment from the chromosome of a
strain belonging to CC30, another epidemic MRSA lineage (21)
(Fig. 1).
Sequential Acquisition of Virulence Genes by Contemporary CC8
Clones. To gain information about the genetic basis for the
pathogenic potential of the major CC8 clones, we assessed the
presence of 43 core genome- or MGE-encoded virulence genes
in representative isolates by PCR (Table S2). With specific
regard to the evolution of the USA300 sublineage, this analysis
led to the key observations that (i) USA500 MRSA does not
contain any known enterotoxin genes typically encoded on
prophages and pathogenicity islands and (ii) USA300 MRSA
evolved from the USA500 background genome, subsequently
acquiring multiple additional virulence determinants from
MGEs, including the arginine catabolic mobile element
(ACME), the staphylococcal pathogenicity island encoding en-
terotoxin K (sek) and enterotoxin Q (seq), and a prophage that
contains the lukSF-PV genes encoding PVL (10, 12).
Significant Differences in Virulence Exist Among CC8 Clones. We next
evaluated virulence of the 6 selected clones using murine models
of bacteremia and skin infection, which are frequent manifes-
tations of disease caused by CA-MRSA (2). USA500 and
USA300 were significantly more virulent in the animal infection
models compared with the other CC8 strains tested (Fig. 2 A and
D). In the bacteremia model, we also measured levels of TNF-?
and IFN-? in blood as markers of inflammation at the time of
death (Fig. 2 B and C). The levels of these cytokines correlated
well with survival curves in the bacteremia model (compare Fig.
2A with Fig. 2 B and C), consistent with the idea that animals
experienced bacterial sepsis. Notably, results for the USA300
and USA500 strains in the infection models were virtually
identical, indicating that the acquisition of virulence determi-
nants on MGEs by USA300 did not lead to a significantly
enhanced virulence potential by comparison. This is especially
noteworthy, given the absence of prophage- and pathogenicity
island-encoded enterotoxins in USA500. The virulence of the
other strains as assessed by the animal infection models was also
similar within the respective sublineages. Notably, in vitro
growth of the tested strains was virtually indistinguishable except
forstrainCOL,whichshowedslightlyslowerinvitrogrowth(Fig.
S1). This indicates that the presence of different staphylococcal
cassette chromosome mec (SCCmec) elements (Fig. 1 and Table
S1) does not have a strong impact on bacterial fitness in the
analyzed strains, as suggested particularly for the SCCmec type
IV element, which is smaller and size and does not lead to
different persistence in a competitive infection model compared
with an isogenic SCCmec-negative strain (12). Finally, distribu-
tion of the 43 virulence genes tested by PCR (Table S2) could
not explain the virulence patterns of CC8 subclones detected in
the animal infection models simply by gene presence. Thus, our
Fig. 1.
inferred by analysis of 7 housekeeping and 7 surface protein genes (Table S1).
Presence of virulence genes was determined by analytical PCR (Table S2).
Strains analyzed in this study are shaded in gray, blue, and red, representing
the 3 different CC8 sublineages, and are labeled with the specific strain
designations. *Single-nucleotide polymorphisms in housekeeping or surface
protein-encoding genes used to infer evolutionary relation (Table S1).
Evolutionary relation of CC8 subclones. The tree architecture was
Fig. 2.
(A) Bacteremia model. CD1 Swiss female mice were infected with 108cfus of
the indicated MRSA strains. Survival curves were compared using log-rank
(Mantel-Cox)tests.ConcentrationofTNF-?(B)andIFN-?(C)inmousebloodat
death. Strains for which statistically significant differences were achieved are
namedabovebars.ComparedwithPBS,valuesweresignificantlydifferentfor
BD02-25, SF8300, HS-522, and BAA43 (TNF-?) and for BD02-25 and SF8300
(IFN-?). n.s., not significant. (D) Abscess model. As a control, SKH1-hrBR
hairless mice were infected with 107cfus of the indicated MRSA strains, and
abscess or dermonecrosis areas were measured each day.
Virulence assessment of CC8 subclones by animal infection models.
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results emphasize the importance of genetic factors intrinsic to
the CC8 genotype rather than specific virulence factors acquired
via horizontal gene transfer from prophages or pathogenicity
islands and suggest that virulence within CC8 is determined to
a considerable extent by differential gene expression.
Interaction with Components of Innate Host Defense.VirulenceofS.
aureus is largely dependent on the interaction with innate host
defense.Neutrophilsarethemostprominentcellularcomponent
of human host defense against S. aureus. Host antimicrobial
peptides (AMPs) are a major component of innate host defense
in neutrophil phagosomes and on epithelial surfaces (22). To
evaluate whether the CC8 strains have differential capacity to
interact with these key components of innate host defense, we
measured the ability of S. aureus exoproteins to cause lysis of
human neutrophils and tested resistance to AMPs. Culture
filtrates from USA300 and USA500 caused significant lysis of
human neutrophils (70–80%) (Fig. 3). This finding is in pro-
nounced contrast to that observed for all other strains (?10% at
most). Additionally, the USA500 and USA300 strains had higher
resistance to dermcidin and indolicidin, 2 AMPs of human/
mammalian origin, whereas resistance to other AMPs was
almost unchanged (Table 1). In summary, these findings indicate
that the USA300/USA500 sublineage has increased capacity to
evade mechanisms of innate host defense.
Expression of key Virulence Genes. To test the hypothesis that
differential expression of virulence determinants dictates patho-
genic potential of MRSA sublineages within CC8, we evaluated
presence and expression of cytolytic toxins, molecules with a key
impact on immune evasion and virulence of S. aureus (23, 24).
We first confirmed that the genes encoding cytolysins were
present in all strains investigated (Fig. 4A and Table S2).
Next, we determined expression of ?-toxin and phenol-soluble
modulins (PSMs), S. aureus cytolytic toxins that kill white blood
cells and strongly influence virulence of CA-MRSA (25, 26).
Among PSMs, those of the ?-type have, by far, the greatest
cytolytic potential toward human neutrophils (25). Importantly,
?-type PSMs are largely responsible for neutrophil lysis, which
is caused by secreted factors of CA-MRSA, including USA300
(25). We detected higher production of ?-toxin (Fig. 4B) and
?-type PSMs (Fig. 4C) in the USA300/USA500 sublineage than
in the other strains. Furthermore, relative production of ?-type
PSMs in comparison to other PSMs with a lower cytolytic
potentialwasincreasedinUSA300andUSA500(Fig.4C).These
findings indicate that the observed increased potential of
Fig. 3.
culture filtrates from CC8 strains was determined by release of lactate dehy-
drogenase (LDH). Values were significantly different for all comparisons ex-
cept BD02-25 vs. SF8300 and all among HS-522, BAA-43, BAA-43, and COL.
Neutrophil lysis by CC8 subclones. Lysis of human neutrophils by
Table 1. Minimum inhibitory concentrations (mg/mL) of different strains to AMPs
AMPsBD02-25SF8300 HS-522BAA-43BAA-44 COL
Dermcidin-1
Indolicidin
Nisin
Gramicidin
Melittin
1.28
0.05
0.004
2.56
0.002
1.28
0.06
0.004
2.56
0.002
0.48
0.01
0.002
1.92
0.002
0.48
0.04
0.002
1.92
0.002
0.64
0.03
0.004
2.56
0.002
0.48
0.01
0.008
1.92
0.002
Fig. 4.
ofcytolysingenesbyanalyticalPCR.(B)Westernblotof?-toxinproduction.(C)
PSM production. PSM production in culture filtrates was determined by
RP-HPLC/electrospray ionization-MS. Relative production of ?-toxin (pink),
other ?-type (red), and ?-type (blue) PSMs is shown in pie diagrams. (D)
Hemolysis.Lysisofredbloodcellswasdeterminedbydroppingequalamounts
of cells on sheep blood agar plates, incubating at 37 °C, and measuring
clearing zones after 24 h. Values were significantly different for all compar-
isonsexceptBD02-25vs.SF8300andBAA-44vs.COL.(E)Proteolysis.Proteolysis
of supernatants concentrated by lyophilization was measured by an agar
diffusion assay. (F) RNAIII expression by qRT-PCR. (E and F) Values were
significantly different for all comparisons except BD02-25 vs. SF8300 and all
among HS-522, BAA-43, BAA-43, and COL.
Expression of virulence determinants in CC8 subclones. (A) Presence
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USA500 and USA300 to lyse neutrophils is largely attributable
to increased expression of ?-type PSMs.
In addition, we determined the capacity of the strains to lyse
erythrocytes, another target cell of cytolytic toxins (Fig. 4D).
Compared with USA500 and USA300, capacities of the Archaic
and Iberian clones to lyse red blood cells were considerably
reduced. However, we did not detect significant differences
between the 2 other sublineages. Nevertheless, these results
revealed differences in hemolytic capacities among strains con-
taining identical hemolysin genes.
S. aureus-secreted proteases have a key role in virulence as a
means to acquire nutrients from host tissues and protect against
AMPs. We found that proteolytic capacity of USA300 and
USA500 culture filtrates was significantly increased compared
with all other strains (Fig. 4E). Most likely, the differences in
AMP resistance that we observed (Table 1) are, in part, attrib-
utable to differential expression of secreted proteases and
differential susceptibility of AMPs to proteolytic digestion. That
is, degradation of dermcidin but not melittin correlated with
increased proteolysis (compare Fig. 4E and Fig. S2).
Thus, production of virulence determinants that are present in
the representative CC8 genomes tested here, particularly those
previously implicated in the virulence of CA-MRSA (25, 26),
correlated with virulence observed in the animal infection
models. These findings underscore the importance of differen-
tialgeneexpressionfortheevolutionofvirulencewithintheCC8
complex.
PSMs. Because PSMs have a demonstrated crucial role in deter-
mining CA-MRSA infection (25), we analyzed expression of
PSMs in more detail. We previously reported that selected
CA-MRSA strains have increased expression of PSMs compared
with representative HA-MRSA (25). Although appropriate for
analysis of the most prominent clones, the number of isolates
analyzed was limited. Therefore, we measured PSM?3 and
?-toxin expression, exemplifying relative expression of all PSMs
(27), in a large subset of S. aureus strains (n ? 88) from the same
geographical origin (San Francisco) (Fig. 5). The strains were
fromindividualswithcarriage,hospital,orcommunityinfections
and MRSA or methicillin-susceptible Staphylococcus aureus
(MSSA). Average PSM production was significantly higher in
USA300 and USA500 clinical isolates compared with that from
the major HA-MRSA lineages, USA100 and USA200. These
results confirm that CA-MRSA strains have increased PSM
production compared with the most prominent HA-MRSA
strains. Consistent with the results obtained using representative
USA500 and USA300 isolates (Fig. 4C), there was no significant
difference in average PSM production between a larger collec-
tion of USA300 and USA500 strains. These data indicate that
high PSM expression was established a priori in USA500, the
USA300 progenitor. Furthermore, average PSM expression in
USA300 and USA500 was not significantly different from that in
a mixture of CC8 MSSA clinical isolates (Fig. 5). This finding
confirms that methicillin resistance does not have an impact
on virulence, as shown recently in the USA300 and USA400
clones (12, 13, 28) and is consistent with reports on comparable
clinical outcomes observed in CA-MRSA and CA-MSSA strains
(29–31). Finally, we did not detect significant differences in
PSM?3 expression between carriage vs. infection isolates, indi-
cating that high expression of this key virulence determinant is
not selected for in strains causing disease but is a general
characteristic of the USA300/USA500 sublineage.
Role of the Global Virulence Regulator agr. Differential expression
of a series of virulence determinants as observed here for
USA300 and USA500 is often caused by changes in activity of
global regulatory systems in charge of virulence control. PSMs,
?-toxin, and secreted proteases are classic examples of virulence
determinants that are under control of the pivotal virulence
regulator agr (25, 32, 33). To evaluate the hypothesis that agr
expression contributes to the differential expression of virulence
determinants in the strains tested, we measured expression of
RNAIII, the major intracellular effector of agr (34). Activity of
agr was significantly increased in USA300 and USA500 (Fig. 4F).
This result indicates that the enhanced virulence potential of
these strains may, at least in part, be attributable to high
expression of this regulatory system.
On the other hand, several phenotypes, such as neutrophil
lysis, hemolysis, and proteolytic capacity, did not completely
correlate with RNAIII expression (Figs. 3 and 4 D, E, and F).
Furthermore, there was a greater difference in production of
PSM?3 (5.93-fold increased for USA300 vs. USA100/USA200
infection isolates; 6.23-fold increased for USA500 infection
isolates) than in that of ?-toxin (2.35- and 2.09-fold, respectively)
between USA300 and USA500 strains vs. USA100/USA200.
Although both the psm? operon and the RNAIII-embedded
?-toxin gene hld are under direct control of the AgrA response
regulator protein (27, 35), we previously found that the inter-
genic region in front of the psm? operon is subject to additional
regulatory influences on psm? expression (27). The findings
presented here are in accordance with these previous results and
indicate that although agr activity plays a major role in defining
the exceptional virulence potential of the USA300/USA500
sublineage, regulatory influences other than agr also have an
impact on expression of key virulence determinants in these
strains.
Fig. 5.
tions. Strains collected at hospitals in the San Francisco area were analyzed by
RP-HPLC/electrosprayionization-MSforPSMproduction.Carriageisolatesare
from nonhospitalized homeless youth and urban poor in the same area.
PSM?3 and ?-toxin levels are shown. Horizontal bars depict the mean. Statis-
tical analysis is by unpaired t tests or one-way ANOVA for comparison of
USA300, USA500, and MSSA carriage or infection isolates, respectively, which
showednostatisticallysignificantdifferences.N.S.,notsignificant.**P?0.01,
***P ? 0.001.
PSM production in HA-MRSA, CA-MRSA, and ST8 MSSA strain collec-
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Discussion
Bacterial strains that cause epidemics, such as USA300, com-
monly combine extraordinary virulence with efficient coloniza-
tion and host-to-host transmissibility. Whereas the analysis of
colonization capacity and transmissibility helps to explain a
pathogen’s persistence and spread, assessment of the virulence
potential allows predictions on the severity of disease that a
pathogen may cause. Distinction between these phenotypes is
crucial, because the underlying molecular factors may be entirely
different, especially in the case of S. aureus, which may colonize
humans in an asymptomatic fashion. Notably, the analysis of
virulence potential is a key prerequisite for endeavors to find
anti–CA-MRSAtherapeutics,particularlythosethatusemodern
target-oriented drug development (36). Therefore, in an effort
to establish a scientific basis for drug development against
CA-MRSA, we focused on the evolution of virulence within
CC8. Analysis of representative strains demonstrated that vir-
ulence was considerably increased in the lineage that comprises
USA300 compared with the other CC8 MRSA lineages. This is
consistentwiththereportedpotentialofUSA300tocausesevere
and widespread disease (37, 38).
MGEs, such as plasmids, prophages, transposons, and patho-
genicity islands, often contribute to bacterial virulence, espe-
cially in S. aureus (39). However, our results indicate that for the
CC8 lineage, particularly for USA300, differential expression of
core genome-encoded virulence factors rather than MGEs may
have a more profound impact on the evolution of virulence. This
is based on our observations that (i) high virulence potential of
USA300 was established in its progenitor strain, USA500 (i.e.,
before the acquisition of additional virulence determinants on
MGEs); (ii) virulence and virulence factor expression were
comparable among clones within the specific CC8 lineages; and
(iii) differential distribution of virulence genes among CC8
strains tested failed to explain the observed differences in
virulence merely by gene presence. Our results are in accordance
with experimental infection studies that indicate there is little or
no contribution of MGEs to USA300 virulence (11–14, 26),
particularly compared with the dramatic influence of the core
genome-encoded ?-toxin and PSMs (25, 26).
This leaves the question of why we do not see severe infections
withUSA500asoftenaswithUSA300.Inthatregard,ithasbeen
speculated that MGEs may contribute to USA300 transmission
rather than virulence, which is, in part, based on the putative
involvement of the ACME element in pH homeostasis (2, 10).
Although this idea remains to be evaluated, it might explain the
more pronounced spread of USA300, which would also cause a
higher frequency of infections by USA300 compared with
USA500. Furthermore, we cannot rule out the possibility that
the toxin genes that are encoded on MGEs and by which
USA300 differs from USA500, namely, the genes encoding PVL
and the enterotoxins K and Q, have a yet unidentified role in
USA300 virulence that is not detectable in mouse infection
models. However, recent epidemiological evidence shows that
CA-MRSA infections by strains other than USA300 may also be
caused by PVL-negative strains (2, 6, 7). This supports the notion
that PVL has a much less significant role in CA-MRSA virulence
than previously assumed.
Our results suggest that the global virulence and quorum-
sensing regulator agr has an important yet not exclusive role in
defining the virulence gene expression pattern resulting in the
increased virulence potential of USA300. Support for a key
function of agr in defining CA-MRSA virulence has come from
a recent study showing that the in vivo gene expression pattern
of USA300 is indicative of a highly active agr system (40).
Furthermore, Montgomery et al. (41) suggested that higher agr
activity of USA300 may have caused the substitution of the early
USA400 CA-MRSA clone by USA300. However, results from
our previous studies indicate that agr activity is not likely
involved in the differential virulence potential of these 2 more
distant CA-MRSA clones (25, 28).
The increasing burden of CA-MRSA, including the ongoing
epidemic in the United States, underscores the need to find
innovative therapeutics for MRSA disease. Although CA-
MRSA isolates are typically susceptible to many non–?-lactam
antibiotics, there is recent emergence of multidrug-resistant
CA-MRSA (42), thus confounding the current serious public
health problem. One major focus in the development of anti-
staphylococcal therapeutics is the design of agents that neutral-
ize virulence determinants (43). For these approaches to be
successful, an in-depth evaluation of the target virulence factors
of S. aureus is critical. Our findings have important implications
for strategies directed to find innovative anti–CA-MRSA ther-
apeutics because they suggest that virulence determinants of the
core genome, such as ?-toxin and PSMs, are more appropriate
for target-oriented drug development than MGEs, on which
manyoftheseendeavorsarecurrentlybeingfocused.Inaddition,
a therapeutic that targets core genome-encoded virulence de-
terminants would have much broader applicability compared
with one aimed at neutralizing virulence determinants limited to
CA-MRSA strains.
In summary, our study establishes that expression of core
genome-encoded virulence genes plays a more crucial role in
CA-MRSA virulence than factors present in MGEs. These
findings represent a substantial change in our notion of how
CA-MRSA virulence evolved and have important implications
for drug development against this leading human pathogen.
Methods
Bacterial Strains, Growth Conditions, and Basic Molecular Biology Methods.
MRSA isolates of sequence types USA100 (ST 5), USA200 (ST 36), USA300 (ST 8),
USA500(ST8),USA1100(ST30),andUSA1000(ST59)wereisolatedfrompatients
atSanFranciscohospitalsorfromnonhospitalizedindividualsintheSanFrancisco
area. BAA-44 (ST 247) and BAA-43 (ST 239) were obtained from American Type
Culture Collection (ATCC). Six ST 239 clones (including HS-522) were randomly
selected from unique patient clinical specimens of the ST 239 sequence type,
which accounted for ?40% of MRSA isolates, at Huashan Hospital, Shanghai,
ChinabetweenJanuary2006andDecember2006.ControlisolatesforPCR-based
assays were obtained from the Network on Antimicrobial Resistance in Staphy-
lococcus aureus (NARSA) and included the 5 reference strains with fully se-
quenced genomes—N315, Mu50, MW2, COL, and NCTC8325. Bacteria were
grown in tryptic soy broth unless otherwise noted. Cultures were incubated at
37 °Cwithshakingat200rpm.DNAmanipulationwasperformedusingstandard
procedures. PCR-based assays for virulence and housekeeping genes were per-
formedusingtheprimerslistedinTableS3.PrimersforDNAamplificationswere
purchased from Sigma Genosys. PCR reactions were performed with Ready-
To-GoPCRBeads(AmershamBiosciences)asrecommendedbythemanufacturer.
DNA was sequenced using Big Dye Terminator cycle sequencing (version 3.0) on
an ABI3700 sequencer (Applied Biosystems). Nucleotide sequences were ana-
lyzed using the program Vector NTI Suite (InforMax).
Mouse Bacteremia and Skin Abscess Models. Outbred immunocompetent CD1
Swiss female mice were used for the bacteremia model, and outbred immuno-
competentCrl:SKH1-hrBRhairlessmice(CharlesRiverLaboratories)wereusedfor
theabscessmodel.Allmicewerebetween4and6weeksofageatthetimeofuse.
S.aureusstrainsweregrowntomidexponentialphase,washedoncewithsterile
PBS, and then resuspended in PBS at 1 ? 108cfus/100 ?L (bacteremia model) or
1?107cfus/50?L(abscessmodel)asdescribed(5).Forthebacteremiamodel,we
injected each mouse with 108cfus of live S. aureus in 0.1 mL of sterile saline into
the retro-orbital vein. Control animals received sterile saline only. After inocula-
tion, mouse health and disease advancement were monitored every 3 h for the
first24handthenevery8hforupto72h.Weeuthanizedthemiceimmediately
if they showed signs of respiratory distress, mobility loss, or inability to eat and
drink.Allsurvivinganimalswereeuthanizedat72h.Atthetimeofdeath,serum
samples were harvested from test animals for the measurement of cytokine
expression with commercial ELISA kits (R&D Systems) according to the manufac-
turer’s instructions. For the abscess model, mice were anesthetized with isoflu-
raneandinoculatedwith50?LofPBScontaining107cfusofliveS.aureusorsaline
alone in the right flank by s.c. injection. Test animals were examined at 24-h
intervals for a total of 14 days; we measured skin lesion dimensions daily with a
Li et al. PNAS ?
April 7, 2009 ?
vol. 106 ?
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caliper.Weappliedlength(L)andwidth(W)valuestocalculatetheareaoflesions
with the formula L ? W. All animals were euthanized after completion of the
entire procedure. Animal studies were approved by the Animal Care and Use
Committee, Rocky Mountain Laboratories, National Institute of Allergy and
Infectious Diseases.
Further detailed protocols are reported in SI Methods.
ACKNOWLEDGMENTS. This work was supported by the Intramural Research
Program of the National Institute of Allergy and Infectious Diseases (NIAID),
NationalInstitutesofHealth(M.O.,F.R.D.);agrantfromtheShanghaiMedical
Key Discipline (to M.L. and Y.L.); Microbial Pathogenesis and Host Defense
Postdoctoral Fellowship 5T32AI060537–02 (to B.A.D.); and U.S. Public Health
Service Grant NIAID R01 AI070289 (to H.F.C.).
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