Infect Dis Clin N Am 16 (2002) 297318

Pathogenesis of streptococcal
and staphylococcal endocarditis
Philippe Moreillon, MD, PhDa,*,
Yok A. Que, MD, PhDa, Arnold S. Bayer, MDb
a

Division of Infectious Diseases, Centre Hospitalier Universitaire Vaudois,

BH19 Rue du Bugnon, 1011 Lausanne, Switzerland
b
HarborUCLA Medical Center, Division of Infectious Diseases, 1000 W. Carson Street,
Building RB2, Room 225, Torrance, CA 90509, USA

Viridans group streptococci and staphylococci (Staphylococcus aureus

and Staphylococcus epidermidis) are the pathogens most frequently responsible for infective endocarditis (IE) [13]. While sharing the same primary
infection site, these organisms often produce dierent disease syndromes.
Viridans streptococcal IE occurs in all populations at risk for this infection
and frequently causes a subacute syndrome with the protracted course of a
chronic wasting disease. In contrast, S. aureus is more common in elderly
patients and in intravenous drug users (IVDUs) [4], and is often responsible
for acute infection with associated rapid valve destruction, sepsis, and
shock. Finally, S. epidermidis IE predominates in the context of infection
of intracardiac prostheses and produces a rather indolent illness.
In the following discussion the authors focus on viridans streptococcal
and S. aureus IE, and describe some of the principal similarities and dierences between these two pathogens in the context of IE. The authors discussion of IE pathogenesis is divided in several distinct and sequential steps that
can be dierentiated in laboratory experiments. The primary event of IE is
bacterial adherence to target tissues. This is completed within minutes during transient bacteremia, and involves both valve tissue and bacterial factors. The second step is establishment and persistence of bacteria in situ
on the valve. The third step is microbial growth with local tissue damage
This work was supported by grants 3200-47099.96 and 3200-0458.95/2 from the Swiss
National Funds for Scientic Research (to P. Moreillon), and grants RO1-AI39108-04 from the
National Institutes of Health (NIAID), USA, and AHA 150699Y from the American Heart
Association (Western Regional Aliate) (to A.S. Bayer).
* Corresponding author.
E-mail address: pmoreill@chuv.hospvd.ch (P. Moreillon).
0891-5520/02/$ - see front matter
2002, Elsevier Science (USA). All rights reserved.
PII: S 0 8 9 1 - 5 5 2 0 ( 0 1 ) 0 0 0 0 9 - 5

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and possibly extension to adjacent tissues. The last step is hematogenous

dissemination to distant anatomical sites, particularly organs that receive
a large portion of the cardiac output (e.g., kidney, spleen, and brain). Viridans streptococcal and S. aureus IE present denable dierences in each of
these pathogenetic steps, as addressed below.
Valve colonization
Predisposing factors of the host
Most patients with IE have pre-existing valve abnormalities. The normal
endothelium is resistant to colonization and infection by circulating bacteria
[5,6]. Any endothelial lesion results in exposure of the underlying extracellular
matrix (ECM) proteins, the production of tissue factor, and the deposition of
brin and platelets as a normal healing process (Fig. 1). Such non-bacterial
thrombotic endocarditis (NBTE) is an ideal nidus for bacterial adherence and
colonization during transient bacteremia [7].
IE can also occur without known or identiable pre-existing valve
lesions. This is particularly true for S. aureus, which has emerged as the leading cause of IE in several recent surveys [2,3]. Thus, there might be at least
two scenarios for primary valve colonization: one requiring a physically
damaged endothelium (or NBTE) favoring infection by most types of
organisms, including viridans streptococci (Fig. 1A), and one occurring
on physically intact endothelia, either caused by virulent, invasive pathogens
(e.g., S. aureus) or by pathogens capable of parasitizing the endothelium, such
as C. burnettii, the agent of Q fever (Fig. 1B).
Colonization of the normal endothelium
Although S. aureus can colonize and invade cultured endothelial cells in
vitro [8,9], several studies indicate that bridging the bacteria to the surface of
endothelial cells with bronectin enhances the phenomenon [10,11]. S. aureus harbors surface-bound bronectin-binding proteins (FnBPA and
FnBPB) that avidly bind to immobilized bronectin [12]. Endothelial cells
may also bind bronectin and respond to cytokines and bacterial stimulation by expressing a variety of molecules, including integrins of the b1 integrins (late antigen or VLA) family that bind bronectin [13,14].
In the elderly population, more than 25% of individuals over 65 years of age
develop some degree of degenerative valve sclerosis [15]. This condition
involves local inammation, micro-ulcers, and microthrombi of the endothelium and is quite similar to arteriosclerosis [16]. Local inammation might
promote endothelial binding of bronectin and other ECM proteins that are
specic ligands for S. aureus adhesins. In IVDUs, repeated injections of
impure materials might also trigger cytokine production and endothelial
inammation and damage. Such a type of priming condition would explain
the clustering of S. aureus IE in elderly and IVDU populations.

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299

Fig. 1. Early steps in bacterial valve colonization. (A) Viridans group streptococci colonize
valves with preexisting damaged endothelia. The exposed underlying stromal cells and
extracellular matrix (ECM) proteins trigger the deposition of brin-platelet clots to which
streptococci bind avidly (upper panel). Fibrin-adherent streptococci attract monocytes and
induce them to produce tissue factor activity (TFA) and cytokines (middle panel). These
mediators activate the coagulation cascade, attract and activate blood platelets, and induce
cytokine, integrin and TFA production from neighboring endothelial cells (lower panel). This
vicious circle encourages vegetation growth, in which the bacteria are protected from cellular
host defense mechanisms. Streptococcal factors implicated in this process are summarized in
Table 1. (B) S. aureus can colonize physically undamaged endothelia. In response to local
inammation (e.g., valve sclerosis) endothelial cells express integrins such as vascular cell
adhesin (VCA) molecules, which bind plasma bronectin. S. aureus adhere to these surfaces via
wall-attached bronectin-binding proteins. Fibronectin-bridging triggers endothelial internalization of the bacteria (upper panel). In response to invasion endothelial cells produce TFA and
cytokines, thus triggering blood clotting and extension of the inammation. These reactions
promote the formation of the vegetation (middle panel). Internalized S. aureus eventually lyse
the endothelial cells by secreting membrane-active proteins (e.g., a-hemolysin) (see Fig. 3).
Tissue invasion is mediated by a variety of secreted enzymes and toxins. Switching between
expression of surface-adhesin molecules (e.g., bronectin-binding proteins) at the colonization
step, and secretion of toxins after internalization is orchestrated by global regulators agr and sar
(lower panel). The endothelium around the lesion consists of senescent cells.

Bacterial adherence
Adherence to NBTE is critical for valve colonization. Numerous studies
have demonstrated a correlation between the ability of bacteria to adhere in
vitro to plateletbrin matrices that mimick NBTE and their propensity to

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induce IE in human and experimental animal models [1719]. In a combined

system of experimental periodontitis and endocarditis in rats, dental procedures were accompanied by polymicrobial bacteremia. Some gingival bacteria were abundant in blood cultures while others were undetectable.
Nevertheless, only bacteria with high adherence phenotypes to platelet
brin clots (e.g., group G streptococci and S. aureus) were responsible for
post-extraction IE, and these strains were not those found most abundantly
in the blood [19]. Thus, there is a hierarchy in bacterial factors promoting
valve colonization, with adherence being among the most important.
Adherence is mediated by bacterial surface molecules engaging in specic
interactions with host ECM molecules. These are collectively called microbial
surface components recognizing adhesive matrix molecules (MSCRAMMs)
[20]. NBTE lesions carry multiple ECM molecules that act as specic
ligands for MSCRAMMs [21]. Likewise, streptococci (Table 1) and S. aureus
(Table 2) carry multiple MSCRAMMs that mediate binding to NBTE and
endothelial cells. Only a limited number of these MSCRAMMs have been
studied to assess their specic relevance to disease pathogenesis in experimental IE, however.
MSCRAMMs of viridans streptococci
Several streptococcal MSCRAMMs have been studied in relation to IE
pathogenesis (Table 1). Glucans are surface polysaccharides produced from
dietary sucrose by bacterial glucosyltransferase (gtf) and fructosyltransferase (ftf) enzymes [22]. Glucan production increases streptococcal adherence
to damaged valves and brinplatelet clots in vitro [7,17]. Their role in
experimental IE was demonstrated by using conditions favoring or not
favoring the production of these surface polysaccharides in vivo as well as
in gtf- and ftf-inactivated mutants of Streptococcus mutans [7,17,22]. Notably, gtf inactivation did not decrease experimental infectivity in Streptococcus gordonii [23]. Glucans might therefore be only one of several
factors required for infection.
Adherence to bronectin is associated with the ability of Streptococcus
sanguis to induce IE in rats [24]. A Tn917-insertional mutant lacking bronectin-binding activity is signicantly less infective than the parent strain.
While the specic adhesin responsible was not identied, binding of viridans
streptococci to bronectin and other ECM proteins is heterogeneous and probably involves multiple specic and non-specic mechanisms [25]. The impact
of other surface adhesins was demonstrated in vitro, including FBP-130 in
S. mutans [26] and CshA and CshB in S. gordonii [27,28] (Table 1). They were
not studied in vivo, however.
Finally, FimA is an important pathogenetic factor in Streptococcus parasanguis [29]. FimA belongs to a family of oral mucosal adhesins including
SsaB of S. sanguis, ScaA of S. gordonii, PsaA of Streptococcus pneumoniae,
and EfaA of Enterococcus faecalis [30]. Inactivation of FimA greatly

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Table 1
Streptococcal adhesins (MSCRAMMs) possibly involved in IE pathogenesis

Adherence
substrate
Fibrinplatelet
clots

MSCRAMMa
Surface glucans
in S. sanguis
and S. mutans
Surface glucans
in S. gordonii

Fibronectin

Gene or
function
Glucosyltransferase
(gtf)
Fructosyltransferase
(ftf)
Glucosyltransferase
(gtf)

Demonstrated
role in
experimental
IE

References

Yes
Yes

[17,22]

No

[23]

Tn917-inactivated
in S. sanguis
FBP-130 in
S. mutans
Adhesion molecules
in S. gordonii

Gene not described

Yes

[24]

Gene not described

No

[26]

Adhesin (cshA
and cshB)

No

[28]

Salivary pellicle
and possibly
ECM
molecules

mA in
S. parasanguis
Homologues in
other streptococci
(ssaB, scaA,
psaA, efaA)

Adhesin (mA)

Yes

[29]

Adhesins (ssaB,
scaA, psaA,
efaA)

No

[30]

Platelets

S. sanguis phase I
and Phase II
antigens
pblA and pblB in
S. mitis
pblT in S. mitis

Binding and
aggregation

Yes

[70,71]

Adhesins (pblA,
pblB)
Solute-binding
protein (pblT)

No

[73,74]

No

Many surface determinants are described in viridans streptococci [30], but few of them
were tested in experimental IE.

decreased the ability of S. parasanguis to adhere to immobilized brin and to

cause IE [29] in rats. Moreover, immunization of animals with FimA conferred
protection against subsequent experimental IE with the original strain [31].
FimA-like proteins were found by cross-hybridization in a number of
streptococci [31], suggesting that immunization could confer cross-protection against several organisms. While this awaits conrmation, it is noteworthy that inactivation of the ScaA homologue of FimA in S. gordonii
did not decrease infectivity in rats with experimental IE [32].
Although streptococcal adherence to the ECM is clearly implicated in IE
pathogenesis, successful infection probably requires the interplay between
several adherence determinants. Determining their nature, regulation, and
ligand specicity will require additional studies.

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Table 2
S. aureus adhesins (MSCRAMMs) involved in IE pathogenesis
Gene
or function

Demonstrated
role in experi
mental IE

References

Clumping factor A
Clumping factor B
Coagulase
Extracellular
brinogen-binding
protein
Fibrinogen-binding
protein A

clfA
clfB
coa
efb (formerly b)

Yes
No
No
No

[45,49,53]
[46]
[45,47]
[106,107]

fbpA

No

[108]

Fibronectin

Fibronectin-binding
protein A and B

fnbA, fnbB

Yes

[53,54]

Collagen

Collagen-binding protein

cna

No

[109]

Broad-spectrum
ECM

MHC class II analog

(map or eap)
Serineaspartate
repeat proteins (SDR)

map or eap

No

[110,111]

sdr gene family

No

[112]

Adherence
substrate
Fibrinogen/brin

MSCRAMM

MSCRAMMs of S. aureus
S. aureus is extremely well equipped with both MSCRAMMs and
secreted factors that mediate tissue colonization and invasion [33]. Most
described MSCRAMMs (Table 2) are covalently attached to the peptidoglycan stem peptide by way of the conserved C-terminal LPXTG and cell wall
attachment sortase system [34]. Some of them, including coagulase [35],
are loosely attached to the wall.
The expression of these factors is orchestrated by the global regulators
accessory gene regulator (agr) [36], staphylococcal accessory regulator (sar)
[37], and potentially by other determinants such as sigB [38]. Together, sar
and agr coordinately regulate the expression of adhesins during exponential
growth and the secretion of soluble factors in the post-exponential growth
phase in vitro [39]. Moreover, they were shown to be critical for infectivity
in experimental IE and other models in vivo [4044]. Thus, S. aureus pathogenesis not only depends on the intrinsic role of individual virulence factors,
but also on their regulated interplay in the hostparasite relationship.
In such an intricate context it is quite dicult to analyze the pathogenic
role of individual gene products by classical gene inactivation and gene complementation experiments. For instance, experiments with adhesin-defective
mutants suggest that clumping factor A (ClfA) had a substantial role in
experimental IE [45], whereas ClfB, coagulase, and bronectin-binding
proteins (FnBPA and FnBPB) had no impact at all [4549]. This was in
contradistinction to the visible coagulase activity of such strains in vitro and

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the fact that ClfA, ClfB, and FnBPs all conferred signicant bacterial adherence to host proteins present in endovascular lesions [12,50,51].
To study the individual importance of these surface adhesions in IE more
precisely, a genetic transfer system was developed to express these adhesin
genes separately or together in a surrogate genetic background lacking the
normal S. aureus MSCRAMM multiplicity and redundancy. This was achieved using both S. gordonii and Lactococcus lactis as recipient organisms
[49,52]. In S. gordonii, expression of ClfA increased the ability of bacteria
to both adhere to plateletbrin clots in vitro and to colonize damaged
heart valves in live rats by about one order of magnitude. On the other
hand, the expression of coagulase either alone or together with ClfA had
no signicant eect.
Since S. gordonii itself may cause IE, it may carry its own pathogenic
determinants and thus mask the eect of transferred genes. The system was
improved by expressing S. aureus genes in the nonpathogenic organism
Lactobacillus lactis [52,53]. Expression of ClfA increased the infectivity of
lactococci by 100 in this setting, thus highlighting the critical role played
by this adhesin. Moreover, expression of FnBPA also increased lactococcal
infectivity by 100, indicating that this second determinant is also critically
important.
This observation helped solve a previously unsettled and contentious
issue in the literature. Using an S. aureus mutant inactivated in its unique
fnb gene, Kuypers et al. demonstrated that bronectin binding was critical
for induction of experimental IE [54,55]. In contrast, using an S. aureus isolate carrying two fnb genes (fnbA and fnbB), Flock et al. observed that inactivation of both of these determinants did not decrease infectivity in rats,
although persistence of the organisms on the valve was reduced [48]. The
results obtained with recombinant lactococci indicated that both determinants are important, but can probably complement each other in singlegeneinactivated mutants. This kind of redundancy might explain some of
the unexpected results in single gene inactivation experiments.
Dierential colonization of NBTE and endothelial cells in vivo
The lactococcal system also helped dierentiate the involvement of ClfA
and FnBP in valve colonization and persistence. Valves of rats with experimental IE were examined by immunohistochemistry and electron microscopy
(EM). After infection with ClfA-positive lactococci, the microorganisms
were strictly localized within the NBTE, but were absent from the adjacent
endothelium. In contrast, FnBPA-positive lactococci co-localized both within
the NBTE and the neighboring aortic endothelium. EM indicated that
FnBPA-positive lactococci were invading the host endothelium and could
divide intracellularly (Fig. 2). The requirement for FnBP to invade endothelial
cells was conrmed in vitro and in vivo for S. aureus [10], thus supporting the
concept depicted in Fig. 1B.

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Fig. 2. Electron microscopy of the endothelium adjacent to aortic vegetations in a rat with
experimental aortic endocarditis. The animal was infected with a recombinant Lactococcus
lactis expressing the bronectin-binding protein A (FnBPA) of S. aureus. The picture illustrates
massive invasion of an endothelial cell (EC) with the recombinant organism. Bacteria can be
seen both at the vascular lumen (L) cell surface and in the cytoplasm, and are actively dividing
in both environments. Smooth muscles (SMC) are not invaded. Controls, wild type L. lactis or
L. lactis expressing other S. aureus adhesins (e.g., clumping factor protein A), were not
internalized (see text for details). Original magnication was 8,300.

Taken together, the studies in L. lactis indicate that both ClfA and
FnbPA are critical factors for IE induction while FnbPA is also a persistence factor. Thus, for future anti-adhesin strategies (e.g., MSCRAMM vaccines), both of these latter adhesins should be targeted simultaneously.

In situ bacterial persistence

After valve colonization, the infecting organism must develop strategies
for local survival and persistence as a prelude to growth and proliferation.
A key event in this process is maturation of the vegetation, within which the
microorganisms become fully enveloped, and thus protected from cellular
and soluble host defense mechanisms. Local production of tissue factor
(TF) and aggregation of platelets are major elements of this process. Both
streptococci and S. aureus present distinct mechanistic dierences in terms
of activating TF and platelets.

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Tissue factor
TF is a 47 kDa integral membrane glycoprotein [56]. Combined with
activated factor VII, it activates factor X, which cleaves prothrombin to
thrombin, which then triggers the polymerization of brinogen into brin.
TF also activates platelets, which are integral components of the vegetation
[5,6,57,58].
TF elaboration is essential for vegetation growth. It is not produced by unperturbed endothelial cells or monocytes, but can be induced by various agonists, including cytokines (IL-1) and bacterial lipopolysaccharides [56,59].
Intra-vegetation TF activity (TFA) was demonstrated in specimens from
experimental IE due to S. sanguis, S. aureus, and S. epidermidis [6062]. The
mechanisms of TFA induction from various host cells diered between these
organisms, however.
TFA induction from the host monocytes
Since TFA is cell membrane-associated, only endothelial cells, broblasts, and monocytes are candidates for its local production in the valve
lesion in IE. Endothelial cells and broblasts are mostly absent from the
early vegetation while scattered monocytes are present [5,6]. Thus, monocyte-related TFA could contribute to building up the vegetation.
Binding of monocytes to brin clots elicited TFA in vitro [63]. Moreover,
addition of S. sanguis to the system increased TFA by circa tenfold. Increased
TFA occurred despite the inability of monocytes to physically engulf and
remove brin-adherent streptococci (referred to as frustrated phagocytosis), suggesting that some extrinsic bacterial factor was involved [64]. Likewise, the presence of bronectin in the clot was necessary for activation.
S. aureus and S. epidermidis can also induce TFA from monocytes [61,62].
Moreover, the contribution of monocytes to intra-vegetation TFA was conrmed in animals with etoposide-induced monocytopenia [6062]. Thus,
bacteria can encourage vegetation growth by subverting monocytes to produce TFA. The process is by no means simple, however, and requires the
presence of both bacterial and ECM molecules.
Eventually, monocytes may downregulate TFA production in response
to endogenous tumor necrosis factor (TNF)-dependent production of IL10 [65]. This further underlines the subtlety of the bacterialhost interaction
in the disease process.
TFA induction from the host endothelial cells
Because S. aureus IE can develop on physically intact endothelia, the
question arises as to whether these organisms can trigger local vegetation
formation by direct induction of endothelial TFA. Veltrop et al. [66] showed
that S. aureusbut not S. epidermidis and S. sanguiscould induce TFA
expression from cultured endothelial cells. Treating the cells with interleukin
1 (IL-1) also induced TFA. However, abrogating the IL-1 response with an

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IL-1-receptor antagonist did not abrogate TFA induction by S. aureus.

Thus, IL-1 and S. aureus induce endothelial TFA by distinct mechanisms.
The additional presence of monocytes is needed to induce endothelial
TFA with S. epidermidis and S. sanguis [14]. This requires endothelial expression of intercellular adherence molecule 1 (ICAM1) and vascular cell
adhesion molecule 1 (VCAM1), and probably the contribution of bacterial
and ECM molecules. While the mechanism of this monocyte-induced
endothelial TFA is unknown, it could bypass the intrinsic inability of S. epidermidis and S. sanguis to induce endothelial TFA directly and promote
vegetation growth of the original NBTE lesion.
Thus, TFA induction seems to be an essential step for in situ bacterial
persistence. In doing so the microorganism can subvert monocytes, a major
player in the innate immune system. Next, as discussed below, the organism
must survive innate host antibacterial systems (e.g., platelet microbicidal
proteins secreted and released locally by activated platelets at sites of
infected vegetations).
The dual role of platelets in IE pathogenesis
The ability of bacteria to trigger platelet aggregation has been classically
considered as a potential pathogenic factor in IE. However, bacteriainduced platelet activation is a double-edged sword. On the one hand, platelets contribute to the formation of the vegetation [5,6,57]. On the other
hand, they also contribute to anti-infective host defenses by releasing antimicrobial peptides and inammation mediators [58]. Interaction of both
streptococci and S. aureus with platelets is briey described next.
Pathogenic role in streptococcal IE
The roles of platelet binding and aggregation in IE pathogenesis were
initially delineated in S. sanguis [67,68]. In this organism, the ability to
induce platelet aggregation is conferred by two surface-expressed antigens
[69,70]. The class I antigen promotes streptococcal adhesion to platelets
(Adh+ phenotype). The class II antigen triggers further platelet aggregation
(Agg+ phenotype). This so-called platelet aggregation-associated protein
(PAAP) contains a collagen-like platelet-interacting domain [69].
Herzberg et al. found that an Agg+ S. sanguis isolate produced more
severe infection and larger vegetations than an Agg) control strain in rabbits with experimental IE [70]. Manning et al. [71] reported similar results
with two unrelated Agg+ and Agg) S. sanguis isolates. Kitada et al. [72],
however, found no clear correlation between platelet aggregation and the
severity of experimental IE in other types of viridans streptococci.
In all of these studies the Agg) comparator strains were either not characterized for other pro-endocarditis factors [70,72] or were less adhesive to
bronectin, brinogen, and plateletbrin clots [71]. No strong conclusions
can therefore be drawn. Moreover, although the Agg+ phenotype might be

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307

a pathogenic factor, not all IE streptococcal isolates demonstrate this phenotype [68]. Additional factors must also be responsible.
Recently, streptococcal platelet binding interactions were further characterized in Streptococcus mitis [73]. Binding was mediated by a complex interaction between at least three loci (pblA, pblB, and pblT). PblA and pblB are
surface structures that are likely to act as adhesins. PblT is homologous to a
membrane transporter of the major facilitator superfamily. Solute-binding
proteins of such transport systems have been shown to mediate adherence
[30]. PblT might therefore be involved in platelet interactivity as well. Mutants in these loci have yet to be tested in vivo in experimental IE. Of note,
pblA and pblB are encoded by a lysogenic bacteriophage (SM1) of S. mitis,
suggesting a mechanism for the dissemination of these potential virulence
determinants to other pathogens [74].
Pathogenic role in S. aureus IE
S. aureus may directly bind platelets in a plasma-independent manner
[75]. After platelet binding S. aureus triggers platelet aggregation by way
of bridging with brinogen, but not bronectin [76]. Adhesion of S. aureus
to activated platelets may also occur by way of thrombospondin, vWF, proteinA, or C1q bridging [77].
The role of the S. aureus platelet aggregation phenotype in experimental IE was studied by Sullam et al. [78]. They compared an Agg+ parent
S. aureus to a Tn551-insertional mutant with decreased platelet binding
and aggregation capacities. Both organisms were equally able to colonize
catheter-induced vegetations 30 seconds after bacterial challenge. At 48 hours,
however, the parent strain was up to 100-fold more infective than the
mutant, as determined by the size of inoculum required to infect the majority of the animals. The organisms were comparable for other relevant virulence properties, including their susceptibility to platelet peptide-induced
killing and their ability to bind to brinogen and bronectin. This provides
strong evidence for the pathogenic role of S. aureus-induced platelet aggregation in IE pathogenesis. The nature of the bacterial factor responsible has
yet to be determined. Recent data from the same laboratory have shown
that the Tn551 insert resided within the clfA locus, further underscoring the
importance of brinogen binding in this process. Moreover, they demonstrated that ClfA bound avidly to a 118 kDa platelet membrane protein
receptor that remains to be identied [79].
The antimicrobial eects of platelets
Aside from their implication in promoting IE pathogenesis, platelets also
play an important role in preventing and mitigating the infection. Platelets
harbor three types of granules (a, d, and k) that release a variety of mediators
involved in adhesion and coagulation (a granules), vascular tone (d granules),
and thrombus dissociation (k granules) [58]. a granules also contain an arsenal

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of antimicrobial peptides, collectively called PMPs (in rabbit platelets) or

thrombocidins (in human platelets). Up to seven PMPs have been described
in rabbit platelets, with selected PMPs being released by specic agonists
(e.g., thrombin stimulation) [80]. In human platelets, two thrombocidins have
been delineated that represent C-terminal truncates of previously described
CXC chemokines, NAP2 and CTAPIII [81]. Such platelet antimicrobial peptides of rabbit and human origin represent one of the rst and most potent
hostdefense barriers that bacteria encounter in their quest to initiate endovascular infections.
Role of platelets in protection against streptococcal IE
Sullam et al. [82] observed that experimental IE due to an S. sanguis
strain was more severe in thrombocytopenic rabbits than in control animals
with normal platelet counts. The infecting organism aggregated platelets
extensively in vitro and was killed rapidly by PMPs in vitro. Dankert et al.
[83] conrmed the role of PMPs in experimental IE by using a series of
PMP-susceptible and PMP-resistant streptococcal isolates. PMP-susceptible
streptococci more eectively colonized damaged valves than PMP-resistant
strains 5 minutes after bacterial challenge. However, PMP-susceptible
organisms were eradicated rapidly from the vegetations during the following
24 to 48 hours. In contrast, PMP-resistant strains persisted at this site and
induced a progressive infection. Thus, although streptococci may subvert
platelets to develop the vegetation [70,71], they may only be able to accomplish this if they initially escape the microbicidal actions of PMPs [70].
Role of platelets against S. aureus infection
The mechanism of action of thrombin-induced PMP-1 (tPMP-1), the
principal PMP released from rabbit platelets, has been claried in S. aureus
[80]. Like other PMPs, tPMP-1 is a cationic molecule of relatively small
size (8.036 Da). It permeabilizes the cytoplasmic membrane in a voltagedependent manner without classic pore formation [81,84]. This diers from
other types of bactericidal peptides such as neutrophil defensins [85]. Most
mutants of S. aureus resistant to tPMP-1 studied to date share a common
phenotypea decrease in membrane potential and an increase in membrane
uidity [84,86]. This supports the notion that the bacterial membrane is a
primary site of action of PMPs.
Several experimental and clinical studies support the protective role of
PMPs in intravascular infections, hence the salutary eect of PMP-resistance
for S. aureus virulence [8791]. PMP-resistant S. aureus strains in rabbits
produced more severe experimental IE than their PMP-susceptible parent
and were more dicult to eradicate by antimicrobial therapy [87,88]. S. aureus isolates from intravascular infections in humans were more often resistant to PMPs than those isolated from other types of infections [90,91].
Despite resistance, however, PMP may play a role in therapy. tPMP-1 was
shown to act synergistically with several antibioticsin particular cell wall

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309

active drugsboth in vitro and in vivo [92,93]. Moreover, this eect was
observed against both PMP-susceptible and PMP-resistant isolates. Thus,
development of PMP or thrombocidin analogs for therapy may be envisioned.
The possible value of anti-platelet therapy
Since platelets are involved in IE pathogenesis and vegetation growth,
investigators have explored the eect of adjuvant anti-platelet therapy. Limited clinical observations have suggested that aspirin might prevent vegetation growth and decrease cerebral septic emboli in human IE [94]. Nicolau
et al. [9597] have studied the role of aspirin and ticlopidine, two anti-platelet drugs with dierent modes of action, in rabbits with S. aureus experimental IE. Both drugs synergistically decreased the vegetation size. They also
moderately decreased the vegetation bacterial titers. The PMP-resistance
phenotype of the infecting organism was not reported, however. Because
blockade of platelet activation (and subsequent release of antimicrobial peptides) could potentially promote IE due to PMP-susceptible bacteria, assessing this phenotype is important.
Kupferwasser et al. [98] have further claried the mechanism of aspirins
benecial eects in IE using the same model of S. aureus IE. Aspirin signicantly decreased vegetation weight, vegetation bacterial titers, and renal septic emboli in vivo. Moreover, pre-exposure of platelets to aspirin in vitro
and pre-exposure of S. aureus to salicylic acid (the principal biometabolite
of aspirin in vivo) decreased both platelet adherence and aggregation and
bacterial adherence to brin and brinplatelet matrices in vitro. Thus,
aspirin reduced several severity factors of experimental IE, and these benets involved the eect of aspirin on both the platelets and the microbe.
Experiments have also indicated that aspirin, but not ticlopidine, was synergistic with vancomycin in decreasing vegetation bacterial densities and
increasing valve sterilization [96,99].
Collectively, these experimental IE studies indicate that aspirin may have
a dual benecial impact on mitigating infection by way of both (1) aspirinmediated reduction in platelet aggregation, and (2) aspirin biometabolite
(salicylic acid)-mediated antistaphylococal eects. The precise mechanisms
by which the direct antimicrobial eects of salicylic acid are mediated
remains to be fully elucidated. Anti-platelet therapy carries the risk of bleeding; therefore, extrapolation of these experimental results to the human
situation must be tempered by the risk of hemorrhage during acute IE and
awaits formal clinical evaluation.

Invasion and dissemination

Local tissue invasion and abscess formation are primary features of
S. aureus infections [33]. S. aureus surface-bound factors are summarized in

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Table 2. Secreted factors include enzymes (membrane-active or proteins) and

super-antigens [33].
Relatively few studies have investigated individual S. aureus factors relevant to valve invasion. Using the L. lactis adoptive pathogenesis system,
Que et al. [10,53] demonstrated that both S. aureus ClfA and FnBPA are
important for early valve colonization. When looking at later stages of infection, however, they observed that ClfA-positive lactococci were progressively
cleared from the vegetation while FnBPA-positive lactococci caused
increased vegetation bacterial titers and induced clinical signs of sepsis [10].
In the light of data cited above indicating the critical role of ClfA in platelet
binding, it is conceivable that ClfA-bearing lactococci may bind more readily
than FnBPA-bearing cells to platelets in vivo, bringing them in close proximity to platelet-released antimicrobial peptides within the vegetative lesion.
In these latter models, disease progression correlated with the ability of
FnBPA-positive recombinants to invade perivalve endothelial cells in vivo
(Fig. 2) [10,11]. Moreover, while FnBPA-positive lactococci could be visualized in intact endothelial cells, similar experiments with S. aureus detected
the microbes in both intact and lysed cells (Fig. 3). This suggests that additional factors produced by staphylococci (e.g., hemolysins) could disrupt the
endothelial envelope and allow further tissue invasion and spread to other
anatomical sites.
This hypothesis ts the regulatory sequence of agr and sar during the natural growth cycle [100]. First, expression of surface adhesins, such as FnBPs,
allows endothelial colonization and invasion. Second, bacterial growth in
situ results in increased bacterial density and quorum- sensingdependent agr
activation. Third, agr activation triggers the secretion of proteins such as
hemolysins that lyse endothelial cells. Alternatively, endothelial cells could
provide a niche in which bacteria could become sequestered, thus escaping
host defenses or antibiotic eects for a prolonged period of time [101].
The impact of the agr locus upon abscess formation was demonstrated
recently in mice [44]. Inactivating agr either by mutation or by treatment
with an inactive analog of the agr-activating peptide drastically decreased
the formation of subcutaneous abscesses. The demonstration of in situ sar
activation during experimental IE also ts this global scenario [102].
Paradoxically, however, over-expression of ahemolysin (an agr-dependent phenotype) decreased the severity of infection in experimental IE
[103]. This was hypothesized to be due to PMP-induced bacterial killing
resulting from activation and lysis of platelets by ahemolysin. This observation further highlights the critical role of gene regulation in pathogenesis.
Inverting the sequence of gene expression or over-expressing toxins such as
ahemolysin might be counterproductive for the bacterium.
The same factors driving both vegetation size and tissue destruction are
likely to be responsible for bacterial dissemination to remote anatomical
sites. This occurs both by the direct impact of circulating bacteriathus
related to the density of vegetation infectionand the fragmentation of the

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311

Fig. 3. Electron microscopy of the endothelium adjacent to aortic vegetations in a rat infected
with S. aureus Cowan. This organism produces bronectin-binding proteins and is internalized
by endothelial cells both in vitro and in vivo (see text for details). The gure depicts an
endothelial cell (EC) that has undergone lysis after S. aureus internalization. The plasma
membrane at the vascular lumen (L) side is disrupted. S. aureus are actively dividing in the
cellular milieu. LB indicates the lamina basalis. Original magnication was 8,300, as in Fig. 2.

vegetation, which depends on multiple factors, including dissolution by proteolytic enzymes. Eventually, colonization of distant target tissues is likely
to involve the same infection cycle, starting with tissue adherence.

Summary
Although streptococcal and S. aureus IE share the same primary site of
infection, their pathogenesis and clinical evolution present several major differences. Streptococci adhere to cardiac valves with pre-existing endothelial
lesions. In contrast, S. aureus can colonize either damaged endothelium or
invade physically intact endothelial cells. These interactions are mediated
by multiple surface adhesins, some of which have been only partially characterized. Streptococci produce surface glucans (gtf and ftf), ECM adhesins
(e.g., bronectin-binding proteins, FimA), and platelet aggregating factors
(phase I and phase II antigens, pblA, pblB, and pblT), all of which have been

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P. Moreillon et al / Infect Dis Clin N Am 16 (2002) 297318

implicated in IE pathogenesis. S. aureus produces clumping factor A (ClfA),

bronectin-binding proteins (FnBPA and FnBPB), and an as-yet undetermined platelet aggregation factor, all of which have been shown to be
important in both valve colonization and invasion.
After valvular colonization, streptococci and S. aureus stimulate vegetation formation and maturation, principally by driving local procoagulant
activity, which in turn protects the organism from cellular and soluble host
defenses. Both viridans streptococci and S. aureus can induce TFA from
brin-adherent monocytes. S. aureus can also induce TFA from intact
endothelia, thus promoting vegetation formation on physically undamaged
valves. Platelets contribute to vegetation growth but also release microbicidal peptides that may inhibit or kill bacteria. Hence, the microorganisms
must be able to withstand platelet-induced killing for successful persistence
and proliferation at sites of endovascular damage in IE.
Following valvular colonization tissue invasion by S. aureus appears to
be critically related to the coordinated expression of surface adhesins and
secreted factors (e.g., enzymes and toxins), which are tightly controlled by
global regulators, agr and sar. Discordant expression of these virulence
determinants (e.g., manifested by excess ahemolysin secretion) may be detrimental for in situ survival of the bacterium.
Thus, bacteria use sophisticated strategies to subvert normal host defenses
and healing processes to promote vegetation formation. Many microbial
components involved in IE pathogenesis have yet to be determined. The recent
availability of bacterial genomes and related new technologies, such as in
vivo expression technology [104], signature-tagged mutagenesis [105], and
adoptive pathogenesis [49,52] will be invaluable in these future studies.
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