platelet activation and biofilm formation by aerococcus urinae, an

INFECTION AND IMMUNITY, Oct. 2010, p. 4268–4275 Vol. 78, No. 100019-9567/10/$12.00 doi:10.1128/IAI.00469-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Platelet Activation and Biofilm Formation by Aerococcus urinae, anEndocarditis-Causing Pathogen�

Oonagh Shannon, Matthias Morgelin, and Magnus Rasmussen*Department of Clinical Sciences, Division of Infection Medicine, Lund University, Lund, Sweden

Received 6 May 2010/Returned for modification 6 June 2010/Accepted 28 July 2010

The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons.Biofilm formation and platelet aggregation are believed to contribute to bacterial virulence in IE. Five A. urinaeisolates from human blood were shown to form biofilms in vitro, and biofilm formation was enhanced by thepresence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet-rich plasmafrom healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets,as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and onplatelet activation since it was sensitive to both trypsin and prostaglandin E1. Plasma proteins at the bacterialsurface were needed for platelet aggregation; and roles of the complement system, fibrinogen, and immuno-globulin G were demonstrated. Complement-depleted serum was unable to support platelet aggregation by Au3and complement blockade using compstatin-inhibited platelet activation. Platelet activation by Au3 wasinhibited by blocking of the platelet fibrinogen receptor, and this isolate was also shown to bind to radiolabeledfibrinogen. Removal of IgG from platelet-rich plasma by a specific protease inhibited the platelet aggregationinduced by A. urinae, and blockade of the platelet FcR�IIa hindered platelet activation induced by Au3.Convalescent-phase serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregateplatelets in an otherwise nonresponsive donor. Our results show that A. urinae exhibits virulence strategies ofimportance for IE.

Aerococcus urinae is a Gram-positive coccus with the capac-ity to cause urinary tract infections (7), infectious endocarditis(IE) (6), and spondylodiscitis (3). A. urinae was recognized asa distinct species in 1992 (1) and can be misreported as analpha-hemolytic streptococcus due to its growth characteristicsand hemolysis pattern. Correct classification is based on se-quencing of the 16S rRNA gene. The bacterium is generallysusceptible to penicillin, vancomycin, and cephalosporins,whereas it is resistant to sulfonamide (8, 28). Patients whopresent with severe infection are typically elderly men withunderlying urinary tract abnormalities. Twenty cases of A. uri-nae IE have been described in the literature, and the casefatality rate is about 50% (10, 15). Despite reports of invasivedisease caused by A. urinae, including IE, nothing is knownabout the potential virulence strategies of the bacterium.

Biofilm formation is believed to contribute to the ability of abacterium to colonize foreign materials, such as urinary tractcatheters. In the biofilm, the bacteria are protected againsthost defenses and against antibiotics. The first step in thepathogenesis of IE is believed to be bacterial adherence todamaged heart valves. This is followed by bacterial accumula-tion and probably also formation of a bacterial biofilm (9). Inaddition, fibrin, neutrophils, and platelets are deposited on theinfected heart valve, leading to the formation of a typical IEvegetation.

The majority of Gram-positive pathogens that cause IE in-

teract with and activate human platelets, and this has beenproposed to contribute to virulence. The molecular mecha-nisms leading to platelet activation have been described indetail for many pathogens (recently reviewed in references 11and 16). Many pathogens activate platelets through bacterialbound fibrinogen in combination with specific immunoglobulinG (IgG) bound to the bacterial surface. This mechanism hasbeen described for Staphylococcus aureus ClfA (19) and fi-bronectin-binding proteins (12), Streptococcus pyogenes (27),and Streptococcus agalactiae (23). Direct interactions betweenbacteria and platelets can also lead to platelet activation (5, 17,21). In addition, Streptococcus sanguis, S. aureus, and Entero-coccus faecalis can activate platelets in the absence of bacterialbound fibrinogen through an IgG- and sometimes comple-ment-dependent mechanism with a longer lag time (13, 19, 20,24). Upon activation, platelets release antibacterial peptides,and resistance to such substances has been linked to the ca-pacity of S. aureus to cause IE (4, 32). Thus, platelet activationmay, on the one hand, contribute to host defense against in-vading bacteria and, on the other hand, contribute to thepathogenesis of IE (31).

Given the potential importance of biofilm formation andplatelet activation in the pathogenesis of IE and the ability ofA. urinae to cause this condition, we investigated the capacityof the bacteria to form biofilms and to activate human plate-lets.

MATERIALS AND METHODS

Bacteria and culture conditions. Five isolates of Aerococcus urinae from hu-man blood cultures were obtained from the accredited routine diagnostic labo-ratory for clinical microbiology of the Skåne University Hospital, Lund, Sweden.The isolates were collected from 2005 to 2008 and were from five distinct

* Corresponding author. Mailing address: Department of Clinical Sci-ences, Division of Infection Medicine, BMC B14, Tornavagen 10, 221 84Lund, Sweden. Phone: 46-462220720. Fax: 46-46157756. E-mail: [email protected].

� Published ahead of print on 9 August 2010.

4268

on Novem

ber 29, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

http://iai.asm.org/

patients. The isolates had been subjected to PCR and sequencing of the 16SrRNA gene to confirm their identity. A. urinae was cultivated on blood agar orin tryptic soy broth (Difco) with 0.5% glucose (TSBG) at 37°C with 5% CO2.Streptococcus pyogenes strain AP1 was grown as described previously (25).

Biofilm assay. Quantification of biofilm formation was performed using apreviously described microtiter plate assay (29), with modifications. Briefly, 20 �lof precultured bacteria was added to the wells of a sterile 96-well flat-bottomedplastic culture plate (Nunclon Surface F; Nunc A/S, Denmark) containing 180 �lof TSBG medium, and the plate was incubated at 37°C for different time periods.The medium alone served as a negative control. The medium was removed, andthe wells were gently washed three times with 200 �l phosphate-buffered saline(PBS). The biofilm was fixed in 200 �l methanol for 10 min, air dried for 15 min,and stained with 160 �l crystal violet (1% [wt/vol] in water; Diagnostica Erlich,Darmstadt, Germany) for 5 min. The staining solution was removed and theplate was washed three times with 200 �l PBS per well, prior to extraction of thedye with 200 �l acetone-ethanol (20/80, vol/vol). One hundred microliters of thissolution was transferred to a new plate and diluted in an equal volume ofacetone-ethanol. The absorbance was measured at 550 nm in a Victor3 1420multilabel counter (Perkin-Elmer Precisely). If the absorbance was above 2.5, thesample was diluted in an equal volume of acetone-ethanol and the absorbancewas measured again. The value obtained was multiplied by the number of timesthat the sample had been diluted, and the absorbance of the negative control wassubtracted. Each isolate was tested in duplicate at least three times.

Platelet preparation. Blood samples were collected from five healthy donorswho had not taken antiplatelet medication in the previous 10 days. Five millilitersof blood was collected into citrated vacuum tubes. Centrifugation at 150 � g for10 min produced an upper layer of platelet-rich plasma (PRP), which wasremoved. Subsequent centrifugation at 1,500 � g for 10 min produced an upperlayer of platelet-poor plasma (PPP). Washed platelets (WP) were obtained from5 ml of PRP, which was supplemented with 1 �M prostaglandin E1 (PGE1;Sigma), followed by centrifugation at 1,000 � g for 10 min. The soft pellet waswashed once in 5 ml washing buffer (26), followed by centrifugation at 800 � gfor 15 min and gentle resuspension in 1 ml HEPES buffer (pH 7.4). Sampling ofblood from healthy volunteers had been approved by the local ethics committee(approval 2008/657).

Aggregometry. Bacteria grown for 16 h in 10 ml of TSBG were harvested bycentrifugation at 2,500 � g for 10 min, washed in PBS, and resuspended in 500�l of the same buffer. The absorbance at 620 nm was determined for a 10-fold-diluted bacterial suspension, and the bacterial concentration was adjusted so thatthe diluted suspension had an optical density at 620 nm (OD620) of 0.59, whichcorresponds to 2.6 � 108 CFU/ml of isolate Au3 and 2.6 � 109 CFU/ml in theundiluted suspension after the corresponding adjustment. Forty microliters ofthe latter bacterial solution was added to 450 �l of PRP, and the plateletresponse was monitored in a Chrono-Log aggregometer for a maximum of 25min. As a positive control, 1 �l of soluble collagen I (Triolab, Sweden) was addedto 450 �l of PRP. To inactivate the IgG in PRP, 5 �g of purified IgG-degradingenzyme of Streptococcus pyogenes (IdeS) was added to 450 �l PRP, and themixture was incubated for 15 min at 37°C. Alternatively, 25 �l of serum wastreated with 0.25 �g IdeS for 15 min at 37°C. IdeS was purified as a fusion toglutathione S-transferase (GST), as described previously (30). For some exper-iments, 0.1 mg of trypsin from bovine pancreas (Sigma) was added to 100 �l ofbacterial suspension (2.6 � 109/ml in PBS), followed by incubation at 37°C for 30min. Trypsin was blocked by the addition of benzamidine hydrochloride (Sigma)to a final concentration of 6 mM. After incubation for 5 min, the sample wascentrifuged at 3,000 � g for 5 min and the bacterial pellet was resuspended in 100�l of PBS. For some experiments, PGE1 was added to a final concentration of 1�M to PRP and the mixture was incubated for 5 min before addition of agonist.Serum was prepared from blood that was allowed to coagulate in glass tubes for1 h, followed by centrifugation at 1,500 � g for 10 min. For inactivation ofcomplement proteins, serum was heated to 56°C for 15 min. Alternatively, serumwas treated with 50 mg/ml of zymosan A (Sigma) at 37°C for 20 min, followed bycentrifugation at 10,000 � g for 2 min (14). To reconstitute serum with fibrino-gen, 50 �l of a solution containing 5 mg of fibrinogen/ml (ICN Biochemicals,Aurora, OH) in PBS was added to 350 �l of serum. Results were analyzed usingAggrolink, version 5.2.1, software.

Determination of platelet activation by A. urinae. Twenty microliters of PRPwas incubated for 25 min at room temperature with 40 �l of HEPES buffer, pH7.4, in either the presence or absence of washed bacteria (approximately 1.2 �107 bacteria). After 25 min, 5 �l fluorochrome-conjugated antibody (CD42-peridinin chlorophyll protein, CD62 phycoerythrin, and PAC-1–fluorescein iso-thiocyanate; all from BD Biosciences) was added, and after 10 min the incuba-tion was stopped by addition of 500 �l of 0.5% formaldehyde in ice-cold PBS.Samples were analyzed using a FACSCalibur flow cytometer in logarithmic mode

with a gate setting for the CD42-positive platelet population. Fifty thousand cellswere acquired and analyzed using Cell Quest software (Becton Dickinson). Inorder to assess the role of plasma factors for platelet activation, PRP was treatedwith specific blockers, 20 �g/ml ReoPro (Eli Lily), 50 �g/ml AT10 (Serotec), or70 �g/ml compstatin (a kind gift from Ulf Sjobring described elsewhere [22]), for30 min at room temperature.

Electron microscopy. For scanning electron microscopy (SEM), 300 �l ofbacterial culture was added to 2.7 ml of TSBG, and the bacteria were allowed toform a biofilm on a heat-sterilized glass coverslip for 72 h in a 12-well cell culturecluster (Costar; Corning Incorporated). The glass was carefully washed threetimes in PBS, followed by incubation in 3 ml of fixation solution (4% formalde-hyde and 2.5% glutaraldehyde in PBS) at room temperature for 12 h. Fixedspecimens were dehydrated for 10 min at each step of an ascending ethanolseries and dried to the critical point in a Balzers critical point dryer in liquidcarbon dioxide using absolute ethanol as an intermediate solvent. Samples wereexamined in a Jeol J-330 scanning electron microscope at an acceleration voltageof 5 kV and a working distance of 10 mm.

Other methods. Human fibrinogen (Sigma) was radiolabeled and tested forbinding to bacteria, as described previously (25). Statistical analysis was per-formed using the GraphPad Prism 4 for Macintosh, version 4.0C, program.Handling of patient data was approved by the local ethics committee (approval2008/657).

RESULTS

Characterization of isolates and patients. Five isolates of A.urinae were identified in blood cultures from 2005 to 2008 atthe diagnostic laboratory for clinical microbiology of SkåneUniversity Hospital. The bacterium was isolated from two ormore flasks, and A. urinae was the only bacterium isolated in allcases. The identity of the isolates was confirmed by sequencingof the 16S rRNA gene. The isolates were from male patientsbetween 82 and 96 years of age. Three patients had received adiagnosis of infectious endocarditis (International Classifica-tion of Diseases, 10th revision, code ICD10 I33.0), but in twoof the patients this diagnosis was delayed by more than 3weeks, as the condition was initially misinterpreted as a urinarytract infection. In one patient an anal abscess was suspected tobe the cause of the bacteremia, and in one patient hydrone-phrosis was the main diagnosis, indicating that the bacteremiawas judged to be secondary to the hydronephrosis.

A. urinae biofilm formation is stimulated by plasma. Biofilmformation was determined for each A. urinae isolate by mea-suring the amount of crystal violet absorbed by the bacteria ina biofilm formed on polystyrene plastic. Biofilm formation wasdetermined after incubation for 24, 48, and 72 h. At 48 and72 h, all isolates formed a robust, macroscopically visible bio-film. The biofilm formation for all isolates at 72 h is illustratedin Fig. 1A. The addition of 10% human plasma to the growthmedium led to an increased amount of dye bound to thebiofilms of all isolates by 30 to 758% (mean, 397%). To inves-tigate if the stimulatory effect of plasma was due to the in-creased adherence of bacteria to the surface, the wells werepretreated with 20% plasma in TSBG for 2 h, followed byremoval of the plasma and determination of biofilm formation.Pretreatment of wells did not increase biofilm formation butrather decreased the biofilm formation of the five isolates from47 to 66% (Fig. 1). This indicates that the stimulatory effect ofplasma on biofilm formation is not explained by the presenceof plasma proteins at the plastic surface. The biofilms formedby two isolates (Au1 and Au3) on glass coverslips in the pres-ence or absence of 10% human plasma were visualized by SEM(Fig. 2). Both isolates formed multilayered bacterial conglom-erates on the glass surface. An intercellular substance covers

VOL. 78, 2010 AEROCOCCUS URINAE VIRULENCE STRATEGIES 4269

on Novem


.org/D

ownloaded from

http://iai.asm.org/

the bacterial aggregates present in the biofilm formed by Au1irrespective of the presence of plasma (Fig. 2A and B). In thebiofilm formed by Au3, the intercellular substance could beseen only on bacteria grown in the presence of plasma (Fig. 2Cand D). This likely reflects the ability of Au1 to produce abiofilm in the microtiter plates irrespective of the plasma con-tent, while formation of biofilm by Au3 in the microtiter plateswas stimulated by plasma (Fig. 1).

A. urinae aggregates human platelets. The five isolates of A.urinae were tested for their ability to induce aggregation ofplatelets from five healthy donors. Washed bacteria were

added to PRP, and the response was monitored for 25 min inan aggregometer. The positive-control bacterium, the AP1strain of S. pyogenes, induced aggregation in all donors within5 min. Platelet aggregation in response to A. urinae occurred inall donors and was induced by two to four isolates, dependingon the donor (Table 1). One isolate (Au3) induced aggregationin all donors, whereas one isolate (Au4) failed to induce ag-gregation in any donor (Table 1). The mean time to aggrega-tion for all isolates was 13 min (range, 6 to 25 min). Typicalaggregation curves for Au3 and Au4 in donor 1 are shown inFig. 3. Treatment of Au3 bacteria with trypsin abolished theability to aggregate platelets in the three donors tested (datanot shown). To exclude an effect of trypsin on platelets, theBef5 isolate of Enterococcus faecalis (24) was used as a control.Trypsin-treated Bef5 bacteria retained the ability to aggregateplatelets in the same donors (data not shown). Platelet aggre-gation by all isolates in the same three donors could be blocked

FIG. 1. A. urinae biofilm formation is stimulated by plasma. Biofilmformation by A. urinae isolates after 72 h incubation on a plasticsurface was determined as the absorbance at 550 nm in the presence ofmedium (black bars) or medium containing 10% human plasma (graybars). Biofilm formation was also determined in wells pretreated withhuman plasma (white bars). The error bars represent the standarddeviations from three independent experiments, carried out in tripli-cate.

FIG. 2. Morphology of the A. urinae biofilm. Scanning electron microscopy was used to visualize biofilms formed by Au1 (A and B) and Au3(C and D) on glass coverslips in medium (A and C) or in medium containing 10% human plasma (B and D). Bar, 10 �m.

TABLE 1. Numbers of donors whose platelets responded withaggregation to the A. urinae isolates

IsolateNo. of donors

responding withaggregationa

Mean (range)lag time (min)

S. pyogenes AP1 5 3 (2–5)A. urinae Au1 3 16 (9–23)A. urinae Au2 2 10 (7–12)A. urinae Au3 5 12 (6–21)A. urinae Au4 0A. urinae Au5 2 19 (12–25)

a PRP was obtained from a total of five donors.

4270 SHANNON ET AL. INFECT. IMMUN.

on Novem


.org/D

ownloaded from

http://iai.asm.org/

by PGE1, indicating that passive agglutination did not explainthe platelet aggregation (data not shown).

A. urinae activates human platelets. Platelet activation inPRP was determined using three-color flow cytometry follow-ing stimulation with washed bacteria or ADP as a positivecontrol. Platelets were identified using a CD42-specific anti-body, the activation status of the fibrinogen receptor (GPIIb/IIIa) was determined using an antibody specific for the activeconformation (PAC-1), and release of the alpha granules wasdetermined using a CD62-specific antibody. As expected,platelets from all donors were negative for CD62 and PAC-1 inthe absence of an agonist and became positive only after acti-vation. After treatment with ADP, medians of 78% (range, 63to 83%) and 68% (range, 57 to 76%) of the platelet populationwere positive for CD62 and PAC-1, respectively. Au3-medi-ated platelet activation occurred in all five donors, rendering amedian of 35% (range, 27 to 48%) of the platelets positive forCD62 and a median of 31% (range, 21 to 41%) of the plateletspositive for PAC-1.

Platelet aggregation and activation by Au3 involves the com-plement system. Some mechanisms described for platelet ag-gregation with a long lag time to aggregation involve activationof the complement system. To determine if this was the casefor A. urinae, Au3 bacteria were pretreated with 100 �l of PPPfor 15 min, followed by addition of 400 �l of PRP from donor1 (Fig. 4A). As a control, Au3 bacteria were added to a mixtureof 100 �l of PPP and 400 �l of PRP from the same donor. Ascan be seen in Fig. 4A, pretreatment of bacteria with plasmasignificantly shortens the lag time to aggregation, suggestingthat Au3 accumulates plasma components which participate inplatelet aggregation.

Next, 100 �l of washed platelets was added to 350 �l ofplasma or serum, followed by the addition of Au3 bacteria(Fig. 4B). As expected, platelets aggregated in plasma but not

in serum, which contains only small amounts of fibrinogen.When fibrinogen was added to serum, Au3 was able to induceaggregation of platelets (Fig. 4B). In contrast, in serum pre-treated at 56°C with added fibrinogen, Au3 was unable toinduce platelet aggregation, indicating a role for the comple-ment system in platelet aggregation. Importantly, the AP1strain of S. pyogenes could aggregate washed platelets sus-pended in heat-treated serum with added fibrinogen (Fig. 4B).In similar experiments, zymosan was used to deplete serum ofcomplement proteins (14). When washed platelets were addedto zymosan-depleted serum reconstituted with fibrinogen, ag-gregation by Au3 still occurred. However, the lag time toaggregation was increased (38, 41, and 78% in three separateexperiments, respectively) compared to the lag time in normalserum reconstituted with fibrinogen (data not shown).

PRP was preincubated with buffer or compstatin, a peptideinhibitor of the complement system, for 15 min, followed bythe addition of Au3 bacteria and determination of plateletactivation by flow cytometry. Compstatin decreased the num-ber of platelets presenting PAC-1 and CD62 in response toAu3 by means of 59% and 63%, respectively (n � 3).

Platelet activation by Au3 is dependent on fibrinogen. Toassess the role of plasma fibrinogen in mediating platelet ac-tivation, the platelet receptor for fibrinogen was blocked usinga monoclonal antibody (ReoPro). Since platelet aggregationdepends on fibrinogen irrespective of which agonist used, weused flow cytometry to monitor activation. In the presence ofReoPro, the PAC-1 antibody can no longer bind to the plate-lets (Fig. 5B), but platelet activation in response to ADP stilloccurred as CD62 presentation was unaffected (data notshown). When the fibrinogen receptor was blocked, activa-tion of platelets by Au3 was significantly decreased in all

FIG. 3. A. urinae stimulates platelet aggregation. Platelet aggrega-tion in PRP from donor 1 in response to A. urinae isolates Au3 andAu4 was determined using a Chrono-Log aggregometer. The relativelight absorption of the sample is shown on the y axis, where 100represents the absorption of PRP after addition of bacteria and 0 theabsorption of PPP. When platelets aggregate the opacity, the lightabsorbance of the sample decreases.

FIG. 4. Platelet aggregation and activation by Au3 is dependent onthe complement system. (A) Au3 was either pretreated with PPP,followed by addition of PRP from donor 1 (curve b), or added to amixture of PPP and PRP from the same donor (curve a), and aggre-gation was determined by aggregometry. (B) Washed platelets fromdonor 1 were added to citrated plasma from the same donor, followedby addition of Au3 bacteria (curve a). Washed platelets were added toserum reconstituted with fibrinogen (curve b) or heat-treated serumreconstituted with fibrinogen (curve c), followed by addition of Au3bacteria. AP1 was able to induce aggregation of washed platelets inheat-treated serum reconstituted with fibrinogen (curve d).


on Novem


.org/D

ownloaded from

http://iai.asm.org/

donors (Fig. 5A and B). This indicates that the fibrinogenreceptor at the platelet surface is involved in platelet acti-vation by Au3.

We next tested the ability of the A. urinae isolates to bind toradiolabeled fibrinogen. The AP1 strain of S. pyogenes, whichbinds to fibrinogen with a high affinity via the M1 protein (2),was used as a positive control. The ability of 109 bacteria/ml tobind to fibrinogen is shown in Fig. 5C. Au3 absorbed a higherproportion of added fibrinogen than the other isolates. Com-petitive binding studies were carried out, where unlabeled fi-brinogen was allowed to compete with radiolabeled fibrinogenfor binding to 108 Au3 bacteria or to 2 � 107 AP1 bacteria (Fig.5D). In both isolates, the unlabeled fibrinogen could competewith the labeled fibrinogen for binding, indicating that bindingwas specific.

A. urinae platelet activation and aggregation is dependenton IgG. To determine the role of plasma IgG in mediatingplatelet activation, the platelet receptor for the Fc part of IgGwas blocked using a monoclonal antibody (AT10). As ex-pected, platelet activation in response to ADP was unaffectedby blockade of the platelet IgG receptor, but AT10 diminishedactivation in response to Au3 in all donors (Fig. 6A and B).This indicates that plasma IgG is involved in mediating plateletactivation in response to Au3.

The highly specific IgG-degrading enzyme IdeS of S. pyo-

genes (30) was used to degrade all IgG in PRP. In samplespretreated with buffer, collagen and Au3 bacteria could induceplatelet aggregation. As expected, collagen retained the abilityto aggregate platelets in IdeS-treated PRP, whereas Au3 failedto induce aggregation of platelets in IdeS-treated plasma.Identical results were obtained for donors 1 and 2, and repre-sentative results from an experiment with platelets from donor1 are shown in Fig. 6C.

Convalescent-phase patient serum from A. urinae infectiontransfers the ability to aggregate platelets. Serum from thepatient infected with isolate Au1 was obtained 4 weeks afterthe appearance of the first symptoms of IE. Twenty-five mi-croliters of this serum or serum from donor 1 was added toPRP from donor 1, and platelet aggregation in response toAu1 was monitored. In PRP from donor 1, Au1 did not causeplatelet aggregation and the addition of serum from the samedonor did not stimulate platelet aggregation (Fig. 6D). Asexpected, Au3 induced aggregation of platelets in PRP fromdonor 1 also after the addition of control serum. Importantly,after the addition of patient serum, Au1 induced platelet ag-gregation in PRP from donor 1 (Fig. 6D). In three separateexperiments the lag times to aggregation were 12, 15, and 16min. respectively. Results from one representative experimentare shown in Fig. 6C. When it was pretreated with IdeS, thepatient serum could not support Au1-mediated aggregation of

FIG. 5. Role of platelet fibrinogen receptor and fibrinogen binding by A. urinae. Au3 was added to PRP from the five donors, and after 25 min,platelet activation was determined using flow cytometry. The percentage of the platelet population positive for CD62P (A) and PAC-1 (B) wasdetermined in the presence of buffer alone (white bars) or an antibody to block platelet fibrinogen binding (ReoPro; black bars). (C) The bindingof radiolabeled fibrinogen to the A. urinae isolates and to the AP1 strain of S. pyogenes is expressed as the percentage of added fibrinogen associatedwith the bacterial pellet (n � 3; bars represent standard deviations). (D) The binding of radiolabeled fibrinogen to Au3 (triangles) and to the AP1strain of S. pyogenes (squares) was determined in the presence of unlabeled fibrinogen. Binding is expressed as a percentage of the binding ofradiolabeled fibrinogen alone. Results are from three independent experiments, and the bars represent standard deviations.


on Novem


.org/D

ownloaded from

http://iai.asm.org/

platelets from donor 1, again suggesting that IgG is the serumfactor that transfers the ability to aggregate platelets.

DISCUSSION

In the present work, we demonstrate that A. urinae canform biofilms and that the bacteria can aggregate humanplatelets. It is the first report of potential virulence mecha-nisms for this pathogen, and both mechanisms may contrib-

ute to the ability of A. urinae to cause IE. A. urinae is welldocumented as a pathogen in IE, though the number ofreported cases is low (10, 15). We identified three cases ofIE and two additional cases of invasive infection with A.urinae over a 4-year period in a population of about 400,000inhabitants. This indicates that the importance of A. urinaeas a pathogen in IE and other invasive diseases has previ-ously been underestimated. In accordance with previousreports, all the patients in this study were older males, and

FIG. 6. Role of plasma IgG in platelet activation and aggregation by A. urinae. Au3 was added to PRP from the five donors, and after25 min, platelet activation was determined using flow cytometry. The percentage of the platelet population positive for CD62P (A) andPAC-1 (B) was determined in the presence of buffer alone (white bars) or an antibody to block platelet Ig binding (AT10; black bars).(C) PRP from donor 1 was pretreated with the IgG-degrading enzyme IdeS or buffer, followed by addition of collagen or Au3 bacteria.Collagen induced rapid aggregation in PRP pretreated with IdeS (curve a) or buffer (curve b). Au3 bacteria failed to induce aggregation inPRP pretreated with IdeS (curve c), whereas aggregation occurred in PRP treated with buffer alone (curve d). (D) Aggregation of plateletsin PRP from donor 1 in response to Au1 and Au3 was determined. In the presence of 25 �l of serum from donor 1, platelets did not aggregatein response to Au1 (curve a), whereas aggregation occurred in response to Au3 (curve d). In the presence of 25 �l of serum from the patientinfected with Au1, the platelets aggregated in response to Au1 (curve b). Patient serum preincubated with IdeS could not mediate plateletaggregation in response to Au1 (curve c).


on Novem


.org/D

ownloaded from

http://iai.asm.org/

though the material was too small to draw any definiteconclusions, A. urinae may be a significant cause of IEamong older men.

The ability of A. urinae to form biofilms may play a roleduring IE caused by this organism, especially since the biofilmformation of many isolates was stimulated by plasma. Impor-tantly, bacteria in biofilms are partially protected against theaction of antibiotics (18), so biofilm formation by A. urinae mayhinder the eradication of the organism with antibiotics. Biofilmformation may also play a role during earlier stages of an A.urinae infection. Many patients with severe A. urinae infectionhave indwelling urinary catheters, and biofilm formation on thecatheter may increase the likelihood of A. urinae bacteremia,which in turn increases the risk for colonization of heart valves.

The interaction between Gram-positive bacteria and plate-lets has been extensively studied, though the importance of thisinteraction for the pathogenesis of IE is less clear. We showthat many isolates of A. urinae induce aggregation and activa-tion of human platelets, and this further underlines the impor-tance of platelet interactions in invasive bacterial infections.The mechanism of platelet aggregation by Au3 was studied indetail, since this isolate induced aggregation of platelets fromall of the donors tested. The mechanism employed by Au3 issimilar to that utilized by many Gram-positive pathogens andwas dependent on complement activation, on fibrinogen, andon IgG.

Four of the other five A. urinae isolates also induced plateletaggregation in sera from some donors. In all cases, biochemicalinactivation of platelets with PGE1 abolished the aggregation,demonstrating an active role for platelets in this process. Theaggregation of platelets by the other isolates could be inhibitedby removal of IgG with IdeS from the serum from the twodonors tested, supporting the role of IgG in platelet aggrega-tion. Significantly, serum from a patient infected with Au1induced platelet aggregation in response to Au1 in a previouslynonresponsive donor. The stimulatory effect of this serum sam-ple was lost after proteolytic cleavage of IgG. Taken togetherwith the results obtained for Au3, our findings imply an essen-tial role for IgG in platelet aggregation by A. urinae.

GPIIb/IIIa activation, perhaps through a fibrinogen bridge,is not necessarily needed for platelet aggregation by all isolatesof A. urinae, since most isolates bound poorly to this plasmaprotein. In addition, we cannot exclude the possibility that adirect interaction between Au3 and platelet GPIIb/IIIa is in-volved in platelet activation by this isolate. Complement acti-vation may also be important for the ability of the other A.urinae isolates to induce platelet aggregation, since a long lagtime was observed for all isolates. In addition to these hostproteins, a bacterial protein structure seems to be involvedbecause platelet aggregation was abolished by treatment of thebacteria with trypsin.

Many molecular mechanisms at the bacterial surface seemto occur when A. urinae enters the bloodstream, and the out-come of these interactions will likely determine if IE is estab-lished. We believe that both platelet activation and biofilmformation participate in the pathogenesis of IE caused by A.urinae. However, further studies on the role of platelet activa-tion by A. urinae and other bacterial species during invasiveinfection are needed. A better understanding of the molecularinteractions between invading bacteria and human platelets

may stimulate the development of novel therapeutic strategiesagainst invasive bacterial diseases such as IE.

ACKNOWLEDGMENTS

This work was financed by Swedish Government Funds for ClinicalResearch (ALF), the Swedish Research Council (projects 21112 and7480), the Swedish Society of Medicine, the Royal Physiographic So-ciety in Lund, and the foundations of Osterlund and Crafoord.

We acknowledge Sara K. Sobirk and Daniel Johansson for impor-tant help and Ingbritt Gustafsson and Maria Baumgarten for excellenttechnical assistance. We thank Rita Wallen from the Department ofCell and Organism Biology, Lund University, for help with electronmicroscopy.

REFERENCES

1. Aguirre, M., and M. D. Collins. 1992. Phylogenetic analysis of some Aero-coccus-like organisms from urinary tract infections: description of Aerococ-cus urinae sp. nov. J. Gen. Microbiol. 138:401–405.

2. Åkesson, P., K. H. Schmidt, J. Cooney, and L. Bjorck. 1994. M1 protein andprotein H: IgGFc- and albumin-binding streptococcal surface proteins en-coded by adjacent genes. Biochem. J. 300(Pt 3):877–886.

3. Astudillo, L., L. Sailler, L. Porte, J. C. Lefevre, P. Massip, and E. Arlet-Suau.2003. Spondylodiscitis due to Aerococcus urinae: a first report. Scand. J. In-fect. Dis. 35:890–891.

4. Bayer, A. S., D. Cheng, M. R. Yeaman, G. R. Corey, R. S. McClelland, L. J.Harrel, and V. G. Fowler, Jr. 1998. In vitro resistance to thrombin-inducedplatelet microbicidal protein among clinical bacteremic isolates of Staphylo-coccus aureus correlates with an endovascular infectious source. Antimicrob.Agents Chemother. 42:3169–3172.

5. Bensing, B. A., J. A. Lopez, and P. M. Sullam. 2004. The Streptococcusgordonii surface proteins GspB and Hsa mediate binding to sialylated car-bohydrate epitopes on the platelet membrane glycoprotein Ib�. Infect. Im-mun. 72:6528–6537.

6. Christensen, J. J., E. Gutschik, A. Friis-Moller, and B. Korner. 1991. Uro-septicemia and fatal endocarditis caused by Aerococcus-like organisms.Scand. J. Infect. Dis. 23:717–721.

7. Christensen, J. J., B. Korner, and H. Kjaergaard. 1989. Aerococcus-likeorganism—an unnoticed urinary tract pathogen. APMIS 97:539–546.

8. Christensen, J. J., H. Vibits, J. Ursing, and B. Korner. 1991. Aerococcus-likeorganism, a newly recognized potential urinary tract pathogen. J. Clin. Mi-crobiol. 29:1049–1053.

9. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mechanisms ofclinically relevant microorganisms. Clin. Microbiol. Rev. 15:167–193.

10. Ebnother, C., M. Altwegg, J. Gottschalk, J. D. Seebach, and A. Kronenberg.2002. Aerococcus urinae endocarditis: case report and review of the litera-ture. Infection 30:310–313.

11. Fitzgerald, J. R., T. J. Foster, and D. Cox. 2006. The interaction of bacterialpathogens with platelets. Nat. Rev. Microbiol. 4:445–457.

12. Fitzgerald, J. R., A. Loughman, F. Keane, M. Brennan, M. Knobel, J.Higgins, L. Visai, P. Speziale, D. Cox, and T. J. Foster. 2006. Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human plate-lets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgGbinding to the Fc�RIIa receptor. Mol. Microbiol. 59:212–230.

13. Ford, I., C. W. Douglas, D. Cox, D. G. Rees, J. Heath, and F. E. Preston.1997. The role of immunoglobulin G and fibrinogen in platelet aggregationby Streptococcus sanguis. Br. J. Haematol. 97:737–746.

14. Ford, I., C. W. Douglas, J. Heath, C. Rees, and F. E. Preston. 1996. Evidencefor the involvement of complement proteins in platelet aggregation by Strep-tococcus sanguis NCTC 7863. Br. J. Haematol. 94:729–739.

15. Kass, M., B. Toye, and J. P. Veinot. 2008. Fatal infective endocarditis due toAerococcus urinae—case report and review of literature. Cardiovasc. Pathol.17:410–412.

16. Kerrigan, S. W., and D. Cox. 2010. Platelet-bacterial interactions. Cell. Mol.Life Sci. 67:513–523.

17. Kerrigan, S. W., I. Douglas, A. Wray, J. Heath, M. F. Byrne, D. Fitzgerald,and D. Cox. 2002. A role for glycoprotein Ib in Streptococcus sanguis-inducedplatelet aggregation. Blood 100:509–516.

18. Lewis, K. 2001. Riddle of biofilm resistance. Antimicrob. Agents Chemother.45:999–1007.

19. Loughman, A., J. R. Fitzgerald, M. P. Brennan, J. Higgins, R. Downer, D.Cox, and T. J. Foster. 2005. Roles for fibrinogen, immunoglobulin andcomplement in platelet activation promoted by Staphylococcus aureus clump-ing factor A. Mol. Microbiol. 57:804–818.

20. Miajlovic, H., A. Loughman, M. Brennan, D. Cox, and T. J. Foster. 2007.Both complement- and fibrinogen-dependent mechanisms contribute toplatelet aggregation mediated by Staphylococcus aureus clumping factor B.Infect. Immun. 75:3335–3343.

21. Miajlovic, H., M. Zapotoczna, J. A. Geoghegan, S. W. Kerrigan, P. Speziale,


on Novem


.org/D

ownloaded from

http://iai.asm.org/

and T. J. Foster. 2010. Direct interaction of iron-regulated surface determi-nant IsdB of Staphylococcus aureus with the GPIIb/IIIa receptor on platelets.Microbiology 156:920–928.

22. Nilsson, M., M. Weineisen, T. Andersson, L. Truedsson, and U. Sjobring.2005. Critical role for complement receptor 3 (CD11b/CD18), but not for Fcreceptors, in killing of Streptococcus pyogenes by neutrophils in human im-mune serum. Eur. J. Immunol. 35:1472–1481.

23. Pietrocola, G., A. Schubert, L. Visai, M. Torti, J. R. Fitzgerald, T. J. Foster, D. J.Reinscheid, and P. Speziale. 2005. FbsA, a fibrinogen-binding protein fromStreptococcus agalactiae, mediates platelet aggregation. Blood 105:1052–1059.

24. Rasmussen, M., D. Johansson, S. K. Sobirk, M. Morgelin, and O. Shannon.2010. Clinical isolates of Enterococcus faecalis aggregate human platelets.Microbes Infect. 12:295–301.

25. Rasmussen, M., H. P. Muller, and L. Bjorck. 1999. Protein GRAB of Strep-tococcus pyogenes regulates proteolysis at the bacterial surface by bindingalpha2-macroglobulin. J. Biol. Chem. 274:15336–15344.

26. Shannon, O., and J. I. Flock. 2004. Extracellular fibrinogen binding protein,

Efb, from Staphylococcus aureus binds to platelets and inhibits platelet ag-gregation. Thromb. Haemost. 91:779–789.

27. Sjobring, U., U. Ringdahl, and Z. M. Ruggeri. 2002. Induction of plateletthrombi by bacteria and antibodies. Blood 100:4470–4477.

28. Skov, R., J. J. Christensen, B. Korner, N. Frimodt-Moller, and F. Espersen.2001. In vitro antimicrobial susceptibility of Aerococcus urinae to 14 antibi-otics, and time-kill curves for penicillin, gentamicin and vancomycin. J.Antimicrob. Chemother. 48:653–658.

29. Stepanovic, S., D. Vukovic, I. Dakic, B. Savic, and M. Svabic-Vlahovic. 2000.A modified microtiter-plate test for quantification of staphylococcal biofilmformation. J. Microbiol. Methods 40:175–179.

30. von Pawel-Rammingen, U., B. P. Johansson, and L. Bjorck. 2002. IdeS, anovel streptococcal cysteine proteinase with unique specificity for immuno-globulin G. EMBO J. 21:1607–1615.

31. Yeaman, M. R. 2010. Platelets in defense against bacterial pathogens. Cell.Mol. Life Sci. 67:525–544.

32. Yeaman, M. R. 1997. The role of platelets in antimicrobial host defense. Clin.Infect. Dis. 25:951–968.

Editor: R. P. Morrison


on Novem


.org/D

ownloaded from

http://iai.asm.org/

platelet activation and biofilm formation by aerococcus urinae, an

Documents