Serodiagnosis of Streptococcus faecalis endocarditis by immunoblotting of surface protein antigens

Immunoblotting of Surface Protein

Antigens

EILEENJ. AITCHISON,' PETER A.

LAMBERT,'*

E. GRACE SMITH,2 AND IAN D. FARRELL2

Microbiology Research Group, Department of PharmaceuticalSciences, Aston University,Aston Triangle, Birmingham B47ET,' and Public Health Laboratory, EastBirminghamHospital, Bordesley Green East, Birmingham B9 5ST,2

United Kingdom

Received 12May1986/Accepted8 October 1986

We describeamethodfortheserodiagnosis of Streptococcusfaecalis ininfective endocarditis which couldbe

of value in culture-negativecases. Serum-growncellsofS. faecalis produced three major characteristicprotein antigens (73,000, 40,000, and 37,000 molecular weight) which were separated by sodium dodecyl

sulfate-polyacrylamidegelelectrophoresis of solubilizedwholecells. Afterelectrophoretic transferto anitrocellulose

membrane,theseantigenswerevisualized by probing withserumfrompatientswithendocarditis caused by S.

faecalis.

Serum from patients with endocarditis caused by other organisms did not react with the S.

faecalis-specificantigens.Thisprocedureshouldfacilitate positive early diagnosis ofS.faecalisendocarditisor

establish its absence in culture-negativecases.

Despite the advent of antimicrobial chemotherapy,

infec-tive endocarditis remains a difficult condition to diagnose

andtreat and still carriesa mortality rate of30% (7, 8, 15, 28). Current trends indicate a rise in the mean age of

endocarditis patients, owing partly to the increased age of

thegeneral population,adecrease in frequency of rheumatic

heartdisease in theyoung,and the earlier surgical correction

ofcongenital heart defects (7, 8, 10, 17, 26, 27).

Streptococ-cusfaecalis endocarditis is becomingmoreprevalent,

espe-cially in the elderly, and is particularly difficult to treat in that it requires a prolonged course of a combination of

antibiotics (22, 25, 26, 29). The current incidence of S. faecalis endocarditis among infective endocarditis cases is

as highas 15% (19,27).

Identification of thecausative organism isolated from the bloodof patients with infectiveendocarditis is ofparamount importance for the selection of appropriate treatment (8). Newrapid techniques that might reliably confirmorexclude

S.faecalis in infective endocarditis would be ofgreatclinical value.

We describeamethodfor theserodiagnosis of S.faecalis

endocarditis which exploits the surface antigens of this organism. S. faecalis was grown in serum to mimic the nutritional conditions found in vivo. This resulted in the expression of several prominent protein antigens (1) which reacted strongly with sera from hyperimmune rabbits and

patients with S. faecalis endocarditis. Three antigens of

73,000 molecular weight (73K), 40K, and 37K were

exclu-sivetoS.faecalis species. The potentialof theseantigensas

serodiagnostic agents was investigated by using serafrom

endocarditis patients to probe immunoblots prepared from

seven Streptococcus species and Staphylococcus aureus.

This technique was used successfully in a blind trial to

diagnose S.faecalis as the infecting organism in six of six patientsandtodiscount S.faecalis astheinfecting organism in sixof sixpatientswith infective endocarditisdueto other species.

* Correspondingauthor.

MATERIALSANDMETHODS

Bacterial strains. Four strains of S. faecaliswereused: a

strain cultured from the blood ofa patient with S. faecalis

endocarditis (EBH1); a strain isolated from a

patient

with

urinary tractinfection (strain 777);a laboratorystrain of S.

faecalis subsp. zymogeneskindlydonatedbyN. J. Parsons atBirminghamUniversity; andareference strain, S. faecalis

subsp. zymogenes NCTC 05957. Other standard strains of

streptococci usedwereStreptococcussanguisNCTC 07863,

Streptococcus salivarius NCTC 08618, Streptococcus milleri NCTC 10708, Streptococcus mutansNCTC 10449,

Strepto-coccus bovis NCTC 11436, and

Streptococcus

faecium

NCTC 07171. A strain of Staphylococcus aureus isolated

from the blood ofapatient with infective endocarditis was

alsoused.

Strainsweremaintainedonnutrientagarslopesat4°C and subcultured monthly. The bacteria were grown in heat-inactivated, mycoplasma-tested horse serum (Gibco

Labo-ratories Ltd., Paisley, United Kingdom) and harvested at stationaryphase by centrifugationat10,000 x g.Wholecells

werewashed three times andsuspendedtoanOD470 of 5.0in

0.01 MTris hydrochloridebuffer, pH7.4.

Hyperimmune rabbit serum. Whole cells of S. faecalis

EBH1 were suspended to an OD470of 1.0 in normal saline.

Bacteria tobe used forimmunization were killed by expo-sureto UV lightfor 10 min. Pairs ofrabbits (3 kg, half-lop)

were injected at weekly intervals with 1 ml of the killed whole-cell suspension at three sites in the neck. After 8 weeks, bloodwas obtainedby cardiacpuncture. Serawere

stored at -20°C untilrequired.

Humanserum. Serumwascollectedby venipuncturefrom patients with infective endocarditis at East Birmingham Hospital. Several serum samples were taken from each

patient duringthe episodeofinfective endocarditis. For the

purposeofthis study, portions ofeach serum sample were

pooledfor individual patients.

SDS-PAGEandimmunoblotting. Thegel systemdescribed

by Lugtenberg et al. (13) was used for sodium dodecyl

sulfate-polyacrylamidegelelectrophoresis (SDS-PAGE)and

211

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immunoblotting.

Wholecells

suspended

to an

OD470

of 5.0 in 0.01 MTris

hydrochloride buffer, pH 7.4,

wereadded toan

equal

volume of

sample

buffer. Solubilization and

denatur-ation was carried out at

100°C

for 10

min,

and then the

denaturedcells

(10

,ul)

were

separated

ona12%

gel

at15 mA

for 90min

(Hoefer

miniverticalslab

gel

apparatus; Bio-Rad

Laboratories,

Rockville

Centre, N.Y.).

The molecular

weight

standards used were

bovine

serum albumin

(66K),

ovalbumin

(45K),

pepsin

(34.7K),

and

trypsinogen

(24K)

(Sigma

Chemical

Co.,

St.

Louis, Mo.).

Separated

cell components were transferred from the

acrylamide gel

onto a nitrocellulose membrane

(pore

size,

0.45 ,um;

Bio-Rad)

by

the method of Towbin et al.

(23).

Electrophoretic

transfer took

place

overnight

at 30 V in an

ice-cooled buffer

(pH

8.3)

containing

25 mM

Tris,

192 mM

glycine,

and 20%

(vol/vol)

methanol

(Bio-Rad

transblot

apparatus).

Transfer of

protein

to the

nitrocellulose

was

determined

by staining

withamido black

(23).

Blots

carrying

the bacterial

antigens

were stored in

airtight

containers at

-20°C

until

required.

After removalfrom storage the

nitro-cellulose blotswere washed

by gentle

agitation

for30 minat

37°C

inTTBS

(0.3%

[vol/vol]

Tween-20,

0.9%

[wt/vol]

NaCl,

and 0.01 M Tris

hydrochloride, pH 7.4)

to block unbound sites on

the

nitrocellulose

(4).

TTBS was

replaced by

TBS

(0.01

MTris

hydrochloride

buffer,

pH

7.4,

containing

0.9%

[wt/vol]

NaCl),

in which the blots were rinsed three times.

Subsequently

the blots were

probed

with either

hyperim-mune rabbit serum or serum from

patients

with infective

endocarditisat adilution of1:50 in

TTBS

by gentle

shaking

at

37°C

for

3 h. Blots were then rinsed three times in TBS

and soaked in

protein

A-peroxidase

conjugate

(1:2,000)

(Sigma)

in TTBS for2 h at 370C.

Antigenic

cell components were visualized after

being

rinsed threetimesin TBS

by

addition ofa

freshly

prepared

solution

containing H202

(0.1%,

vol/vol)

and

4-chloro-1-naphthol (25

,ug/ml)

in 0.01 MTris

hydrochloride, pH

7.4.

Identification of

glycosylateçd

antigens.

Several S.

faecalis

antigens

were revealedtobe

glycosylated

by

being

suscep-tibletooxidation withsodium

periodate

or

by

ligand blotting

with lectins. For sodium

periodate

oxidation,

immunoblots

ofS.

faecalis antigens

were

prepared,

andunbound siteson

thenitrocellulose wereblocked withTTBSasstatedabove.

The blots were then incubated in 0.5 M sodium

periodate,

pH 4.0,

for 1 h at

37°C.

After a wash and

incubation

in

serum,

the blotswere visualized with

chloronaphthol-H202

asusual. Control blotswerepreparedinwhichthe

periodate

incubation

stage was omitted.

Ligand blotting

with lectins

followedthesamebasic

procedure

as

immunoblotting.

How-ever, instead of

incubation

in serum for 3 h the blots were

incubated in

peroxidase-conjugated

lectinsdiluted1:2,000in

TTBS for 2 h at

37°C.

The lectin

conjugates

used were

soybean

agglutinin-,

wheat germ

agglutinin-,

asparagus pea

lectin-,

and concanavalin A-peroxidase (all from Sigma).

Serodiagnosis

ofS.faecalis

endocarditis.

Serodiagnosiswas

performed

blind inthat serumwasobtainedfrom12 patients

withinfective

endocarditis

caused

by

organisms

unknown to

the authors.These

organisms

wereidentified separatelywith the API 20 STREPsystem (Appareils etProcédés

d'Identi-fication,

A.P.I.

Laboratory

Products

Ltd.,

Basingstoke,

United

Kingdom).

Patient sera were used to probe

im-munoblots prepared from seven Streptococcus species and

Staphylococcus

aureus,including

clinical

isolatesand

refer-ence strains. Blots probed with each

serum

included the

separated

cellular

components

of theorganism isolated from

theblood of thatpatient.

After

visualization

of antigens the immunoblots were

1 2 3 4 5 6 7 8 9 10 Il

73

-56----

-53 40---

37--66

-45 -34-7

---24 FIG. 1. Immunoblotanalysis of the antigens of different strepto-coccal species and Staphylococcus aureus aftergrowth in serum. Bacterial components were separated by SDS-PAGE and electro-phoretically transferredonto anitrocellulose membrane. Lanes: 1, S.faecalissubsp.zymogenes;2, 777; 3, EBH1; 4,S.faecalis subsp. zymogenes NCTC05957; 5,S.faecium NCTC07171; 6, S.milleri NCTC 10708; 7, S. mutansNCTC10449;8, S. sanguisNCTC 07863; 9, S. bovis NCTC 11436; 10, S. salivarius NCTC 08618; 11, Staphylococcus aureus (endocarditis isolate). The blot was reacted withhyperimmune rabbit serum raised against S. faecalis EBH1 grownin serum.Numbers tothe left andright indicate themolecular weight (103) of bacterial antigens. Numbers to the right indicate positionsQf molecularweightmarkers.

examined for the presence or absence of the major serum-inducedS. faecalis antigens (73K,40K, and 37K). Reaction of the serum with these antigensindicated infection with S. faecalis. Conversely, nonreaction of serum with these anti-gens allowed exclusion of S. faecalis as theinfecting orga-nism.

RESULTS

The antigenic profiles of the Streptococcus species and

Staphylococcus aureusaftergrowthin serum are shown in

Fig. 1. Major antigens of73K,56K,53K, 40K, and 37Kwere commontothe four strains of S.faecalis (lanes 1 to 4). The

73K, 40K, and 37K antigens were exclusive to S.faecalis

species and were not recognized by hyperimmune rabbit serumin the other streptococcalspecies or the

Staphylococ-cus aureus strain (lanes 5 to 11). The 56K and 53K S.

faecalis antigensdid notappear to havediagnosticpotential,

sincehyperimmune rabbit serum reacted with theseantigens

in all the streptococcal species (Fig. 1, lanes 5 to 10).

Both the 73K and37Kantigensareglycosylatedproteins.

Theyact as receptorsfor wheat germagglutinin, asparagus

pea lectin, concanavalin A, and soybean agglutinin (1).

Periodate oxidationof the blotpriortoincubation in serum

(0.5M sodiumperiodate, pH 4.0, for 1 h at 37°C) abolished the reaction of hyperimmune rabbit serum with the 37K

antigen (Fig. 2).

SerumfromapatientwithknownS.faecalisendocarditis was used toprobe a blot prepared from the same panel of

organisms (Fig. 3) plus two strains from other infective

endocarditis patients (lanes 5 and 6). This S. faecalis

endocarditis serum reacted strongly with all of the S.

faecalis antigens (lanes 1 to 4) recognized by the

hyperim-mune rabbit serum but not with antigens from the other streptococcal species (lanes 7 to 12). The Staphylococcus aureus strain wasalso recognized by this serum(lane 13).

Serafrom12patientsintheblindstudy were used to probe

replicateblots.Serafromsixpatientslater confirmed to have

S.faecalis endocarditisrecognized one or more of the 73K,

40K, and 37K antigens in the S.faecalis strains. The six

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-z,756

N137

73-56

--40- -FIG. 2. Immunoblot analysis of serum-grown S. faecalis

anti-gens after separationby SDS-PAGE and electrophoretic transfer ontonitrocellulose. Lanes1and 3 contain strain EBH1, and lanes 2 and 4 contain S. faecalis subsp. zymogenes. The blot was reacted withhyperimmune rabbit serum raised againstserum-grownEBH1, with (lanes 3 and 4) or without (lanes 1 and 2) prior incubation in 0.5 M sodium periodate. Numbers show molecular weight(103).

patients with non-S. faecalisendocarditis did not recognize

any S.faecalis antigens. Serum from all patients strongly

recognized antigens in the Staphylococcus aureus strain. S.

faecalis endocarditis patientseradid niot recognize

Staphy-lococcusaureus antigens ofsimilar molecularweightto the

S.

faecalis-specific

antigens, the major Staphylococcus

au-reus antigens being 82K, 70K, 66K, 58K, and 43K. An

immunoblot obtained with serum from a patient with S.

faecalis endocarditis is shown in Fig. 4. The 73K and 40K

antigenswere recognizedin the fourstrainsofS.faecalis.In

addition the serum reacted with the 56K antigen in the S.

faecalis strains and in the infecting strain of the patient. Itis

interesting thatalthough the S.faecalis strain infecting the

patient in Fig. 4 (lane 5) only weakly expressed the S.

faecalis-specific antigens, this serum still recognized the

equivalent antigens intheother S.faecalis strains(lanes 1 to

4).

Figure5shows animmunoblot probed with serumfroma

patient who had had twoepisodes of endocarditis. Thefirst

episode in February 1985 resulted in the isolation oftwo

organisms with the same API identification number but

different colonialappearance. Two

different

organisms were

isolatedfromthesecondepisode in October1985.Theserum

used toprobe the blotwas

collected

duringthesecond attack

FIG. 4. Immunoblot analysis of separated antigens of strepto-coccal species and Staphylococcus aureus. Lanes: 1, S. faecalis subsp. zymogenes; 2, 777; 3, EBH1; 4, S. faecalis subsp. zymogenes NCTC 05957; 6, S. faecium NCTC 07171; 7, S. milleri NCTC 10708; 8, S. mutans NCTC 10449; 9, S. sanguis NCTC 07863; 10, S. bovis NCTC 11436; 11, S. salivarius NCTC 08618; 12, Staphylococcus aureus (endocarditis isolate). Lane 5 contained a non-S. faecalis strainisolatedfrotnthe bloodofapatientwithendocarditis. The blot was reacted with serum collectedfrom this patient. Numbersshow molecularweight (103).

of endocarditis. It is interesting that the serum from this patient, in addition to recognizing the characteristic S. faecalis antigens (indicating an infection by this organism),

recognizeda67Kantigen present in theinfecting strain (lane 5) and in the reference S. bovis strain (lane 11). The first endocarditis episode of this patient was caused by an S. faecalis infection and the second by S. bovis.

Figure 6 showsanimmunoblot probed withserumfroma patient withStaphylococcus aureus endocarditis. Only the antigens of hisownisolate(lane 5) and the reference strain of Staphylococcus aureus (lane 12) were recognized by the serumfrom this patient.

DISCUSSION

We have previously shown that protein antigens of S. faecalis can be visualized on immunoblots with antiserum (1). The antigenic profile obtained isstrongly dependent on how the organism is grown. In the present study we have usedgrowth in serum to simulate the transient bacteremia which precedes colonization of the heart valve in infective 1 2 3 4 5 6 7 8 9 10 Il 12 13

73- -- - .

56-40- _

37---l

73-

67-40

37

--FIG. 3. Immunoblot analysis of separated antigens of strepto-coccal speciesandStaphylococcusaureusgrowninserum. Lanes: 1through 4,asforFig. 1;lane 5,strain ofS.faecalisisolatedfrom the bloodofapatient withendocarditis; lane6, anon-S.faecalis

endocarditisisolate;7through 13,asforlanes5through11in

Fig.

1. The blot was reacted with serum from apatient with S.faecalis endocarditis. Numbers show molecularweight (103).

1 2 3 4 5 6 7 8 9 10 11 12 13

1

4-k

FIG. 5. Immunoblot analysis of separated antigens of

serum-grownstreptococcalspeciesandStaphylococcusaureus.Lanesare

as for Fig. 3 except for lanes 5 and 6, which contain organisms isolated fromthebloodofapatientduringthe first oftwoepisodes ofendocarditis(seetext).Theblotwasreacted withserumfrom this patient. Numbersshowmolecularweight (103).

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2 3 4 5 6 7 8 9 10 Il 12

73

---

40---37--:3`:i

FIG. 6. Immunoblot analysis of separated antigens of

strepto-coccalspeciesandStaphylococcusaureus.Lanes: 1to4and6to12,

as for Fig. 4. Lane S contains a Staphylococcus aureus strain

isolatedfromtheblood ofapatient with endocarditis (notthe same

patient or strain as inlane 12). The blot was reacted with serum

collected fromthispatient. Numbers show molecular weight(103).

endocarditis. Results from this blind trial have shown that

serafrom sixofsixS.faecalisendocarditis patients reacted with three prominent protein antigens (73K, 40K, and 37K)

producedbyS.faecalis after growth inserum. Itis

interest-ing that these proteins are only weakly expressed in cells

grown in acomplexlaboratory medium such as brain-heart

infusion (1).

Sera from these patients did not react with antigens of similar molecularweights in any of the other streptococcal

species examined. Furthermore, sera from patients with

endocarditis due to other species (Staphylococcus aureus,

Staphylococcus epidermidis, S. sanguis, and S. bovis) and from a patient with cultùre-negative endocarditis did not

react with antigens in any of the S. faecalis strains. We

recently extended this studytoincludeserumsamplestaken

from patients with a wide range of infections. First, sera

from 26 patients with endocarditis caused by a range of

organisms other than S.faecalis all gave anegative result.

Likewise, sera from 34 patients with septicemia caused by

organismsotherthan S.faecaliswerenegative.Sera from 10

patients undergoing continuous ambulatory peritoneal dial-ysis and likely to exhibit high antibody titers to bacteria because of recurrent infections were all negative. Finally,

sera from 48 patients with various S. faecalis infections

(including 28 urinary tract infections) but not endocarditis

gave negative results. To date, only serum from one

nonendocarditis patient has produced a partially positive

reaction, recognizing the 73K antigen.This patienthad had repeated isolations ofS.faecalisover severalmonths from

nephrostomy fluid.

Thisstudytherefore demonstratesthe diagnosticpotential

ofthetechnique, which shouldenableapositive diagnosisof

S.faecalis infection to be made even in culture-negative

cases.Equallyimportant,itshould bepossibletoruleoutS.

faecalisastheinfectingorganism inendocarditis causedby

otherspecies. As thisserodiagnostictechniqueis both rapid

and sensitive, it will permit promptinitiation ofthe

appro-priate antibiotic chemotherapy. The test takes

approxi-mately 5 h to complete with a prepared nitrocellulose

im-munoblot.This isaconsiderableimprovementovermethods

whichrequire isolationand cultureof theorganism.Itmight

alsobepossibletoexpand this techniquetoinclude

diagno-sis of other streptococcal species that exhibit

species-specific characteristicantigens. For example,S. bovis (Fig.

5) produces a67K

protein

antigen

whichwas detected

only

by the serum of the

patient

infected with this

organism.

It is

becoming

increasingly

apparent that surface

proteins

ingram-positive cocci playan

important

part in

pathogenic-ity; for example, M protein in group A

streptococci (5,

14),

fibronectin receptors in staphylococci and

streptococci

(6,

11, 12, 20), and surface proteins on oral

streptococci (2,

16,

18). Ourdemonstration ofanumber of surface

proteins

in S.

faecalis which are

strongly expressed

in simulated in vivo

conditions leads us to

speculate

ontheir role in

pathogenic-ity. We are currently investigating their

possible

involve-mentin adhesionto valvularvegetations vialectinlike

inter-actions with receptors on host tissues

(3, 9,

21, 24)

and

bindingtofibronectin (6,20). Isolation and

purification

ofthe

proteins is a first step to the further

exploitation

of their

diagnostic value and also tothe assessment of their

protec-tive capacity.

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