Rapid screening assay for toxic shock syndrome toxin production by Staphylococcus aureus

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Rapid

Screening

Assay

for Toxic Shock Syndrome Toxin Production

by Staphylococcus

aureus

LANA S. WECKBACH,l* MICHAEL R. THOMPSON,2 JOSEPH L. STANECK,1 AND PETER F. BONVENTRE3 Departments of Pathology and Laborato,y Medicine,1 InternalMedicine, Division ofDigestiveDiseases,2 and

Microbiology

and

Molecular Genetics,3

University

of

Cincinnati,

Ohio 45267-0714

Received 9 January 1984/Accepted 16 March 1984

Arapid immunoblotassay (TST-blot)wasdeveloped and usedtoscreenStaphylococcusaureusisolates for

toxic shock syndrome toxin (TST)production.Growth froman18-hstab inoculum of S.aureusonbrain heart

infusionagar wastransferred directlytoanitrocellulose sheet.Nonspecificprotein binding siteswereblocked with bovineserumalbumin, and the nitrocellulose sheetwasincubated withaffinity-purifiedantibodytoTST, followed by incubation with horseradishperoxidase-conjugated protein A. Toxinwasvisualized by detection of

the peroxidase-conjugated protein A-anti TST-TST complex with 4-chloro-1-napthol. The sensitivities and specificities of the TST-blotandOuchterlony microslide immunodiffusionassaywerecompared byscreening

141S. aureusisolates for TSTproduction.Inbothassays,53of 141 isolates produced detectable levels of TST, whereas 88 isolates producednotoxin.A 100%concordancewasfoundbetweenthetwoassays.TheTST-blot

yielded thesameresultsin less than 24 hasthoseyielded by the 3-day immunodiffusionassay.Thus, this rapid

method fordetection of TSTin multiple samplesappearstobe well suited fordiagnostic and epidemiological studies. Furthermore,itwould appeartobe ideal forusein TST geneticsresearch.

Toxic shock syndrome (TSS), a clinical entity first

de-scribed by Todd et al. (10) in 1978, is a manifestation of

Staphylococcusaureusinfection.Thusfar, theonly reliable marker for identifying S. aureus associated with TSS has

been the production ofa toxin designated staphylococcal enterotoxin F (1) or

pyrogenic

exotoxin C (9). Recent publications (2, 3)haveindicatedthattheseareprobablythe same toxin, and, therefore, the designation toxic shock

syndrome toxin(TST) will be used in this paper.

Three methods for identifying TST are Ouchterlony immunodiffusion (1), isoelectric focusing (9), and Western

blot (2). All three methods require

specialized

instrumenta-tion,and 3 daysormore are required forcompletion ofthe assay. Arapid immunoblotassay(TST-blot) todetect

TST-producing S. aureus has been developed. The TST-blot is

sensitive andspecificandyields interpretable results in less than 24 h.

MATERIALSAND METHODS

Source of isolates. The S.aureusisolates used in this

study

were classified (i) genital non-TSS, (ii) acute TSS, (iii) recoveredTSS, (iv) clinicalnongenital, and (v) food

entero-toxigenic.

The

genital

non-TSS strainswere collected from

the labia, fornix, or endocervix of

nonsymptomatic

women

in a S. aureus prevalence survey described

previously

(5).

The acute TSS isolates were genital, surgical, or from a

wound and were obtained from hospitalized patients

diag-nosedasconfirmedorpresumptiveTSS. Thesewere

provid-ed by the University of Cincinnati TSS Study Group; W.

Altemeier, University of Cincinnati; and H. Pickrum,

Procter and Gamble Co., Cincinnati, Ohio. The recovered TSS isolatesweregenitaloranteriornarescultures obtained from women cultured 3 months to 2 years after recovery from TSS andwerecollected inconjunctionwith the preva-lence study (5). The clinical nongenital isolates were from

staphylococcal infections andwere

provided

bythe Clinical

*Correspondingauthor.

Microbiology Laboratory, University of Cincinnati Medical

Center. The food enterotoxigenic isolates originally were

implicated in food poisoning outbreaks and were proven

enterotoxin producers; they were provided by R. Bennett, Food and Drug Administration, Washington, D.C. All S. aureusisolateswerecodedand frozen in brain heart infusion

(BHI) broth with 20% glycerol before testing for TST

production by microslide immunodiffusion and bythe

TST-blot.

Affinity-purified antiserum for TST. Purified TST and

rabbit antiserum forTST werekindly provided by D. Arch-er, Food and Drug Administration, Cincinnati, Ohio.

Puri-fied TST was coupled to CNBr-activated Sepharose 4B

(Pharmacia FineChemicals) by using theinstructions of the

manufacturer for coupling. The column was prepared by

coupling5 mgofpurifiedTSTto1 gof swollen gel. To obtain

affinity-purified antibody to TST, 7 ml of high-titer rabbit

anti-TST was diluted to a volume of 50 ml in 20 mM

phosphate-bufferedsaline(pH 7.2) and then pumped through thecolumn overnight at 4°C. After the binding ofanti-TST

antibodyto thecolumn, thespecific anti-TSTantibody was eluted with 5 M KI in 50 mM Tris (pH 7.4) at room temperature. The eluted affinity-purified antibody was dia-lyzedextensively against phosphate-bufferedsaline and then

divided and frozen(-80°C).

Immunodiffusion assay. TST was produced for the immunodiffusion assay by the membrane-over-agar method (7),usingBHI agar (DifcoLaboratories). Briefly, S. aureus isolateswereinoculated into4mlof BHI broth and incubat-ed without shaking at 37°C in 7 to 10% CO2 overnight. An inoculum of0.1 ml of each broth culture wasused on agar

platesoverlaid withdialysismembrane (Spectropor12-14K; Fisher Scientific Co.). Inoculatedplates were

incubated

for 18 h at37°C in 7to 10% C02;then 2.5mlof 0.01 Msodium

phosphatebufferwasappliedtotheplates.Aftersuspending

the bacterial mass, organisms were removed by centrifuga-tion,and the supernatantwastestedfor the presence of toxin

by microslide immunodiffusion as previouslydescribed (5).

TSTwasusedat aconcentration of1 ,ug/ml, andantiserum for TST was used at 1:4 dilution. Twenty microliters was

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RAPID ASSAY FOR TOXIC SHOCK SYNDROME TOXIN 19

added per well. Standards of TSTandanti-TSTwere provid-ed by M. Bergdoll, University ofWisconsin, Madison.

TST-blot. The following reagents were prepared for the TST-blot:a stocksolutionof 20 mMTris-150mMNaCl(pH

7.4) at 25°C (Tris-saline) and 5% (wt/vol) bovine serum albumin (BSA)-Tris-saline were prepared as described by

Towbin et al. (11). Horseradish peroxidase conjugated to protein A (Protein A POD conjugated; Boehringer

Mann-heim Biochemicals) (protein A-POD) was

used

at a 1:1,000

dilution in 5%BSA-Tris-saline. A modification of the

chro-magen visualization procedure described by Hawkes et al. (4) wasused. A stocksolution of3 mg of4-chloro-1-napthol

per ml of methanol was stored at 4°C in the dark and was

diluted with 5 volumes of 50 mM Tris (pH 7.4) and H202

added to 0.015% concentrationjust before use. Protein A-POD was stored at -20°C inportions and diluted just before

use. All other reagents were stored at 4°C. The affinity-purified antiserumforTSTwasdiluted 1:20 in 5% BSA-Tris-salineand stored at

-200C

or4°C,dependingonthe

frequen-cy ofuse. Theantiserumwas stabletofreezing and thawing

and was reused several times.

The TST-blot was performed by stab inoculating an ac-tivelygrowing culture from agar orbrothonto BHI agar (Fig. 1). Toscreenorganismsfortoxin production, amaximumof 36 isolates wasinoculated perplate. Plates were incubated

for 18 h at 37°C in 7 to 10% CO2. All additional TST-blot procedures were at room temperature. A cut sheet of nitrocellulose separating paper (blue waxed paper) was placeddirectlyonthe agarsurface andthenquickly lifted to remove mostof the culture growth fromtheagar. Asheet of nitrocellulose (Millipore Corp.) was placed directly on the

agarfor10min, removedwithforceps, and thenplacedagar side upinanemptypetri dish. The sheetwas marked before

removal from the agar to aid in alignment with the culture

plate afterdevelopment.

The nitrocellulose was rinsed three times with 15 ml of 0.05% BSA-Tris-saline with gentle shaking to remove any

adhering growth. Then the nitrocellulose was incubated in

5% BSA-Tris-saline for 30 min without shaking to block nonspecific protein binding sites. The blocking solution was

discarded, affinity-purified antiserum for TST (diluted 1:20 in 5% BSA-Tris-saline) was added, and the sheet was

incubated for 2 h without shaking. The

nitrocellulose

sheet was rinsed in 200 to

400

mlof

Tris-saline

withgentleshaking for 15minto remove unbound antibody and then

incubated

with 15 ml of protein A-POD (1:1,000) for 1 h without shaking. The rinsingprocedure was repeated to remove the residual unbound proteinA.

Finally,

the nitrocellulosesheet was placed in a clean petri dish with freshsubstrate. Areas in which TSTimmunocomplexwas bound to thenitrocellulose

werevisualizedby ablue-purplecolordeveloping within 5to

15 min. After color development, the substrate

was

thor-oughlyrinsed fromthenitrocellulose withwater, andthetest was readimmediately. Any visible colordevelopment (blue-purple) was scored as a positiveTSTproducer. Known

TST-positive

(tox+)

and TST-negative (tox-) isolates were

in-cludedwith each assay as controls.

RESULTS

The sensitivity and specificity of theTST-blot were influ-enced by the type ofmedium, concentration ofBSA,

condi-tions ofincubation, and dilution of anti-TST. To determine

anoptimum culture medium for

the

TST-blot, four commer-cially available agarmedia (tryptic soy plus 5% sheep blood

[BAP],

Columbia [CNA], tryptic soy [TSA], and BHI) were

eachinoculated with 36 S. aureusisolates,and toxin

produc-InoculateBHI ager incubate 370C, 18 hr

Overlaywithnitrocellulose (NC)

10'

room

temperature

(RT,

250C)

Wash .05%BSA-Tris-Saline, 5'with

shaking

IncubateNC with5% BSA-Tris saline

30',

RT,

stationury

IncubateNC with antiTST

2hr,RT,

stationary

Wash Tris saline

15'

RT

IncubateNC with protein A-POD I hr RT

Wash Tris saline 15' RT

Transfer NCtofreshpetridish,incubate in substrate

5-15'

RT

Rinse tapwater/blot dry- store indark FIG. 1. Flowdiagram of TST-blot procedure.

tion was determined (Fig. 2). Only the S. aureius isolates which were strong TST producers on TSA and BHI were detected onCNA and BAP. TSA gave greatercolor intensity

but

occasionally yielded

false-positives.

BHI

yielded

the

most reliable and unambiguousresults.

Concentrations of 1 and 5% BSA

blocking

buffers were

compared for

efficiency

in

blocking nonspecific

protein bindingsites. The5% BSA

yielded

moreconsistentresultsin

the assay. The optimum conditions of incubation were at roomtemperature(25°C)andwithout

shaking.

Incubationat 4 and 42°C in anti-TST and protein A-POD decreased sensitivity, and incubationat37°Cdecreased

specificity.

The shaking ofthese reagents during incubation increased the

nonspecific binding, thus

decreasing

the

specificity

of the

assay. Affinity-purified anti-TST also was requiredto elimi-nate the nonspecific cross-reactivity observed with unpuri-fied antitoxin. Adequate color development was observed with antibody dilutions of 1:5 and 1:20 within the 2-h incubation time, but noappreciable color developmentwas found with a 1:50dilution of anti-TST within the sametime

period. TST transferred to the nitrocellulose was easily observed as ablue-purple color on a white background.

Enterotoxigenic food isolateswere usedto testthe

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20 WECKBACH ET AL. #."#e ;: ,,,:f' ,4r:: .s :-X>., ,. .g : |,' .,'S: :s: , es: ''.:. ;V:: *x:.'., A

ttsci<

s ; * ,pt vr., ?r .re .;r. 'S :@ 8

,$S, :@ :*

.4 : : oW':_r, J \<; 4' .SR., ,,, B: :.:. ... .rr s-.. .,., ,,r a^L

.E, , _

'R wr.

i: _,"',*:.ew.

.,, :. ={ ,

*..L'_:

w.. '' SF 0.

FIG. 2. Comparison of TST-blot for36 S. aureus isolate

inoculated on four types ofagar culture medium. Nitrocel

from TST-bloton(A) CNA,(B)BAP,(C)BHI, (D)TSA.Dar

areasindicate thepresenceof bound TST immune complex.

ficity of theassay. These 24isolates wereknowntoprc oneormoreof enterotoxinA-E. No cross-reactionwas

withanyofthese isolates in the TST-blot. Asafurthe

for specificity, nitrocellulose was inoculated with pu staphylococcal enterotoxin A, staphylococcal entero

B, and TST. When the TST-blotwas performed, only

gave a positive reaction. No cross-reaction was seen

staphylococcal enterotoxins A orB.

The sensitivities of TST-blotandOuchterlony immur fusionwerecompared since immunodiffusion isan acc

methodfordetecting TST-producing S.aureus(1, 2). A

of 141 S. aureus isolates categorized as genital non-acuteTSS,recoveredTSS, clinical nongenital non-TSS foodenterotoxigenicweretestedby toxin productionin assays (Table 1). A 100% concordance was found bet

theTST-blot and immunodiffusion. WhenaS.aureusis wastox+ inoneassay,itwasalsopositive in the othera

Therefore, under these conditions the TST-blot and (

terlony immunodiffusion wereconsidered equivalent ir sitivity (6 ng) and specificity.

The results of a TST-blot of 36 S. aureus isolate

shown in Fig. 3. The areas ofimmunoprecipitation v

ized as blue-purple spots on nitrocellulose indicates

producing S. aureus. In this assay, 10 of 36 isolates TSTpositive. A reproducible phenomenon observed i TST-blot was that when some S. aureus isolates pros

TST which diffused into themediumsurroundingthec

with otherisolates, TSTwasdetected onlydirectly be

the colony. The feasibility ofdetecting tox+ isolates mixed with (tox-) S. aureus isolates was determine

surface

streaking

BHI agar with an

appropriate

diluti tox+ and tox- S. aureus in BHI broth to yield isc colonies. When the areas of immunoprecipitation o

nitrocellulosewerecomparedtothediscrete coloniesc

BHIagar, only colonieswith pigmentation characteristic of the

tox+

isolate was positive by the TST-blot (Fig. 4).

Therefore, tox+ colonies could be distinguished from tox

coloniesby the TST-blot.

IDISCUSSION

Several methods have been developed to identify TST-producing strains of S. aureus. These include Ouchterlony

immunodiffusion (1), isoelectric focusing(9),andan

electro-phoretic transfer procedure (Western blot) (2). All three methodsare relatively time consuming, require specialized

instrumentation or radiolabeled reagents or both, and

re-quireatleast 3 days for interpretable results. The TST-blot utilizes commercially available medium and reagents and does not require any specialized equipment. At least 36

isolates can be tested on a single plate compared with 1

isolateperplate by the membrane-over-agar method. In the

clinical laboratory, an isolated S. aureus colony from a

suspected TSS patient could be used to directly inoculate BHI agar, and TST production could be determined within

24h.Since inaclinical setting onlyoneortwoisolates would be tested ata time, the procedurecan be adapted to small strips of nitrocellulose and can be performed in test tubes with smaller volumes ofreagents (unpublished data). The

s stab sensitivity and specificity of the TST-blot are equivalent to

[lulose

those ofthe immunodiffusion assay.

^kened Factors which were controlled inoptimizing theTST-blot procedure were media, incubation conditions, and

concen-tration of anti-TST. When f3AP, CNA, TSA, and BHI agar

werecompared, onlyisolateswhichshowedstrongreactions

on TSAand BHI were also detectedon BAP and CNA by

the TST-blot. TSA (Fig. 20)) gave a more intense color

)duce reaction than did BHI (Fig. 2C), but it also occasionally seen yielded false-positives. BHI gave the most reproducible

rtest result and, therefore, was the medium used in subsequent rified assays. This difference in toxin detection between media )toxin may be a function of a specific nutrient requirement by

TST selected isolatesfor TSTproductionormaybe the resultofa

with component in the media interfering with the assay. Other investigators (8) have shown thatvaryinggrowth conditions nodif- can influence the amount of TST produced by selected S. epted aureusisolates. Another characteristic of TSTproduction by

i total the S.aureustested inthe TST-blotwasthat insomeisolates

-TSS, a halo of toxin surrounded the colonial growth and others

5,and only had toxin under the colony (Fig. 3). This phenomenoh

iboth may reflect hypertoxin or hypotoxin production orperhaps ween

solate issay

:)uch- TABLE 1. Comparisonof TST-blot and immunodiffusionfor

ncn detection ofTST

i sen-s are isual- TST-were in the Juced olony neath when -d by ion of Mlated in the )nthe TSTassays' Source of isolates

Immunodiffusion TST-blot

Genitalnon-TSS' 5/43 5/43

TSS,acutec 42/42 42/42

TSS,recoveredd 3/8 3/8

Clinicalinfectionse, non-TSS 1/24 1/24

Foodenterotoxigenicf 2/24 2/24

aDataarepresentedasnumberofpositives/numberofisolatestested. bGenital carriage isolates were obtained during a prevalence survey conductedbytheTSSStudyGroupof theUniversityofCincinnati(5).

'S. aureus wasisolatedfromclinicallyconfirmed TSS.

d S. aureus isolates werefrom women several months to 2years after clinical TSS.

eClinical isolateswerefrom diverse fociofinfectionfrompatientsatthe University Hospital.

fEnterotoxigenicS. aureus wereincriminated in foodoutbreaks. J. CLIN.MICROBIOL.

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RAPID ASSAY FOR TOXIC SHOCK SYNDROME TOXIN 21

&

-

1~

3

J. I

lI)

1,

/0

9 I

'

1S*iiI~'(lDt' 34

,

1

E?

A;

14-

1

13

0.

34 .23 RX

it

AD I

i

$o

2f

4 .L7

A

tE

*

3G

3f

43

SX * i

FIG. 3. Thirty-six S. aureusisolates screened for TST production by TST-blot.(A)Stab-inoculatedBHIagar;(B)TST-blotnitrocellulose of plate Awith 10tox+ and 26tox- isolates.

may be areflection ofcell-associated versus freely soluble toxin.

Theconditions ofincubation werecontrolledon thebases oftime, temperature, and shaking versus not shaking. The time of incubation for each step is flexible; however, the times given yielded optimum results. When the time of

incubation in BSA was decreased to less than 20 min, an

increase in nonspecific binding was evident. Adecrease in

incubation timeinanti-TSTdecreased sensitivity until weak-erpositives were notevident.Increasingthe

incubation

time

in anti-TST did not appreciably increase sensitivity.

De-creasing the time of incubation in protein A-POD also

decreased theintensity of the color reaction. The tempera-tureofincubationwasoptimumat25°C (roomtemperature).

At 4 and 42°C only strong positives were detected by the

TST-blot. Incubation at37°C resulted innonspecific binding

and thus

yielded

several

false-positives.

A factor which

appeared

to be

mandatory

was not

shaking

the solutions

during

incubation.

Shaking

increased

nonspecific

binding,

yielding

many

false-positives.

Theconcentrationof the

anti-TSTwasanother

limiting factor;

asthe concentration of

anti-TSTwas

decreased,

the

intensity

of the reaction decreased

until no

positives

were evident withinthe 2-h time

period.

Using

Western blot

analysis,

we have observed that a

number ofthese S. aiureius isolates

produced

common pep-tides which cross-react with the non-affinity-purified

anti-TST

(unpublished

data).

Thus,

an

affinity-purified

anti-TST

immunoglobulin

was needed to achieve

specificity

in the

TST-blot. The anti-TST was reused several times without

lossof

activity. Therefore,

asmallvolume of

affinity-purified

antiserum could be used to test hundreds ofisolates. The

specificity

ofanti-TSTwas shown when no cross-reactions

A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

L

FIG. 4. Equalvolumesof BHIbrothsinoculated with tox+and tox-S.aureus andincubatedat37°C for 18 hwerecombined, diluted,and surfacestreaked on BHIagar. (A)Discrete coloniesoftox+and tox- S.aureuson BHIagar;(B)nitrocellulose from TST-blotofplateAwith tox+ colonies visualized.

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with staphylococcal enterotoxins occurred in the food

en-terotoxigenic isolates. Previous studies (6, 7) have shown

that theenterotoxins areproduced in BHI agar and also can be produced simultaneously with TST (1). Thus although oneormoreenterotoxinswereprobably present in the agar, no false-positives wereobtained with the food isolates.

Several advantages of the immunoblot assay are

envi-sioned. First, theTST-blot may provide a valuable tool in epidemiological studies since large numbers of S. aii-eiis

isolates can be screened rapidly for TST production with minimum handling. Second, in geneticresearch,selectionof

tox+ and tox- isolates or hypertoxin producers is feasible

with thisassay. Finally, clinical laboratories can determine within 24 h whether a suspected TSS patient is colonized with a TST-producing strain of S. aureus.

LITERATURECITED

1. Bergdoll, M.S.,B. A.Crass, R.F. Reiser,R. N. Robbins,and J. P. Davis. 1981.A newstaphylococcal enterotoxin, enterotox-in F, associated with toxic-shock-syndrome. Lancet i:1017-1021.

2. Bonventre,P.F., L.Weckbach, J. Staneck,P. M.Schlievert,and M. Thompson. 1983. Productionofstaphylococcal enterotoxin Fandpyrogenic exotoxin Cby Staphylococcusaureusisolates from toxic shocksyndrome-associatedsources.Infect.Immun. 40:1023-1029.

3. Cohen, M.L., L. M.Graves,P. S. Hayes, R.J. Gibson, J. K.

Rasheed,andJ. C. Feeley.1983.Toxic shocksyndrome: modifi-cation andcomparison of methods fordetecting marker proteins in Staphylococcus aureus. J. Clin. Microbiol. 18:372-375. 4. Hawkes, R., E. Niday, and J. Gordon. 1982. A dot

immunobind-ing assay for monoclonal and otherantibodies. Anal. Biochem. 119:142-147.

5. Linnemann, C. C., J. L. Staneck, S. Hornstein, T. P. Barden, J. L. Rauh,P. F. Bonventre, C. R. Buncher, and A. Beiting. 1982. The epidemiology of genital colonization with Staphvlo-coccus aureus. Ann. Intern. Med. 96:940-944.

6. Meyer, R.F., and M. J. Palmieri. 1980. Single radial immunodif-fusion method for screening staphylococcal isolates for entero-toxin. Appl. Environ. Microbiol. 40:1080-1085.

7. Robbins, R., S. Gould, and M. Bergdoll. 1974. Detecting the enterotoxigenicity of Staphylococcusaureusstrains.Appi. Mi-crobiol.28:946-950.

8. Schlievert, P. M., and D. A. Blomster. 1983. Production of staphylococcalpyrogenic exotoxin type C: influence of physical and chemical factors. J. Infect. Dis. 147:236-242.

9. Schlievert,P.M., K. N.Shands,B. B.Dan, G.P.Schmid,and R. D. Nishimura.1981.Identification and characterization ofan exotoxin from Staphylococcus aureus associated with toxic shocksyndrome. J. Infect. Dis. 143:509-515.

10. Todd, J., M. Fishaut, F. Kapral, and T. Welch. 1978. Toxic shock syndrome associated with phage-groupIstaphylococci. Lancetii:1116-1118.

11. Towbin,H.,T.Staehelin,andJ.Gordon. 1979.Electrophoretic transferofproteins from polyacrylamide gelstonitrocellulose sheets: procedure andsomeapplications. Proc. Natl. Acad. Sci. U.S.A. 76:14350-14354.