Evaluation of Sulfamethoxazole Trimethoprim Blood Agar Plates for Recovery of Group A Streptococci from Throat Cultures

Vol. 9, No. 2 0095-1137/79/02-0189/05$02.00/0

Evaluation of Sulfamethoxazole-Trimethoprim

Blood Agar

Plates

for Recovery of Group

A

Streptococci from Throat

Cultures

TERRENCE A. KURZYNSKI* AND CHERYLL K. MEISE

State Laboratory of Hygiene,Madison, Wisconsin53706 Received forpublication20November 1978

Wecompared the selective bloodagarmedium of Gunnetal.(J.Clin.Microbiol.

5:650-655, 1977) which containssulfamethoxazole plus trimethoprim (SXT-BA)

to the conventional bloodagarsurface plate (SBA) and a modified blood agar

pourplate plus brothmethod for therecoveryofgroupAstreptococcifromthroat

swabs. The influence of CO2 and ambient air incubation of the SXT-BA and SBA

plateswasalso evaluated. A total of696throatswabsfrom symptomatic children

werecultured simultaneously by the five methods and observed after overnight

incubation; 204 positive cultureswere detected overall. Recoveryratesof each

individual methodwere:SXT-BA (CO2),90.7%; SXT-BA (air), 87.7%;pourplate

plus broth, 83.3%; SBA(CO2),79.4%; and SBA (air) 77%. Approximatelyone-half

ofthe false-negative cultures in the SXT-BA (C02) and SXT-BA (air) methods

had colonycountsof -10to100coloniesperplate. Incontrast,for theSBA(CO2),

SBA (air), andpourplate plus broth methods, approximately 70% of the

false-negative cultures had colony countsof 210to100/plate. False-positive cultures

obtained by the SXT-BA (002) and SXT-BA (air) methodswere11 and 12.7%,

respectively-one-halfashigh astheratesobtained by the remainingmethods.

Beta-hemolyticstreptococci, groups C, F, and G, areinhibited onthe SXT-BA

plates andweretheprimarycause ofthe higher false-positiveratesonSBA and

pourplate plus broth methods. An additional 3% positive cultureswereobtained

by incubatingSXT-BA (002) platesupto48 hbefore discardingasnegative. We

recommend either the SXT-BA (002)ortheSXT-BA(air)method withupto48

h of incubationfor routineusein throat cultures.

Recently, Gunn et al. (6) introduced a new

selective surface

plating

medium, which contains

sulfamethoxazole plus trimethoprim (SXT-BA),

for the enhanced recovery ofgroup A

strepto-cocci from throat cultures. We have compared

the SXT-BA

plate

to the conventional sheep

bloodagar(SBA) method andto amodification

ofapourplate plus broth (PP+B) method.We

also found the SXT-BA

plate

tobe

superior

to

the SBA

plate

in this and in a previous study

(8a). TheSXT-BAwasalsomoresensitive than

themodifiedPP+Bmethod for the isolationof

group Astreptococci.

MATERIALS AND METHODS

SXT-BA and SBAplatesweremadeinour labora-toryby usingTrypticase soy agar(Baltimore

Biologi-cal Laboratories [BBL]).Thestock solution of sulfa-methoxazoleplustrimethoprim (BurroughsWellcome

Co.)wasmadeasoriginallydescribed(6),exceptthat

thefinal volumewasmadeupto 1literinsteadofthe recommended100ml.This greaterdilutionwasused toavoidworkingwith thesuspensionof thetwodrugs

that occurs with lesser dilution. Quantities of

sulfa-methoxazole-plus-trimethoprim stock solution were storedat-20°C until the day of use. For the SXT-BA plates, 10ml of stock solution was added per liter of agaraftermelting but before autoclaving. After

auto-claving and cooling to 55°C, 7% sheep blood was

added, and 15 ml of SXT-BA was dispensed into plastic petriplates (100by15mm). SBAplateswere prepared in thesamemanner,butwithout the sulfa-methoxazole plus trimethoprim. Plates were stored under refrigeration for up to 1 week, but we have found themtobe stable foratleast2weeksifkeptin aplastic bag.

A total of696throat swabs (cotton tipped) from symptomatic children between the ages of 2 and 15 yearsoldwerecultured.Specimensweresubmittedin amodified Stuarts transport medium by physicians

locatedthroughoutWisconsin. Most specimenswere intransit only 1 day, butsome were2days old upon culture.

Each swabwasfirst inoculated upontwoSBAand then upon two SXT-BA plates which were then streaked for isolation.Several stabsweremade in both theprimary andsecondaryareasofstreakingto

pro-mote betahemolysis. Onesetofplates consisting of

anSBA andanSXT-BAplatewasincubatedat37°C

under 5 to 10% CO2 [SBA (CO2) and SXT-BA 189

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(CO2)1; the secondsetwasincubated at370C under ambient atmosphere [SBA (air) and SXT-BA (air)]. Thiswasdonetodetermine whether theC02 incuba-tion originally used (6) was necessary. Swabs were

thenused for the PP+B culture method.

It has been determined that by swabbingasingle

plate directly with the swab, approximately3to5% of theorganismspresentareremoved from the swab(2).

Since fourplateswereswabbed, approximately 12to

20% of the organisms had been removed before the swab was used in the PP+B method. Practically

speaking, performing thePP+B method last should have had a negligible effect on the detection and

quantitation ofgroup A streptococci by the PP+B method.

The PP+B method usedwas a modification

rec-ommendedtoourlaboratory byElaineUpdyke (then atthe Center forDisease Control, Atlanta, Ga.).We didnotuseeither preincubationof theswabs in broth asisnowrecommended forgreatersensitivity (4),or

surfacestreakingofplates.Swabswereplacedin 3 ml

of Todd Hewitt broth and mixed vigorously on a

Vortex mixer for5s.Next, asterile 6-inch (ca.

15.2-cm) applicator stick wasinserted into thebroth,

re-moved, and reinserted into a tube of melted veal

infusionagar (15ml/tube; BBL). Theagarhad been cooledto 550C and contained 7% defibrinated sheep blood.Approximately0.001 mlof inoculumwas trans-ferred from the broth to the melted agar.The agar tubewasthen mixedby inversion, pouredintoplastic petri plates, and allowedtosolidify.Pourplatesand correspondingbroth cultureswereincubated at37°C

overnightinroomair.

Allthroat swabs were cultured before nooneach

dayand observed forbeta-hemolytic streptococci-like coloniesby 8:30 a.m.thefollowing day. Plates were observedby usinga fluorescentlight sourcelocated behind theupheld plate.

All cultures positive for beta-hemolytic strepto-cocci-like colonieswerescreened forgroupA strepto-cocciby using astandardfluorescent-antibody (FA) technique (9).Smearsweremade fromthe broth cul-ture used in the PP+B method, asis usual for that

method, or by making a directsmear from isolated

colonieson oneof the surface streak plates (3). The

LancefieldprecipitintestwasusedtoconfirmourFA resultswhenever differenceswerenoted in the ability ofthe various methods to recover groupA

strepto-cocci.

In general, for all methods group A streptococci

colonieswerequantitatedonthe basisof thefollowing

categories: negative; <10;10to100;and >100 colonies perplate. However,if <10 coloniesperplatewereonly

visibleon asurface streaked plate but they occurred

in the area of the secondary streaking, the colony countwas recorded asbeing 10to 100 colonies per plate. Additionally, if1to100colonieswerevisibleon

the streakplatebuttheyoccurred in theareaof the

third or fourth cross streak, the colony count was

recordedasbeing>100perplate.Thispolicywasfelt tobe themostconsistent andrepresentativebut it did result in reporting higher colony counts than were actuallyobserved with the SBA(002) and SBA(air) methods.

The quantitativerecoveryratesarepresentedas a

rough comparison byusinganonstandardized

inocu-lum for each of the five media. However, preliminary studies in our laboratory have shown that when a

throat swabisplatedtothe surface of sixplates,there was no detectable difference in either the types of bacteria isolatedorintheir relative numbers.

RESULTS

Of the 696 throat swabscultured, 204 (29.3%)

werepositive forgroupAstreptococci byatleast

oneof the five methods used for eachspecimen.

Nosingle method recoveredallof the 204group

Astreptococci recovered by all five methodsas

awhole(Table 1). The SXT-BA (CO2) method

yielded the highest recovery rate (90.7%)

fol-lowedby the SXT-BA (air) (87.7%), the PP+B

(83.3%), the SBA (CO2) (79.4%), and the SBA

(air) (77%) methods.

Colony counts obtained by surface streak

plate methodsweregenerallyhigher than those

obtainedbythe PP+B method (Table 1). This

was to beexpected, however, from the

consid-erably larger inoculum size used in the former

methods and is an advantage of the surface

streakplate methods over the PP+B method.

It isimportantto notethat the colony

count-ing procedure described in Materials and

Meth-ods resulted in our reporting higher colony

countsby the SBA methods thanwereactually

visible. If, however, the actual number of

colo-nies presentare tabulated, the enhanced

sensi-tivity of the SXT-BA method is even more

TABLE 1. Recovery rates ofgroup Astreptococci from 696 throat cultures by five culture methods

Colony count of No. of positive throat culturesfor group A streptococci as detected by each culturemethod' groupA

strepto-cocci SXT-BA

(002)

SBA

(002)

SXT-BA (air) SBA (air) PP+B

<10 12 27 10 11 42

lOto100 28 12 30 14 27

>100 145 123 139 132 101

Totalpositive 185 (90.7) 162 (79.4) 179 (87.7) 157 (77) 170 (83.3)

cultures

aValues in parentheses indicate percent based on atotal of 204 cultures positive for group A streptococci as detectedby all five methods as a whole.

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EVALUATION OF SXT-BA PLATE 191

apparent:therewere77instances noted in which

the SXT-BA (002) method raised the colony

count by one or more quantitation categories

over that obtained by the SBA (002) method.

Similarly, therewere52such instances noted in

which theSXT-BA (air) method raised the

col-ony count by one or more quantitation

cate-gories over that obtained by the SBA (air)

method.

Table 2 shows thelevel of colonycountswhich

wentundetected byeach method (false-negative

cultures). Significantly, only about one-half of

the false-negative cultures which occurred for

theSXT-BA (002) andSXT-BA (air) methods

showed high colony counts (i.e., 10 to 100 or

>100 colonies perplate). In contrast,

approxi-mately 70% of the false-negative cultures which

occurred for the SBA (002), SBA (air), and

PP+Bmethodswereinthe highrange.Thatis,

theSXT-BA (002) and SXT-BA (air) methods

werethe least likely methodstoyield

false-neg-ativeresults when the colonycountswerehigh.

False-negative cultures where the colonycount

was <10 colonies perplate probably represent

chance variation expected when the swab

con-tains lownumbers ofgroup Astreptococci; we

have, therefore, excludedthem from

considera-tion.

Throat cultures whichyielded beta-hemolytic

streptococci-like coloniesthatprovednot to be

groupAstreptococciweredesignated

false-pos-itive cultures. The SXT-BA(002)andSXT-BA

(air) methods yieldedthe lowest rates of

false-positive cultures, 11% and 12.7% respectively

(Table 3). In contrast, for theSBA (CO2), SBA

(air), and PP+B methods, the rates of

false-positive cultures were 27%, 23.8%, and 22.7%,

respectively. Both the SXT-BA (002) and the

SXT-BA(air)methods reducedbyone-half the

number of unnecessary FA staining tests

per-formedonnon-groupAbeta-hemolytic

strepto-cocci-like colonies.

Beta-hemolyticstreptococci,groups

C,

F,and

G,areinhibitedontheSXT-BAplatesandwere

the primary cause of the higher false-positive

rates on SBA and PP+B plates. In addition,

beta-hemolytic gram-negativerods and

beta-he-molytic staphylococci also wereresponsible for

significantnumbers offalse-positivecultures by

the PP+Bmethod.

Inaseparate study using the SXT-BA

(GO2)

method alone, wefound that the isolation rate

of group A streptococci was significantly

in-creased byincubating plates up to 48 h before

discarding asnegative. Of 2,536 SXT-BA

(GO2)

plates which were negative after overnight

in-cubation, an additional 77 isolates (3%) were

positive afteranadditional day of incubation. In

three-fourths of the cases, the colony counts

were >10 per plate.

DISCUSSION

It is clear that a positive throat culture for

group Astreptococciis not adefinite indication

of streptococcal pharyngitis even in

sympto-maticpatients(12). Inmost cases of

streptococ-cal pharyngitis, a positive throat culture

will

show colonycounts of >100 colonies per plate

by the direct surface streak plate technique.

TABLE 3. False-positive cultures obtained by each culture method

Method Total no. of false-positive culturesa

SXT-BA(002) 23 (11)

SBA(002) 60(27)

SXT-BA (air) 26(12.7)

SBA(air) 49(23.8)

PP+B 50(22.7)

aAfalse-positive culture is a culture which yielded

beta-hemolyticstreptococci-likecolonies thatproved

not tobe group Astreptococci.Valuesinparentheses indicatepercent,determinedby dividing the number of instances in whichagivenmethoddetected beta-hemolytic streptococci-likecolonies into the number of instances in which the isolates were foundto be otherthan group Astreptococci,and thenmultiplying

by100.

TABLE 2. Magnitude of colonycountofgroup Astreptococciwhichwereundetectedbyeach culture method Corresponding highest No. offalse-negativeculturesa

colonycountofgroup Astreptococci

ob-servedbyapositive SXT-BA(GO2) SBA(002) SXT-BA(air) SBA (air) PP+B method

<10 10 12 11 12 10

lOto100 5 17 8 21 20

>100 4 13 6 14 4

Total no. of false- 19(9.3) 42 (20.6) 25(12.3) 47 (23) 34 (16.7)

negative cultures

aAfalse-negative culture is onewhichisnegative for group A

streptococci

by

the methodindicated but

whichwaspositivebyatleastoneoftheremaining4methods. Values inparenthesesindicatepercent.

9,

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192

However, occasionally the same colony counts

maybe found incultures from carriers of group

A streptococci (12). Practically speaking,

how-ever, ifapositivecultureis fromasymptomatic

patient,mostphysicianswill treat,regardless of

theamountofgroupAstreptococcifound.

The use of sulfamethoxazole plus

trimetho-prim in throat culture media to reduce the

in-hibitoryeffect of normal throat florabacteriaon

the growth and/or beta hemolysis ofgroup A streptococci wasexcellently discussed byGunn

et al. (6). This selectivity of SXT-BA platesis

the reason for their superiority over the SBA

plate. The superiority of the SXT-BA method

over the PP+B method that we found in this

studyisdue both to theselectivityofthe

SXT-BA method and to the larger inoculum size it

employs. The minimal inoculum size

recom-mended for the PP+B method is based upon

two assumptions. First, that use ofa small

in-oculum size will prevent normal flora bacteria

fromovergrowing and,thereby,frompreventing

detection of group A streptococci which can

occurbyusingaheavier inoculum size. Second,

that colonycountswillalways be highincases

of streptococcal pharyngitis. From ourown

ex-perience with the PP+B method, the first

as-sumptionappearstobe correct.Inmanythroat

culturesthere is averyheavygrowth of

alpha-hemolytic streptococci, facultative

gram-nega-tive rods, and Staphylococcus aureus. These

organisms oftenovergrowandpreventdetection

of the group A streptococci unless a smaller

inoculum size is used, asin the PP+B method

(10). The second assumption, however,

contra-dictsthe previousreportofKaplanetal.(7)who

found that one-third of thepatients with

phar-yngitis had lessthan 10colonies perplate, but

hadaserologicalresponseindicative of infection.

Ourdata indicate that the PP+B method with

its lowinoculum size isobviouslytooinsensitive

to detect these cases. In fact, Pike (10) has

calculated that there would have to be 4,000

colony-forming units ofapure culture of

strep-tococci permlofbroth before therewas a 98%

probability that a 0.001-ml portion contained

streptococci. Preincubation of swabs for 2 h in

broth followed by plating has been shown to

yieldup to 17%morepositive culturesthancan

be obtained by immediate plating (9). We did

notusethisenrichmenttechnique, sincewefeel

thatit istoo expensiveandimpracticalfor

rou-tine useandthat itmayyield misleading

infor-mation by failing to differentiate between the carrier and the infected state (11). This differ-entiation might ultimately be made by direct,

selectivequantitation ofgroupAstreptococciin

throat cultures, such as by using the SXT-BA

(GO2)

method. In this manner, it may bepossible

toshowacloser correlationbetween

high

colony

countsanda positive serologicalresponse than

observed

by Kaplan

et

al.,

who useda

nonselec-tive

plating

method.

However,Gordisetal.(5)found in their

study

that one-third of thepatientswhodevelop

rheu-maticfever had beenrecently cultured and had

negative results. The negative cultures were

probablyduetovariables suchaspoorcollection

technique,

inadequate survival and/or

over-growthintransportmedia,andpoor culture and

isolation techniques.Thisindicates that

micro-biologists should do all they can to recover as

many group A streptococcias

possible.

Although

the 11.4% greater recovery rate of

group Astreptococciobtained with the SXT-BA

(C02) method as compared to the SBA (GO2)

wassignificant, itwas not asgreat as the28%to

42% valuespreviously reported (6). The reason

forthese differences may be related to the fact

that ourthroat cultures wereall fromchildren,

whereas60% of thepatients studied by Gunnet

al. were adults. It ispossible there may be

dif-ferences between the concentrations of normal

flora and group A streptococci

colony-forming

units found in infected children and infected

adults. Morerecently,Gunn hasfound that the

differences in his isolation rates, using SXT-BA

(GO2) and SBA (GO2) plates, are now running

between 11 to 20%-much closer to what we

encountered in this study (personal

communi-cation).

Anapparentparadox of the SXT-BA method

isthat92% ofgrou,pAstreptococci, when tested

in thymidine-free media, cannot grow in the

presence of the 25 ,ig of

sulfamethoxazole-tri-methoprimper ml used intheSXT-BAmedium

(1). However, the Trypticase soy agar used for

the SXT-BA plates contains thymidine, which

group A streptococci are able to utilize,

circum-venting the metabolic inhibitory effect of 25 ig

ofsulfamethoxazole-trimethoprim per ml.

Viri-dansstreptococci and other normal throat flora

bacteria are less able to use the thymidine and

are thusgenerally inhibited (S. R. M. Bushby,

personalcommunication).

Unfortunately, the precise amount of

thymi-dine required to achieve the optimal selective

effect is unknown. Various commercial media

havebeen shown to varymarkedly in thymidine

content (8), and only Trypticase soy agar has

beenproven to be reliable for theSXT-BA

(O2)

method (6). Of some concern is the finding that

various lots of Trypticase soy broth also vary

considerably in thymidine content (S. R. M.

Bushby, personal communication). Our data

showed that whenincubation was limited to 24

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EVALUATION OF SXT-BA PLATE 193

h, in 9 instances the SXT-BA (CO2) method

failedtodetect group A streptococci in

quanti-ties of -10 to 100 colonies per plate, a

false-negative rate of 4.4%.Gunn et al. (6) reported a

false-negativerate of 1.4%(7/508) for the

SXT-BA(CO2)method. Hisvalue is considerably less

than what we observed and may indeed be due

to differences in thymidinecontent among

dif-ferent lots ofcommercial Trypticase soy agar

used.

Wefound,however, that incubating our

SXT-BA (CO2) plates for up to 48 h resulted in an

additional 3% recovery of group A streptococci. The 1.4% false-negative rate we could expect,

then, after 2 days of incubation is identical to

that reported by Gunn et al. using 24 h of

incubation of SXT-BA (CO2) plates.Thus,

pro-longing the incubation of SXT-BA (CO2) plates

to 48h mayreducethe effects of variable

thy-midinecontentin lots of Trypticasesoy agar. In

viewof thealready discussedimportance of

us-ing as sensitive a culture method as

possible,

microbiologists may wish to include an SBA

(CO2) platetodetectanadditional 1.4% positive

cultures.

In our experience, use of Streptococcus

py-ogenes ATCC 19615

(Bactrol,

Difco

Laborato-ries)to serve as apositive control for growthand

for beta

hemolysis,

ashas been

suggested (6),

is

satisfactory. It may be

possible

and

desirable,

however,to

accurately

establish theamount of

thymidinenecessary toaddto a

thymidine-free

basal mediumtomaximize inhibition of normal

throat floraby

sulfamethoxazole-trimethoprim,

withoutinhibitinggrowth ofany group A

strep-tococci.

Ourlaboratoryprocessesanaverageof

37,000

throatculturesperyear.By

using

theSXT-BA

(CO2) method instead of thePP+B method in

ourlaboratory, we have calculated aminimum

savings of $5,000peryear. This

savings

isbased

upon elimination of the

accompanying

broth

culture andextramaterials neededinthe PP+B

methodandupon

saving

anaverage ofatleast

8 sof worktimeperculture.TheSXT-BA

(CO2)

method is also

considerably

less tedious and

requires less hand

manipulations

than the PP+B

method.

Additionally,

wefindthatsmearsmade

directly

from colonies onSXT-BA

(CO2) plates

for FA

staining

generally

showgreater numbers

offluorescing

cells,

and individual cell walls stain

more

uniformly

positive

than with the PP+B

method (3). Based upon the substantial

eco-nomic and logistic advantages and upon the

superior sensitivity for detection of group A

streptococci,we recommendthe SXT-BA(GO2)

throat culturemethod of Gunn et al. (6).

Hope-fully, commercial media producers will offer

SXT-BA plates for laboratories unable to

pre-paretheirown.

ACKNOWLEDGMENTS

We thank Phil Wand for usefulsuggestions, Dennis Maki and ArlanHelstad for reviewing the manuscript, and Marcia Kirkegaard for technical assistance.

LITERATURE CITED

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3. Ederer, G. M., and S. S. Chapman. 1972. Simplified fluorescent-antibodystaining method for primary plate isolates of group Astreptococci. Appl. Microbiol. 24: 160-161.

4. Facklam, R. R. 1974. Streptococci, p. 96. In E. H. Len-nette, E. H.Spaulding, and J. P. Truant (ed.), Manual ofclinicalmicrobiology,2nd ed. AmericanSocietyfor Microbiology, Washington, D.C.

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6. Gunn,B.A., D. K. Ohashi, C. A. Gaydos, and E. S. Holt. 1977. Selective and enhanced recovery of group Aand B streptococci from throat cultures with sheep blood agarcontaining sulfamethoxazole and trimetho-prim. J. Clin. Microbiol. 5:650-655.

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8. Koch,A.E.,and J. J. Burchall.1971.Reversal of the antimicrobialactivity of trimethoprim by thymidine in commerciallypreparedmedia.Appl. Microbiol. 22:812-817.

8a.Kurzynski, T.,C.Meise,R.Daggs,and A.Helstad. 1979.Improved reliabilityof theprimary plate bacitra-cintest on throatcultures with sulfamethoxazole-tri-methoprimbloodagarplates.J.Clin. Microbiol. 9:144-146.

9. Moody, M. D., A. C. Siegel, B. Pittman, and C. C. Winter. 1963. Fluorescent-antibody identification of group Astreptococci from throat swabs.Am.J. Public Health53:1083-1092.

10. Pike, R.M. 1945.The isolation ofhemolytic streptococci

fromthroat swabs:experimentswith sodium azideand crystalvioletinenrichment broth.Am.J.Hyg. 41:211-220.

11. Stollerman, G.H. 1962. Therole of the selectivethroat culture for betahemolyticstreptococciinthediagnosis

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